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https://openalex.org/W2097538272
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https://www.nature.com/articles/srep05498.pdf
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English
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The image-scratch paradigm: a new paradigm for evaluating infants' motivated gaze control
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Scientific reports
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cc-by
| 6,875
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* These authors
contributed equally to
this work. OPEN SUBJECT AREAS:
COGNITIVE
NEUROSCIENCE
HUMAN BEHAVIOUR Michiko Miyazaki1,2*, Hideyuki Takahashi1,3*, Matthias Rolf3, Hiroyuki Okada1 & Takashi Omori1 Received
28 October 2013
Accepted
12 June 2014
Published
30 June 2014 1Brain Science Institute, Tamagawa University, 2School of Social Information Studies, Otsuma Women’s University, 3Graduate
School of Engineering, Osaka University. Human infants show spontaneous behaviours such as general movement, goal-directed behaviour, and
self-motivated behaviour from a very early age. However, it is unclear how these behaviours are organised
throughout development. A major hindrance to empirical investigation is that there is no common
paradigm for all ages that can circumvent infants’ underdeveloped verbal and motor abilities. Here, we
propose a new paradigm, named the image-scratch task, using a gaze-contingent technique that is adaptable
to various extents of motor ability. In this task, participants scratch off a black layer on a display to uncover
pictures beneath it by using their gaze. We established quantitative criteria for spontaneous eye-movement
based on adults’ gaze-data and demonstrated that our task is useful for evaluating eye-movements motivated
by outcome attractiveness in 8-month-olds. Finally, we discuss the potential of this paradigm for revealing
the mechanisms and developmental transitions underlying infants’ spontaneous and intentional
behaviours. I I
nfants show various spontaneous behaviours from early in life, such as general movement1, self-exploration2,
goal-directed behaviour3,4, motivated behaviour5,6, and intentional behaviour7,8. In this article, we refer to some
behaviour as ‘spontaneous’ when it is independent of external inputs but is driven by lower- or higher-level
internal (e.g. non-reflective, habitual, motivated, or intentional) states. This is a broad term including various
behaviours from non-meaningful bodily movement to intentional behaviour. In the early stages of life, infants
move their hands and arms arbitrarily and independently of external stimuli, which is referred to as general
movement1. Several months later, infants gradually begin to generate actions that are driven by their own
motivation or intention8 (i.e. intentional control). Clarifying when and how these spontaneous behaviours pass from lower to higher levels is one of the great
issues of developmental science. In particular, it is important to illustrate using quantitative measurements how
these behaviours develop. In the case of experiments with adults, verbal reports or complex hand manipulation
are usually used to evaluate the inner state of the subject9,10. However, infants’ large variance of motor abilities throughout various developmental stages prevents us from
using such measures for cross-age comparisons11. Correspondence and
requests for materials
should be addressed to
H.T. (hideyuki@ams.
eng.osaka-u.ac.jp) OPEN The number of those who detected the gaze-contingency was 27. Twenty-four of them were controlling the red circle intentionally. Questionnaire report of spontaneous eye movement in adults. We
categorized those individuals who satisfied the following conditions
as the spontaneous group: the individual who felt they were control-
ling their gaze intentionally, plus they detected the gaze-contingency. The number of those who detected the gaze-contingency was 27. Twenty-four of them were controlling the red circle intentionally. The ratio of adults showing spontaneous scratching in the with-
gaze-point condition (16 out of 19) was significantly higher than that
observed in the no-gaze-point condition (8 out of 17; Fisher’s exact
test; x2 5 5.57, df 5 1, p 5 0.033, Q 5 0.23; Table 1). y
g
y
The ratio of adults showing spontaneous scratching in the with-
gaze-point condition (16 out of 19) was significantly higher than that
observed in the no-gaze-point condition (8 out of 17; Fisher’s exact
test; x2 5 5.57, df 5 1, p 5 0.033, Q 5 0.23; Table 1). screen, by comparing the infant data to adults’ experimental results
on the same task. We established quantitative measures for spontan-
eous gaze control based on the adults’ experimental results. We then
evaluated the eye movements of the 8-month-old infants by these
measures (Experiment 1). Eye movements reflecting spontaneity in adults. The eye movements
of adults showing spontaneous scratching contrasted with the eye
movements of adults showing passive viewing (typical eye move-
ments of both groups are shown in Supplemental Videos S1 and
S2). Figure 2 shows typical eye movement trajectories of adults show-
ing spontaneous and passive viewing. The left and middle panels of
Figure 2 show that the exposed areas in the second phase (Phase II)
are larger for the spontaneous adult than for the passive adult. The
right side of Figure 2 depicts samples of the gaze trajectories of both
an adult showing spontaneous scratching and an adult showing
passive viewing during the interruption phase. The spontaneously
scratching adult looked at the unscratched black layer and moved
her/his gaze rapidly, whereas the adult showing passive viewing only
gazed at the already exposed area. We interpret rapid, black-directed
eye movement during the interruption phase as exploratory beha-
viour caused by a prediction error that indicates acquisition of the
gaze-scratch contingency. OPEN A non-verbal experimental method and criterion that is
adaptable to various ages, producing comparable measurements across ages, is required for a precise understand-
ing of the process of emergence of spontaneous behaviour. Here, we propose a new experimental task using the gaze-contingent technique (i.e. online interactive eye
tracking) to quantify spontaneous behaviour, including higher-order processing (e.g. motivation or intention), in
infants. The task was named the image-scratch task (Fig. 1A). In this task, colourful, attractive pictures covered by
a black layer are presented on a display equipped with an eye tracker, and participants are able to scratch off the
black layer by gaze control and expose the picture beneath the layer. We hypothesised that, if the participants
become aware of the novel contingency between their gaze coordinate and the change of stimulus, they will try to
scratch off the black layer by controlling their gaze, being motivated by exploring the hidden, colourful picture. The gaze-contingency paradigm has the advantage of being able to investigate the development of spontaneous
behaviours in infants regardless of the variance of motor abilities in infancy because voluntary gaze control
develops earlier than limb control12–15. This task is expected to adapt to various ages in the same manner and
become a promising method for quantitative cross-age comparison of spontaneous behaviour in infants. To demonstrate the usefulness of the image-scratch task for evaluating the extent of spontaneous, motivated
behaviour, we examined whether 8-month-olds can control their gaze spontaneously to scratch the layer from the SCIENTIFIC REPORTS | 4 : 5498 | DOI: 10.1038/srep05498 1 www.nature.com/scientificreports www.nature.com/scientificreports Figure 1 | The image-scratch task. (A) Participants are able to scratch off
the black layer using their gaze. This photo courtesy of Michiko Miyazaki
with permission of the participants. (B) Two experimental conditions. In
the with-gaze-point (WGP) condition, a small red circle is presented
simultaneously at the point where the participants’ gaze is. In the no-gaze-
point (NGP) condition, the red circle is not presented. (C) The task
includes five trials; each displaying one picture for 30 s. Pictures in trials 1
and 4 are solid simple geometric patterns (Phases I and II). To reveal
subjective prediction errors indicating acquisition of the gaze-scratch
contingency, a 10-s interruption phase, in which eye movements cannot
scratch off the black layer, is inserted in the middle of the fifth trial. OPEN We define the exploration rate during this
phase as the proportion of gazes aimed at the black layer in relation to
all gazes directed towards the display. Several previous studies have used the gaze-contingent tech-
nique16,17. However, none of these studies has managed to distinguish
infants spontaneous gaze control from reflex-based eye movements
induced by the mere visual saliency of the stimuli. To evaluate
whether the eye movements of infants are truly driven by outcome
attractiveness regardless of the visual saliency factor, we conducted
two additional control experiments. Firstly, we reduced the outcome
attractiveness in Experiment 2 by using monochromatic (greyscale)
images as hidden pictures instead of colourful pictures (See
Supplemental Information) in order to investigate whether the
infants were truly motivated to scratch off the black layer and to
expose the hidden pictures. Secondly, to examine whether infants
were truly influenced by the gaze-scratch contingency instead of
mere visual saliency, we performed a yoked control experiment using
the presentation of a movie that showed exactly what had been on the
screen of other infants classified as showing spontaneous scratching
(See Supplemental Information, Experiment 3). Figure 3 shows individual plots of the exploration rate during the
scratch interruption phase for each participant group (adults/
infants) and each condition (no-gaze-point/with-gaze-point). In OPEN To collect sufficient samples of adults in both spontaneous
scratching and passive viewing, we prepared two conditions that
differed in difficulty (easy/difficult-to-detect contingency). In the
easy condition, a small red circle was presented simultaneously at
the point where the participants’ gaze was, making it easier for the
participants to find the gaze–scratch contingency (the with-gaze-
point [WGP] condition). In the difficult condition, the red circle
was not presented (the no-gaze-point [NGP] condition) (Fig. 1B). Forty adults participated in this study. The adults were instructed
to sit in front of the display and look at it, with no further instructions
regarding the task. In the image-scratch task, five pictures were pre-
sented one-by-one on the display for 30 s each (Fig. 1C). In the last
trial, a 10-s interruption phase was inserted within 20 s of normal
operation, the onset of the interruption being chosen randomly for
each participant between 7 s and 10 s after beginning of the trial. In
this phase, the eye movements of participants did not scratch off the
black layer, even if a fixation was detected. Instead, the image
remained unchanged. Before and after the interruption phase, gazes
scratched off the image as in all other trials. After the task, the adults
were asked to complete a questionnaire asking whether they noticed
the gaze–scratch contingency and scratched off the black layer
intentionally. Figure 1 | The image-scratch task. (A) Participants are able to scratch off
the black layer using their gaze. This photo courtesy of Michiko Miyazaki
with permission of the participants. (B) Two experimental conditions. In
the with-gaze-point (WGP) condition, a small red circle is presented
simultaneously at the point where the participants’ gaze is. In the no-gaze-
point (NGP) condition, the red circle is not presented. (C) The task
includes five trials; each displaying one picture for 30 s. Pictures in trials 1
and 4 are solid simple geometric patterns (Phases I and II). To reveal
subjective prediction errors indicating acquisition of the gaze-scratch
contingency, a 10-s interruption phase, in which eye movements cannot
scratch off the black layer, is inserted in the middle of the fifth trial. Questionnaire report of spontaneous eye movement in adults. We
categorized those individuals who satisfied the following conditions
as the spontaneous group: the individual who felt they were control-
ling their gaze intentionally, plus they detected the gaze-contingency. Results Therefore we tested a range
of different thresholds and evaluated the rate of correct classification
for each threshold value. In order to estimate the optimal threshold
we interpolated the resulting curve by means of a third order poly-
nomial and searched for its peak value within admissible thresholds. The optimal performance was reached at 88.9% correct classifica-
tions for a threshold value 0.187. Hence, defining a threshold of 0.187
on the exploration rate allows to distinguish adults showing spon-
taneous scratching from adults showing passive viewing with 88.9%
accuracy. We verified our method ex post by means of a leave-one-
out cross validation, which showed the same classification rate
(88.9%) as the direct data-driven estimation, which shows the
robustness of the estimation. the two plots on the left side of Fig. 3, the dark-orange plots refer to
the exploration rates of the spontaneously scratching adults. The
dark-green plots refer to those of the passively viewing adults. We
combined the data from the no-gaze-point and with-gaze-point con-
ditions based on these latter two categories. Results show that the
exploration rates of the adults showing spontaneous scratching are
significantly higher than those of the adults showing passive viewing
(t[33] 5 27.54, p 5 0.00000001, r 5 0.80). To find a criterion that
provides the best discrimination of adults showing spontaneous
scratching from adults showing passive viewing, we tested how well
the exploration rate could predict the questionnaire outcomes by
means of a simple threshold decision. Therefore we tested a range
of different thresholds and evaluated the rate of correct classification
for each threshold value. In order to estimate the optimal threshold
we interpolated the resulting curve by means of a third order poly-
nomial and searched for its peak value within admissible thresholds. The optimal performance was reached at 88.9% correct classifica-
tions for a threshold value 0.187. Hence, defining a threshold of 0.187
on the exploration rate allows to distinguish adults showing spon-
taneous scratching from adults showing passive viewing with 88.9%
accuracy. We verified our method ex post by means of a leave-one-
out cross validation, which showed the same classification rate
(88.9%) as the direct data-driven estimation, which shows the
robustness of the estimation. showing spontaneous scratching and passive viewing, the index does
not exhibit an optimal threshold that would allow a prediction of the
questionnaire reports with high accuracy. Results Taken together, the results obtained in the adult experiments show
the existence of two indices for spontaneous eye movement: size of
scratched area and exploration rate. Among them, the exploration
rate in the interruption phase has the best threshold for a high-
accuracy prediction of the questionnaire reports (88.9%). On the
other hand, the size of the scratched area has no threshold allowing
a better than 80%-accurate prediction of the questionnaire reports. Thus, we conclude that the exploration rate is the best predictive
indicator of spontaneous eye movement. Estimation of eye movement in infants. Twenty-two 8-month-old
infants participated in either the with-gaze-point or the no-gaze-
point condition. We adopted the threshold that was established in
the adult experiments to evaluate the eye movements of the infants
(see Fig. 3; above threshold, infants classified as showing spon-
taneous scratching; below threshold, infants classified as showing
passive viewing). Typical eye movements of spontaneous- and pass-
ive-classified infants can be seen in the Supplemental Informa-
tion (Supplemental Videos S3 and S4). The ratio of infants showing
spontaneous scratching in the with-gaze-point condition (8 out of
12) is significantly higher than that in the no-gaze-point condition (1
out of 9; see Table 1; Fisher’s exact test; x2 5 7.25, df 5 1, p 5 0.011, Q
5 0.58). This pattern is consistent with that observed in the exam-
ination of adults. This result suggests that most infants in the with-
gaze-point condition show eye movement similar to the adults who
executed the image-scratch task spontaneously. To validate our find-
ings further, we analysed whether the size of the scratched area sup-
ports our categorisation of infants. As mentioned above, the typical
adult showing spontaneous scratching scratched off a larger amount
of the black layer by shifting her/his fixation quickly compared to the
typical adult showing passive viewing (see Fig. 2). We found similar
tendencies in the size of the scratched area between the spontaneous
and passive participants in both the adult and infant groups. Cases of high exploration rates could also be explained by the
scratched area being rather small at the onset of the interruption. To examine this issue, we tested whether the exploration rate was
correlated to the size of the scratched area at the onset of interrup-
tion. We found no correlation between the scratched area and the
exploration rate both in adults (r 5 2.11) and infants (r 5 2.02). Results The
exposed areas are represented by the white circles. The exposed areas in
Phase II are larger for the spontaneous adult than for the passive adult. The
fixation duration is represented by the size of circles, with larger circles
indicating longer fixation time. The fixation circles obtained in Phase II are
smaller for the spontaneous adult than for the passive adult. In the
interruption phase, the spontaneous adult gazes at the unscratched black
layer and moves her/his gaze rapidly, whereas the passive adult only gazes
at the exposed area. Figure 2 | Typical eye-movement trajectories (A) adult showing
spontaneous scratching and (B) adult showing passive viewing. The
exposed areas are represented by the white circles. The exposed areas in
Phase II are larger for the spontaneous adult than for the passive adult. The
fixation duration is represented by the size of circles, with larger circles
indicating longer fixation time. The fixation circles obtained in Phase II are
smaller for the spontaneous adult than for the passive adult. In the
interruption phase, the spontaneous adult gazes at the unscratched black
layer and moves her/his gaze rapidly, whereas the passive adult only gazes
at the exposed area. Figure 3 | Exploration rate during scratch interruption. Individual plots
of gazing at the black area during the scratch interruption phase for each
participant group (adults/infants) and condition (no-gaze-point/with-
gaze-point). The dark-green, dark-orange, light-green, and light-orange
plots indicate adults in passive viewing, adults in spontaneous scratching,
infants in passive viewing, and infants in spontaneous scratching,
respectively. The dotted line refers to the threshold established for the
discrimination of intentionality in adults, which is used for the estimation
of spontaneity in infants. the two plots on the left side of Fig. 3, the dark-orange plots refer to
the exploration rates of the spontaneously scratching adults. The
dark-green plots refer to those of the passively viewing adults. We
combined the data from the no-gaze-point and with-gaze-point con-
ditions based on these latter two categories. Results show that the
exploration rates of the adults showing spontaneous scratching are
significantly higher than those of the adults showing passive viewing
(t[33] 5 27.54, p 5 0.00000001, r 5 0.80). To find a criterion that
provides the best discrimination of adults showing spontaneous
scratching from adults showing passive viewing, we tested how well
the exploration rate could predict the questionnaire outcomes by
means of a simple threshold decision. Results Experiment 1: efficient measurement of spontaneous eye move-
ment for scratching in adults and evaluation of eye movement in
8-month-old infants. To establish a measure of spontaneous gaze
control in adults, we compared the eye movements of those adults
who subsequently reported that they had discovered the action–
outcome contingency and scratched the layer with intention
(adults showing spontaneous scratching) with the behaviour of
those who reported that they did not notice the rule and simply
looked at the display without any intention (adults showing
passive viewing). Subsequently, we evaluated the eye movement of
infants using this measure. Table 1 | The numbers of adult and infant participants assigned to
spontaneous and passive groups
Condition
Spontaneous
scratching
Passive
viewing
Adults
with-gaze-point (n 5 19)
16
3
no-gaze-point (n 5 17)
8
9
Infants
with-gaze-point (n 5 12)
8
4
no-gaze-point (n 5 10)
1
9 Table 1 | The numbers of adult and infant participants assigned to
spontaneous and passive groups Table 1 | The numbers of adult and infant participants assigned to
spontaneous and passive groups SCIENTIFIC REPORTS | 4 : 5498 | DOI: 10.1038/srep05498 2 Figure 3 | Exploration rate during scratch interruption. Individual plots
of gazing at the black area during the scratch interruption phase for each
participant group (adults/infants) and condition (no-gaze-point/with-
gaze-point). The dark-green, dark-orange, light-green, and light-orange
plots indicate adults in passive viewing, adults in spontaneous scratching,
infants in passive viewing, and infants in spontaneous scratching,
respectively. The dotted line refers to the threshold established for the
discrimination of intentionality in adults, which is used for the estimation
of spontaneity in infants. www.nature.com/scientificreports Figure 2 | Typical eye-movement trajectories (A) adult showing
spontaneous scratching and (B) adult showing passive viewing. The
exposed areas are represented by the white circles. The exposed areas in
Phase II are larger for the spontaneous adult than for the passive adult. The
fixation duration is represented by the size of circles, with larger circles
indicating longer fixation time. The fixation circles obtained in Phase II are
smaller for the spontaneous adult than for the passive adult. In the
interruption phase, the spontaneous adult gazes at the unscratched black
layer and moves her/his gaze rapidly, whereas the passive adult only gazes
at the exposed area. Figure 2 | Typical eye-movement trajectories (A) adult showing
spontaneous scratching and (B) adult showing passive viewing. SCIENTIFIC REPORTS | 4 : 5498 | DOI: 10.1038/srep05498 Discussion The present study proposes a new paradigm named the image-
scratch task. It uses a gaze-contingent technique for quantifying
spontaneous behaviours by young infants to adults by means of
common measurements. Quantified behaviours include those driven
by higher-order processes (i.e. motivation or intention). Based on
results from adults, we have established a quantitative measure of
spontaneous gaze control in infants and have demonstrated that at 8
months, an infant’s gaze is driven, at least partially, by their motiva-
tion (see details in Supplemental Information, Experiment 2). We
found that the exploration rate during the interruption phase is an
efficient measure for detecting spontaneous gaze in adults. We
optimised a threshold on this rate for predicting participants’ ques-
tionnaire reports, with good results. Interestingly the ratio of
spontaneous adults was significantly higher in the with-gaze-point
condition than in the no-gaze-point condition. A seminal study by Wang et al. using the gaze-contingent para-
digm has addressed the quantification of infants’ gaze control using a
similar approach16. In their task, infants could press the button on an
eye tracker’s display by their gaze to play an attractive movie. The
authors demonstrated that 6- to 8-month-old infants would choose a
controllable button over an uncontrollable one, and that their eye
movements are similar to the eye movements of adults who realise
the contingency between their gaze and movie playing. Wang et al.’s
approach is innovative; however, the similarity of eye movements
between the infants and the adults who detect the contingency may
be no more than a superficial approximation and may not reflect a
common mechanism. Our findings suggest that the 8-month-olds can control their gaze,
which is driven by their motivation. However, our current findings
have not decided the issue of whether infants actually have an explicit
expectation of the outcome of their own behaviour17–19. Several pre-
vious studies have shown that younger infants can execute motivated
behaviours such as goal-directed behaviours20,21. However, such
behaviours are not necessarily to be interpreted as being accompan-
ied by explicit expectations22. Instead, they can be explained as habit-
ual responses23. For example, after learning that pulling a supporting
blanket is needed for reaching an out-of-reach toy, 16- and 24-
month-old infants soon stop pulling the blanket when the toy is gone;
in contrast, 8-month-old infants keep pulling the blanket5. Results These results suggest that high exploration rates were not caused by
the larger black area at the onset of interruption. The analysis of the adult eye movements yielded another signifi-
cant index of spontaneous eye movement in addition to the explora-
tion rate. This index is the difference in the size of the scratched area
from the first trial (Phase I) to the fourth trial (Phase II), which is here
termed the ‘‘intra-individual change of gaze control’’ from former to
latter trials (see details regarding the estimation of eye movement in
infants). As can be seen in the left and middle panels of Fig. 2, the
exposed areas in Phase II are larger for adults showing spontaneous
scratching than they are for adults showing passive viewing. Although the scratched area clearly differentiates between the adults A two-by-two, (Spontaneity: spontaneous vs. passive) 3 (Phase:
Phase I vs. Phase II) ANOVA analysis was conducted on the size of
the scratched area, revealing a significant interaction between
Spontaneity and Phase in both adults and infants (adults, F [1, 34]
5 4.77, p 5 0.036, g2 5 0.14; infants, F [1, 20] 5 6.29, p 5 0.02, g25 SCIENTIFIC REPORTS | 4 : 5498 | DOI: 10.1038/srep05498 3 www.nature.com/scientificreports Figure 4 | Mean proportion of the scratched area in Phases I and II. (A) In adults and (B) infants. The error bars indicate standard error. Figure 4 | Mean proportion of the scratched area in Phases I and II. (A) In adults and (B) infants. The error bars indicate standard error. 0.32; see Fig. 4). From the simple main effect test, the effect of
Spontaneity in Phase I is not significantly different between the
two groups (adults, F [1, 34] 5 2.12, p 5 0.21, g2 5 0.06, non-
significant [n.s.]; infants, F [1, 20] 5 1.94, p 5 0.24, g2 5 0.09,
n.s.), whereas in adults, this effect is significantly different between
the groups in Phase II. In infants, this effect is marginally significant
(adults, F [1, 34] 5 16.34, p 5 0.001, g2 5 0.32; infants, F [1, 20] 5
3.21, p 5 0.18, g2 5 0.14). Results The effect of Phase in Passive group is not
significantly different between the two groups (adults, F [1, 34] 5
0.07, p 5 0.80, g2 5 0.002, n.s.; infants, F [1, 20] 5 0.07, p 5 0.79, g2
5 0.003, n.s.), whereas this effect is significantly different between
the groups in the Spontaneous group (adults, F [1, 34] 5 8.02, p 5
0.016, g2 5 0.19; infants, F [1, 20] 5 14.52, p 5 0.004, g2 5 0.42). in Supplemental Information, Experiments 2 and 3). In Experiment
2, we evaluated whether infants’ eye movements were truly derived
from the motivation for exploring attractive pictures. If infants’ eye
movements were not passive, but spontaneous, the size of the
scratched area should be reduced when the attractive colourful pic-
tures were replaced by monotone grayscale images (non-attractive
condition). The results suggest that the size of the scratched area is
indeed reduced in the non-attractive condition, whereas the explora-
tion rate remains high. In Experiment 3, we examined the possibility
that infants’ spontaneous eye movements are driven by reflex-based
gaze evoked by the saliency of visual images. Most infants categorised
as spontaneous were continuously exposed to a red circle indicating
their gaze point during the image-scratch task. The movement and
visual saliency of the red circle possibly evoked reflex-based gaze and
led to their different gaze pattern. To exclude this possibility, we
performed a yoked control using playback movies of the screen
contents seen by the spontaneous infants, but in a non-contingent
condition. The result supports the contention that the spontaneous
infants controlled their gaze independently of the saliency of the
visual stimulus. Taken together, these findings suggest that 8-
month-old infants spontaneously control their gaze, which is driven
by motivation in our task. Furthermore, the proportion of short fixations shows similar tend-
encies in both adults and infants (see Supplemental Information,
Figure S1). In the second phase, the proportion of short fixations is
higher in the spontaneous group than in the passive group. SCIENTIFIC REPORTS | 4 : 5498 | DOI: 10.1038/srep05498 Discussion Finally, in the last trial, another colourful picture was pre-
sented, and a 10-s interruption phase was inserted into 20 s of normal operation, the
interruption onset chosen randomly for each participant between 7 s and 10 s after
the beginning of the trial. In this phase, the eye movements of participants did not
scratch off the black layer, even if a fixation was detected (Fig. 1C). Instead, the image
remained unchanged. Before and after the interruption phase, gazes scratched off the
black layer as in all other trials. Several studies have shown that infants older than 1.5 years can
control their actions depending on goal evaluation7,8,23; these infants
increase/decrease the pulling action depending on the attractiveness
of the reward toy. However, such a task cannot be applied to younger
infants because of their immature motor ability. Our task may be a
useful tool for examining goal evaluation in young infants. Although
in the present study we simply demonstrate that the difference in
outcome attractiveness leads to different behaviour, we can in the
future conduct goal evaluation experiments with infants younger
than 1.5 years using our paradigm. To get the participants’ attention, a movie with a voice saying ‘Look! Look!’ was
presented for 3 s before the presentation of each picture. Pleasant background music
was played while the eye tracker collected the eye-movement data. The music faded
out when the participant was not looking at the display. Both adults and infants
executed the same task. Another interesting topic is that the explicit visual presentation of
the gaze point as a red circle enhances spontaneous gaze control in
both adults and infants. It will be important to reveal the reason
underlying this finding, considering that both conditions are fun-
damentally identical with regard to the presence of an action–
outcome association. We consider that the visual feedback of the
gaze point may not only be a controllable object, but may also be
an aid to attributing the effect to the self-generated action (i.e. an
attribution of agency). Future studies should aim to clarify the rela-
tionship between the visual feedback of eye movement, the detection
of an action–outcome contingency, and the sense of agency in both
adults and infants. Procedure. In all experiments, participants were seated approximately 60 cm away
from the display and eye tracker. Discussion In the
latter case, the mere presence of the blanket is sufficient to evoke
the pulling behaviour that has been reinforced previously. If the
action of infants is based on explicit expectations, their behaviour
should instead be controlled according to behavioural goals. Their
behaviour would be exhibited/inhibited depending on the expected We adopted this threshold to estimate whether an infant scratched
the black layer off by motivation. The exploration rate of half of the
infants was over the threshold in the with-gaze-point condition (but
not in the no-gaze-point condition) and the validity of our method
was further supported by the mean size of the scratched area in
spontaneous infants, which was significantly higher than the size
of the area scratched by passive infants (see Experiment 1). From
these results, we can conclude that 8-month-old infants can control
their gaze for exploring the picture behind the black layer. Our method is based on a direct comparison of eye movements
between infants and adults. However, a possible limitation of this
direct comparison is that infants’ eye movements could be only
superficially similar to those of adults. That is, similar eye move-
ments could be driven by mechanisms (e.g. reflective eye movement)
different from those of adults, mechanisms that are not directly
accessible since infants cannot report directly as adults can. To check
for this issue, we conducted two additional experiments (see details SCIENTIFIC REPORTS | 4 : 5498 | DOI: 10.1038/srep05498 4 4 www.nature.com/scientificreports with a black layer. When an eye gaze was detected as a point on the display, a circular
area with a radius of 50 pixels was scratched off, and the corresponding area of the
picture became visible (Fig. 1A). There was no requirement of gaze fixation for the
scratching. the value of the outcome7,23. In the near future, we intend to test with
our image-scratch task whether infants’ spontaneous behaviour is
accompanied by explicit expectations. Verification would be inter-
esting because it strongly relates to the big issue of developmental
science: how self-consciousness and self-agency emerge through the
developmental process. Figure 1C shows an actual visual stimulus used in Experiment 1. To compare eye
movements between the first and fourth trials, the same pictures of solid simple
geometric patterns were presented in the first (Phase I) and fourth (Phase II) trials. To
increase the participants’ rate of exploration, colourful pictures were presented in the
second and third trials. Discussion The adults were only instructed to look at the
display, with no further instructions regarding the rules of the task. The infants, who
were fastened in a baby carrier to prevent them from standing up, sat on their
mothers’ lap, facing the display, and completed the same task. Stimuli were presented
immediately after the 9-point calibration was executed. After task execution, adult
subjects were asked to complete a questionnaire asking whether they had noticed the
gaze–scratch contingency and scratched off the black layer intentionally. Data rejection. We rejected data from those participants who gazed outside the
display for more than 80% of the mean time from the first to the fourth picture and
during the interruption phase. In Experiment 1, 5 adults and 16 infants were excluded
from further analyses. In addition, 13 infants were excluded because they did not
complete the task. We believe that the image-scratch task has great potential in devel-
opmental science because it permits quantification of infants’ spon-
taneous behaviour accompanied by motivation regardless of
differences in motor ability. We believe that our task design can be
refined further and can become a powerful tool for quantifying vari-
ous infants’ spontaneous behaviours, including those that are orga-
nised by higher-order cognitive processes. Furthermore, our results
seem consistent with results and concepts developed within con-
structive disciplines such as machine learning and cognitive devel-
opmental robotics24. Several studies have highlighted the importance
and functional significance of intrinsically motivated behaviour25,26,
such as behaviour driven by acquisition of novel information27 and
goal-directed behaviour driven toward task fulfilment despite initial
failure28,29. Integrating such findings into a cross-disciplinary con-
ceptualisation seems to be a promising goal for future work,
and could lead to improved hypothesis formulations and refined
experimental designs. For such progress, we must evaluate the
image-scratch task from an inter-disciplinary viewpoint, in light of
behavioural psychology, neuroscience, philosophy, and constructive
robotics. 1. Prechtl, H., Fargel, J., Weinmann, H. & Bakker, H. Postures, motility and
respiration of low-risk pre-term infants. Dev. Med. & Child Neurology 21, 3–27;
DOI:10.1111/j.1469-8749.1979.tb01577.x (1979). 2. Rochat, P. & Striano, T. Emerging self-exploration by 2-month-old infants. Dev. Sci. 2, 206–218; DOI:10.1111/1467-7687.00069 (1999). 3. Butterworth, G. & Hopkins, B. Hand-mouth coordination in the new-born baby. Br. J. Dev. Psychol. 6, 303–314; DOI:10.1111/j.2044-835X.1988.tb01103.x (1988). 4. van der Meer, A. L., van der Weel, F. R. & Lee, D. N. The functional significance of
arm movements in neonates. Discussion Science 267, 693–695; DOI:10.1126/science.7839147
(1995). 5. Frye, D. in: Children’s Theories Of Mind: Mental States And Social
Understanding. (eds Frye, D. & Moore, C.) 15–38. (Lawrence Erlbaum Associates,
1991). 6. Willatts, P. Development of means-end behavior in young infants: pulling a
support to retrieve a distant object. Dev. Psychol. 35, 651–667; DOI: 10.1037/
0012-1649.35.3.651 (1999). 7. Klossek, U. M. H., Russell, J. & Dickinson, A. The control of instrumental action
following outcome devaluation in young children aged between 1 and 4 years. J. Exp. Psychol. Gen. 137, 39–51; DOI:10.1037/0096-3445.137.1.39 (2008). 8. Klossek, U. M. H. & Dickinson, A. Rational action selection in 1K- to 3-year-olds
following an extended training experience. J. Exp. Child Psychol. 111, 197–211;
DOI:http://dx.doi.org/10.1016/j.jecp.2011.08.008 (2012). 9. Fourneret, P. & Jeannerod, M. Limited conscious monitoring of motor
performance in normal subjects. Neuropsychologia 36, 1133–1140; DOI:http://dx. doi.org/10.1016/S0028-3932(98)00006-2 (1998). Author contributions M.M. and H.T. developed the study concept and design. Testing, data collection, and
analysis were performed by M.M. and H.T. M.M., H.T., M.R., H.O. and T.O. wrote the
paper and approved the final version of the paper for submission. M.M. and H.T. developed the study concept and design. Testing, data collection, and
analysis were performed by M.M. and H.T. M.M., H.T., M.R., H.O. and T.O. wrote the
paper and approved the final version of the paper for submission. p
22. Bargh, J. A. Goal and intent: goal-directed thought and behavior are often
unintentional. Psychol. 1, 248–251; DOI:10.1207/s15327965pli0103_14 (1990). 23. Kenward, B., Folke, S., Holmberg, J., Johansson, A. & Gredeback, G. Goal
directedness and decision making in infants. Dev. Psychol. 45, 809–819;
DOI:10.1037/a0014076 (2009). Acknowledgments 17. Deligianni, F., Senju, A., Gergely, G. & Csibra, G. Automated gaze-contingent
objects elicit orientation following in 8-month-old infants. Dev. Psychol. 47,
1499–1503; DOI:10.1037/a0025659 (2011). This study was supported by the H. Nakayama Foundation for Science &Technology grant
to M.M., MEXT KAKENHI ‘‘Origins of the Social Mind’’, JSPS KAKENHI (23700322) to
M.M., and (23700321) to H.T. This study was also supported in part by MEXT KAKENHI
‘‘Constructive Developmental Science’’ (25119510), and JSPS KAKENHI ‘‘Constructive
Developmental Science Based on Understanding the Process from Neuro-Dynamics to
Social Interaction’’ (24000012). We are deeply thankful to K. Shibata, K. Matsumoto, K. Samejima, M. Sakagami, S. Shimojo and K. Izuma for their helpful comments; to C. Murai,
J. Kanero, A. Isawa, and K. Takata for research assistance; to I. Iguchi and M. Saito for data
collection; and to all the parents and infants who participated in this study. 18. Kenward, B. 10-Month-olds visually anticipate an outcome contingent on their
own action. Infancy 15, 337–361; DOI:10.1111/j.1532-7078.2009.00018.x (2010). f
y
j
19. Verschoor, S. A., Weidema, M., Biro, S. & Hommel, B. Where do action goals
come from? Evidence for spontaneous action–effect binding in infants. Front. Psychol. 1, 201; DOI:10.3389/fpsyg.2010.00201 (2010). y
p yg
20. Lewis, M., Alessandri, S. M. & Sullivan, M. W. Violation of expectancy, loss of
control, and anger expressions in young infants. Dev. Psychol. 26, 745–751;
DOI:10.1037/0012-1649.26.5.745 (1990). 21. Kalnins, I. V. & Bruner, J. S. The coordination of visual observation and
instrumental behavior in early infancy. Perception 2, 307–314; DOI:10.1068/
p020307 (1973). Methods Participants. Forty adults and 22 infants participated in Experiment 1: 21 adults were
included in the with-gaze-point condition (mean age 6 SD, 21.7 6 3.7 years; 12
women and 9 men) and 19 adults were included in the no-gaze-point condition
(mean age, 20.7 6 1.2 years; 13 women and 6 men); 12 infants participated in the
with-gaze-point condition (mean age, 8.6 6 0.4 months; 5 females and 7 males) and
10 participated in the no-gaze-point condition (mean age, 8.9 6 0.5 months; 4
females and 6 males). 10. David, N., Newen, A. & Vogeley, K. The ‘‘sense of agency’’ and its underlying
cognitive and neural mechanisms. Conscious Cogn. 17, 523–534; DOI:10.1016/
j.concog.2008.03.004 (2008). 11. Rovee-Collier, C. The development of infant memory. Curr. Dir. in Psychol. Sci. 8,
80–85; DOI: 10.1111/1467-8721.00019 (1999). 12. Aslin, R. N. in: Eye Movements: Cognition And Visual Perception (eds Fisher, D. F.,
Monty, R. A. & Senders, J. W.) 31–51. (Erlbaum, 1981). All participants were recruited from the participant pool of Tamagawa University
(Tokyo, Japan) via telephone calls or email. All adults and parents of infants gave
written informed consent before participating in the study. This study was carried out
in accordance with the guidelines approved by the Ethics Committee of Tamagawa
University. 13. Johnson, M. H. in: The Cognitive Neurosciences (ed Gazzaniga, M. S.) 735–747. (MIT Press, 1995). 14. Morrone, M. C. & Burr, D. C. Evidence for the existence and development of visual
inhibition in humans. Nature 321, 235–237; DOI:10.1038/321235a0 (1986). 15. van de Weijer-Bergsma, E., Wijnroks, L. & Jongmans, M. J. Attention
development in infants and preschool children born preterm: a review. Infant
Behav. Dev. 31, 333–351; DOI:http://dx.doi.org/10.1016/j.infbeh.2007.12.003
(2008). Apparatus and stimuli. Eye gaze was measured using a Tobii near-infrared eye
tracker (T120; Tobii Technology AB) which was integrated with a 17-inch LCD
monitor. The display resolution was 1024 3 768 pixels and the sampling rate was
60 Hz. A standard 9-point calibration was used. The task programming was
completed in Visual Basic 6.0 and Tobii Eye Tracker SDK. 16. Wang, Q. et al. Infants in control: rapid anticipation of action outcomes in a gaze-
contingent paradigm. PLoS One 7, e30884; DOI:10.1371/journal.pone.0030884
(2012). In the image-scratch task, five pictures were presented sequentially on the display
for 30 s each. Just after the presentation of a picture, the whole display was covered SCIENTIFIC REPORTS | 4 : 5498 | DOI: 10.1038/srep05498 5 Additional information 24. Asada, M. et al. Cognitive developmental robotics: a survey. Auton. Ment. Dev. IEEE Trans. 1, 12–34; DOI: 10.1109/TAMD.2009.2021702 (2009). Supplementary information accompanies this paper at http://www.nature.com/
scientificreports 25. Schmidhuber, J. Formal theory of creativity, fun, and intrinsic motivation (1990–
2010). Auton. Ment Dev. IEEE Trans. 2, 230–247; DOI: 10.1109/
TAMD.2010.2056368 (2010). Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Miyazaki, M., Takahashi, H., Rolf, M., Okada, H. & Omori, T. The
image-scratch paradigm: a new paradigm for evaluating infants’ motivated gaze control. Sci. Rep. 4, 5498; DOI:10.1038/srep05498 (2014). How to cite this article: Miyazaki, M., Takahashi, H., Rolf, M., Okada, H. & Omori, T. The
image-scratch paradigm: a new paradigm for evaluating infants’ motivated gaze control. Sci. Rep. 4, 5498; DOI:10.1038/srep05498 (2014). 26. Oudeyer, P. Y., Kaplan, F. & Hafner, V. V. Intrinsic motivation systems for
autonomous mental development. Evol. Comput. IEEE Trans. 11, DOI: 265-286;
10.1109/TEVC.2006.890271 (2007). 27. Gottlieb, J. Attention, learning, and the value of information. Neuron 76, 281–295;
DOI: http://dx.doi.org/10.1016/j.neuron.2012.09.034 (2012). This work is licensed under a Creative Commons Attribution 4.0 International
License. The images or other third party material in this article are included in the
article’s Creative Commons license, unless indicated otherwise in the credit line; if
the material is not included under the Creative Commons license, users will need
to obtain permissionfrom the licenseholderin order toreproduce the material. To
view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Von Hofsten, C. An action perspective on motor development. Tre 28. Von Hofsten, C. An action perspective on motor development. Trends. Cogn. Sci. 8, 266–272; DOI: http://dx.doi.org/10.1016/j.tics.2004.04.002 (2004). 29. Rolf, M. Goal Babbling For An Efficient Bootstrapping Of Inverse Models In High
Dimensions. PhD thesis, Bielefeld University, (2012). SCIENTIFIC REPORTS | 4 : 5498 | DOI: 10.1038/srep05498 6
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Research trends, challenges, and emerging topics of digital forensics: A review of reviews
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Aalborg Universitet Research Trends, Challenges, and Emerging Topics in Digital Forensics
A Review of Reviews
Casino, Fran; Dasaklis, Thomas K.; Spathoulas, Georgios P.; Anagnostopoulos, Marios;
Ghosal, Amrita; Borocz, Istvan; Solanas, Agusti; Conti, Mauro; Patsakis, Constantinos
Published in:
IEEE Access Citation for published version (APA):
Casino, F., Dasaklis, T. K., Spathoulas, G. P., Anagnostopoulos, M., Ghosal, A., Borocz, I., Solanas, A., Conti,
M., & Patsakis, C. (2022). Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of
Reviews. IEEE Access, 10, 25464-25493. https://doi.org/10.1109/ACCESS.2022.3154059 Citation for published version (APA):
Casino, F., Dasaklis, T. K., Spathoulas, G. P., Anagnostopoulos, M., Ghosal, A., Borocz, I., Solanas, A., Conti,
M., & Patsakis, C. (2022). Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of
Reviews. IEEE Access, 10, 25464-25493. https://doi.org/10.1109/ACCESS.2022.3154059 Aalborg Universitet Aalborg Universitet General rights
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Casino, F., Dasaklis, T. K., Spathoulas, G. P., Anagnostopoulos, M., Ghosal, A., Borocz, I., Solanas, A., Conti,
M., & Patsakis, C. (2022). Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of
Reviews. IEEE Access, 10, 25464-25493. https://doi.org/10.1109/ACCESS.2022.3154059 FRAN CASINO
1,2, (Member, IEEE), THOMAS K. DASAKLIS3, GEORGIOS P. SPATHOULAS
4,
MARIOS ANAGNOSTOPOULOS
5, AMRITA GHOSAL
6, ISTVÁN BO ¨RO ¨CZ7,
AGUSTI SOLANAS
1, (Senior Member, IEEE), MAURO CONTI
8,9, (Fellow, IEEE),
AND CONSTANTINOS PATSAKIS
2,10 FRAN CASINO
1,2, (Member, IEEE), THOMAS K. DASAKLIS3, GEORGIOS P. SPATHOULAS
4,
MARIOS ANAGNOSTOPOULOS
5, AMRITA GHOSAL
6, ISTVÁN BO ¨RO ¨CZ7,
AGUSTI SOLANAS
1, (Senior Member, IEEE), MAURO CONTI
8,9, (Fellow, IEEE),
AND CONSTANTINOS PATSAKIS
2,10 1Department of Computer Engineering and Mathematics, Universitat Rovira i Virgili, 43007 Tarragona, Spain
2Information Management Systems Institute, Athena Research Center, 151 25 Marousi, Greece
3Hellenic Open University, 570 01 Patras, Greece
4Norwegian University of Science and Technology (NTNU), 2802 Gjøvik, Norway
5Aalborg University, 9220 Copenhagen, Denmark
6CONFIRM Centre, University of Limerick, Limerick, V94 T9PX Ireland
7Vrije Universiteit Brussel, 1050 Brussels, Belgium
8Department of Mathematics, University of Padua, 35122 Padua, Italy
9Faculty of Electrical Engineering, Mathematics and Computer Science, Delft University of Technology, 2628 CD Delft, The Netherland
10Department of Informatics, University of Piraeus, 185 34 Piraeus, Greece
Corresponding author: Constantinos Patsakis (kpatsak@unipi.gr) 1Department of Computer Engineering and Mathematics, Universitat Rovira i Virgili, 43007 Tarragona, Spain
2Information Management Systems Institute, Athena Research Center, 151 25 Marousi, Greece
3Hellenic Open University, 570 01 Patras, Greece
4Norwegian University of Science and Technology (NTNU), 2802 Gjøvik, Norway
5Aalborg University, 9220 Copenhagen, Denmark
6CONFIRM Centre, University of Limerick, Limerick, V94 T9PX Ireland
7Vrije Universiteit Brussel, 1050 Brussels, Belgium
8Department of Mathematics, University of Padua, 35122 Padua, Italy
9Faculty of Electrical Engineering, Mathematics and Computer Science, Delft University of Technology, 2628 CD Delft, Th
10Department of Informatics, University of Piraeus, 185 34 Piraeus, Greece
Corresponding author: Constantinos Patsakis (kpatsak@unipi.gr) This work was supported in part by the European Commission under the Horizon 2020 Programme (H2020), as part of the projects
LOCARD under Grant 832735, HEROES under Grant 101021801, and the CyberSec4Europe under Grant 830929; and in part by the
European Commission (call ISFP-2020-AG-TERFIN) as part of the CTC Project under Grant 830929. The work of Fran Casino was
supported by the Beatriu de Pinós programme of the Government of Catalonia under Grant 2020 BP 00035. ABSTRACT Due to its critical role in cybersecurity, digital forensics has received significant attention from
researchers and practitioners alike. The ever increasing sophistication of modern cyberattacks is directly
related to the complexity of evidence acquisition, which often requires the use of several technologies. To date, researchers have presented many surveys and reviews on the field. However, such articles focused
on the advances of each particular domain of digital forensics individually. Received January 22, 2022, accepted February 16, 2022, date of publication February 24, 2022, date of current version March 10, 2022. eceived January 22, 2022, accepted February 16, 2022, date of Digital Object Identifier 10.1109/ACCESS.2022.3154059 FRAN CASINO
1,2, (Member, IEEE), THOMAS K. DASAKLIS3, GEORGIOS P. SPATHOULAS
4,
MARIOS ANAGNOSTOPOULOS
5, AMRITA GHOSAL
6, ISTVÁN BO ¨RO ¨CZ7,
AGUSTI SOLANAS
1, (Senior Member, IEEE), MAURO CONTI
8,9, (Fellow, IEEE),
AND CONSTANTINOS PATSAKIS
2,10 Therefore, while each of these
surveys facilitates researchers and practitioners to keep up with the latest advances in a particular domain of
digital forensics, the global perspective is missing. Aiming to fill this gap, we performed a qualitative review
of all the relevant reviews in the field of digital forensics, determined the main topics on digital forensics
topics and identified their main challenges. Despite the diversity of topics and methods, there are several
common problems that are faced by almost all of them, with most of them residing in evidence acquisition
and pre-processing due to counter analysis methods and difficulties of collecting data from devices, the cloud
etc. Beyond pure technical issues, our study highlights procedural issues in terms of readiness, reporting and
presentation, as well as ethics, highlighting the European perspective which is traditionally stricter in terms
of privacy. Our extensive analysis paves the way for closer collaboration among researcher and practitioners
among different topics of digital forensics. I. INTRODUCTION under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/
VOLUME 1 Downloaded from vbn.aau.dk on: October 24, 2024 Downloaded from vbn.aau.dk on: October 24, 2024 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/b I. INTRODUCTION principle is relatively straightforward, it is difficult in many
cases to apply. This is why Locard introduced forensics labs
in Law Enforcement Agencies (LEAs) over the first decade
of the 20th century [3]. According to Edmond Locard’s exchange principle, in every
crime, the perpetrator will alter the crime scene by bringing
something and leaving something else [1], [2]. Therefore,
these changes can be used as forensic evidence. While this While procedures that resemble digital forensics are men-
tioned in computer science literature quite early, the domain
was not fully defined until 1980s when it started to gain
attention. The introduction of the IBM PC generalised the The associate editor coordinating the review of this manuscript and
approving it for publication was Ilsun You
. 25464 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews use of computing machines; thus, more interest was focused
on digital evidence and many people came together and cre-
ated a digital forensics community, which eventually became
more formal in 1993 when the FBI hosted the First Interna-
tional Conference on Computer Evidence [4]. Initially, the
main activity was examining standalone computers to recover
deleted or destroyed files from the disks. However, since
the early 2000s, the digital forensics domain has expanded
steadily, maturing along with regulations [5], [6]. Nowadays,
users tend to utilise multiple digital devices and access tenths
of digital services per day [7], [8]. The digital footprint of
our everyday life has become enormous, and accordingly
the probability that illegal activities leave digital evidence
behind is very high. The need for forensic investigators has
increased, and this have led to multiple academic education
and certification programs related to digital forensics [9]. Additionally, the complexity of the tasks to be carried out
and the required compliance with law and courts’ regulations
has led to the establishment of strict protocols and proce-
dures to be followed [10]–[12]. The continuous appearance of
new forms of cybercrime also requires adaptive investigation
process models, new technology, and advanced techniques to
deal with such incidents [13]–[15]. Moreover, research directions and approaches, that could
be applied in several domains, remain explored in a topic-
wise manner, lacking interoperability, and denoting a lack
of collaboration between researchers in different forensics
domains. 1https://ec.europa.eu/commission/presscorner/detail/en/MEMO_18_
3345 B. CONTRIBUTION According to a thorough methodological research, we collect
all relevant surveys and reviews in the field of digital foren-
sics, analyse them, and answer a set of research questions, listed in Table 1, by performing the following actions: • Analysing the current state of the art and practice, and
identifying the challenges of each domain individually. • Assessing whether the current state of the art is aligned
with the technological evolution in digital forensics. • Using the previously collected information to identify
common issues, gaps, best strategies and key focus
areas in digital forensics, trying to span across different
domains. • Assessing technological advances to highlight emerging
challenges in digital forensics. • Assessing technological advances to highlight emerging
challenges in digital forensics. Beyond the rise of cybercrime, where the evidence is
expected to be digital, digital evidence is underpinning almost
all modern crime scenes. For instance, mobile devices have
become a primary source of digital evidence as almost all
our communications are performed through them [6]. In fact,
according to EU,1 the bulk of criminal investigations (85%)
involve electronic evidence. Thus, emails, cloud service
providers, online payments, and wearable devices are often
used to extract digital evidence in various circumstances. In addition to suggesting promising research lines in the
field based on the above analysis, we cover other dimensions
of digital forensics, including frameworks and process mod-
els, standardisation, readability and reporting, as well as legal
and ethical aspects. To the best of our knowledge, this is the
first review of reviews covering the state of the art in digital
forensics and showcasing the actual state of practice from a
global perspective. The remainder of the article is organized as follows:
Section II details our research methodology, providing a
descriptive analysis of the retrieved literature, which is
then complemented with a taxonomy of digital forensics in
Section III. Section IV analyses the current state of practice
regarding forensic methodologies and their phases, standards,
and ethics. Relevant open issues, trends, and further research
lines are discussed in Section V. The article concludes in
Section VI with some final remarks. I. INTRODUCTION We sustain that the above is a serious gap in current
literature, and we aim to fill it in this article. To this end,
we present a review of reviews in the field of digital forensics. A. MOTIVATION Digital evidence has become a norm and underpins most
modern crime investigations. However, there are digital evi-
dence to which different methods and methodologies apply. Some principles may remain the same; however, they cannot
be applied to all types of evidence. For instance, collect-
ing evidence from the Cloud bears no resemblance to IoT
forensics or image forensics. This has led to a huge amount
of research, which addresses the challenges raised in each
domain individually, with the bulk of the work devoted to
the development of novel tools and algorithms to extract
digital evidence and intelligence from heterogeneous sources. Currently, investigators devote many efforts to provide a
systematic overview of the literature and the advances in
each domain, with focused surveys and reviews. Despite the
importance of these surveys, an analysis considering the chal-
lenges and issues of the different digital forensics domains
as a whole is still missing. In other words, each of these
surveys is focused on a specific domain and, as a result,
common issues, challenges and methods are not identified. used to bring together, appraise, and synthesise the results of
related systematic reviews when multiple systematic reviews
on similar or related topics already exist [17], [18]. Therefore,
a review of reviews or an umbrella review compiles evidence
from multiple reviews or survey papers into a single docu-
ment. Syntheses of previous systematic reviews are known by
a variety of names, one of which is an umbrella review. Other
descriptions include the terms (‘‘review of reviews,’’ ‘‘sys-
tematic review of reviews,’’ ‘‘review of systematic reviews,’’
‘‘overviews of reviews,’’ ‘‘summary of systematic reviews,’’
‘‘summary of reviews,’’ and ‘‘synthesis of reviews’’) [19]. FIGURE 1. Detail of the research methodology steps. Despite their growing popularity, no standardized report-
ing guidelines currently exist for umbrella reviews. How-
ever, various multidisciplinary teams around the globe work
together to develop relevant standardized reporting guidelines
that will soon be available [20]. In our case, we rely upon
an entirely systematic way to conduct our umbrella review. In particular, we have used various features of the approach
presented in [21] to conduct our review of reviews and pro-
vide a transparent, reproducible and sound overview of the
scientific literature on digital forensics from a global perspec-
tive. Our review protocol consists of five steps, as shown in
Figure 1: 1) Planning the review 2) Defining research ques-
tions 3) Searching literature databases 4) Applying inclusion
and exclusion criteria and 5) Synthesising and reporting the
results of the literature analysis. FIGURE 1. Detail of the research methodology steps. TABLE 1. Summary of research questions and the corresponding sections devoted to answer them. TABLE 1. Summary of research questions and the corresponding sections devoted to answer them. TABLE 1. Summary of research questions and the corresponding sections devoted to answer them. TABLE 1. Summary of research questions and the corresponding sections devoted to answer them. s and the corresponding sections devoted to answer them. II. RESEARCH METHODOLOGY In recent years, academic publishing has significantly
increased both in terms of volume and speed. At the same
time, new channels for publication, such as conference pro-
ceedings, open archives and numerous scientific journals,
are rapidly expanding, thus allowing today’s researchers to
publish their work in a multitude of venues [16]. According
to recent studies, approximately 22 new systematic reviews
are published daily [17]. New methodological approaches
for synthesising this evidence have been developed to keep
up with the proliferation of systematic reviews across dis-
ciplines. Besides, conducting reviews of existing systematic
reviews has become a logical next step in providing evidence
in domains where a growing number of systematic reviews is
available. Overviews or umbrella reviews are most commonly 25465 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews B. SELECTION OF STUDIES We used various pre-defined exclusion and inclusion crite-
ria as described in Table 2 to assess the eligibility of the
retrieved literature; both academic and grey. Some exclu-
sion criteria were used before introducing the literature into
the bibliographic manager (language, subject area and doc-
ument type restrictions). It is also worth noting that we
have only examined review papers and reports written in
English. The distribution of publications over time is depicted in
Figure 2. In particular, Figure 2 shows a year-by-year analysis
of the selected papers. It is worth noting that the number of
publications has increased significantly after 2017. Until the
end of 2017, there were only about 38 review papers address-
ing issues of digital forensics. However, from 2017 onwards,
the number of reviews published in the scientific literature
has risen to nearly 70. As a result, over the last four years,
research in the area of digital forensics has slowly but steadily
increased. This upward trend reflects the key public and
policy impact of digital forensics nowadays. Our overall selection process steps are the following:
(i) We initially evaluated the relevance of the titles of all
scientific articles and reports. Articles/reports fulfilling one
of the exclusion criteria were removed from the analysis and
sorted according to the reason for their removal, (ii) In the
sequence, we evaluated the relevance of all paper abstracts
and report introduction sections (grey literature). Articles
and/or reports that met one of the defined exclusion criteria
were excluded from the analysis, and we documented the
reason for exclusion, (iii) We also did a full-text reading, and
some additional articles/reports were excluded and sorted by
reason of exclusion during this step. We resolved any poten-
tial disagreements among authors about the relevance of the
retrieved articles/reports through discussion until reaching a
unanimous consensus. We omitted several studies because
they were not reviews or surveys (for example, papers rel-
evant to financial forensics investigation, business forensics). We also discarded from the analysis articles that did not meet
the inclusion criteria. Figure 2 also shows the domain-specific distribution
of the 109 review papers included in our analysis. It is
worth noting that we have identified seven (7) prevalent
areas of research interest in digital forensics: Blockchain,
Cloud, Filesystem and databases, Multimedia, IoT, Mobile,
Networks. D. BIBLIOGRAPHIC ANALYSIS In this section, we present a descriptive analysis of the
scientific papers included in the challenges-based and
domain-specific classification (see Figure 2). The descrip-
tive analysis includes 109 research papers published from
2006 until the end of November 2021. The purpose of the
descriptive analysis presented is three-fold: We discovered additional studies using the so-called
snowball effect (backward and forward), which involved
searching the references of key articles and reports for addi-
tional citations [23]. For instance, additional grey litera-
ture was discovered by manually searching the reference
lists in several reports, particularly research and committee
reports or policy briefs from private and public sector institu-
tions/organizations. For this study, we take into consideration
109 research papers and 51 reports. The 109 papers are used
for identifying relevant challenges/trends across different dig-
ital forensics domains (see Section III ). The 51 reports were
used to derive further insights about the state of practice
regarding digital forensics methodologies, practices and stan-
dards, as well as discussing future trends and open challenges
from a policy perspective (see sections IV and V). 1) It enhances the statistical description, aggregation, and
presentation of the constructs of interest or their asso-
ciations of the relevant literature (publications per year
and domain etc.). 2) It contains insights to current research trends in the
area of digital forensics and a critical discussion of the
challenges identified. It, therefore, supports the classi-
fication structure presented in Section III 3) It allows us to visually demonstrate the diverse research
approaches used up to this point in the scientific lit-
erature regarding the proliferation of digital forensics
review papers. C. ANALYSIS AND REPORTING Electronic searches using Google also turned up relevant
grey literature, such as unpublished research commissioned
by governments or private/public institutions. In particular,
we looked at the first 200 Google results for the queries
digital forensics and reviews and digital forensics and surveys
to find the published grey literature. It is worth noting that
we used Google searches as a supplement to our primary
search strategy (especially for streamlining the assessment),
and Scopus and WoS were our primary source for finding
scientific-related literature. Furthermore, compared to the
bibliography retrieved from Scopus and WoS, the total num-
ber of documents retrieved from Google was relatively low. All articles and/or reports that met the inclusion criteria were
analyzed (in emerging themes) using a qualitative analysis
software (MAXQDA11). The authors carried out the the-
matic content analysis independently. We applied various
qualitative analysis methods (such as narrative synthesis and
thematic analysis) to classify and synthesise the extracted
data in a sound and comprehensive manner. The results of
our analysis are presented in sections III and IV. A. SEARCH STRATEGY As previously stated, our overall survey process is based
on several predefined research questions relevant to the dig-
ital forensics literature. We conducted extensive research
addressing the various technical/functional/security chal-
lenges of the digital forensics literature guided by these
research questions. To this end, we performed a systematic
literature search without time constraints in May 2021 which
was subsequently updated in November 2021. The main
search engines used were Web of Science (WoS), Scopus and Google. Scopus and WoS were used to locate all scientific-
related literature due to their multidisciplinary coverage and
scope [22], while Google was used to locate relevant stan-
dards and best practices (grey literature). We queried Scopus
and WoS using the terms ‘‘digital forensics and review or
survey’’ in the title, keywords, and abstract of all articles. It is
worth noting that first bulk search query yielded 536 unique
results (combining both sources). 25466 25466 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews A. CLOUD represent research conducted in areas such as social media,
smart grid, unmanned aerial vehicles and etc. Researchers, as well as government agencies, have thor-
oughly explored many of the challenges in cloud forensics,
though some challenges still remain to be addressed. For
example, the diversity of embedded OSs with shorter product
life cycles, as well as the numerous smartphone manufactur-
ers around the world present, are challenges in this research
area. In the literature, we can find research works that have
addressed challenges in cloud forensics and their solutions
from different perspectives. Purnaye et al. [7] explored the
different dimensions of cloud fornesics and categorised the
main challenges of this topic. Alex et al. [24] discussed chal-
lenges in cloud forensics related to data acquisition, logging,
dependence on cloud service providers, chain of custody,
crime scene reconstruction, cross border law and law presen-
tation. Khanafseh et al. [25] pointed out several challenges in
cloud forensics, such as the unification of logs format, miss-
ing terms and conditions in Service Level Agreement (SLA)
regarding investigations where service level agreement is the
main point and condition between the user and the cloud B. SELECTION OF STUDIES Multimedia forensics attracts most of the cur-
rent digital forensics research (38 out of the 109 review
papers), followed by Filesystem and database forensics
papers (18 out of 109). Both streams justify that the
widespread use of mobile devices with lower-cost storage and
increased bandwidth has resulted in a massive generation of
multimedia-related content. Furthermore, various miscella-
neous review papers (applications that do not fit into any of
the above categories) demonstrate the digital forensics mul-
tidisciplinary nature. These multidisciplinary review papers 25467 25467 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews TABLE 2. Selection criteria of the retrieved literature. TABLE 2. Selection criteria of the retrieved literature. FIGURE 2. Year-wise analysis of the selected literature per domain. FIGURE 2. Year-wise analysis of the selected literature per domain. FIGURE 2. Year-wise analysis of the selected literature per domain. III. TAXONOMY OF CHALLENGES-BASED DIGITAL
FORENSICS RESEARCH In this section, we summarise the surveys/literature reviews
collected following a rigorous statistical methodology based
on the literature, as described in Section II. The topics of this
classification have been systematically selected according to
the contents of reviewed literature, and thus reflect the digital
forensics research landscape and illustrates with high fidelity
the heterogeneity of digital forensic solutions. The classifi-
cation of digital forensics topics is graphically represented
in Figure 3. In each case, we discuss the main limitations
and challenges proposed in the literature. More precisely,
we extract the challenges at a research field domain level
(i.e., we group in a higher hierarchical level, when possible,
the limitations of the methods presented in the surveys) to
give a more comprehensive perspective and to enable further
cross-topic comparisons in Section III-I. 25468 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews FIGURE 3. Challenges-based and domain-specific mindmap abstraction of digital forensics topics identified
in the literature. FIGURE 3. Challenges-based and domain-specific mindmap abstraction of digital forensics topics identified
in the literature. forensic challenges based on the cloud forensic process
stages. Amminezhad et al. [31] described the different chal-
lenges in cloud forensics that were addressed by other authors
by performing an exploratory analysis. Rahman et al. [32]
broadly classified the existing challenges in cloud forensics,
classifying the literature into three categories, namely, multi-
tenancy, multi-location and scope of user control. Finally, the
authors in [33] identified and discussed the major challenges
that occur at each stage of the cloud forensic investigation,
according to well-known forensic flows. service provider, lack of forensics expertise, decreased access
to forensic data and control over forensics data at all level
from the customer side, lack of international collaboration
and legislative mechanism in cross-nation data access and
exchange, and lack of international collaboration and legisla-
tive mechanism in cross-nation data access and exchange. Pichan et al. [26] considered the Digital Investigative Pro-
cess (DIP) model [27] for describing the challenges emerging
at each phase of the digital investigation process and pro-
vided solutions for the respective identified challenges. The
challenges identified by the authors in cloud forensics are
unknown physical location, decentralized data, data dupli-
cation, jurisdiction, encryption, preservation, dependence on
CSP, chain of custody, evidence segregation, distributed stor-
age, data volatility and integrity. Similar to the works of
Khanafseh et al. B. NETWORKS Data monitoring and acquisition from network traffic are
mandatory to prevent most of nowadays cyber-attacks
[36]–[38], including, but not limited to, Distributed Denial
of Service (DDoS), phishing, DNS tunnelling, Man-in-the-
middle (MitM) attacks, SQL injection and others [39], [40]. Regardless of the orchestration mechanism behind them
(i.e., single attackers or orchestrated botnets), the analysis
and mitigation mechanisms rely on the proper monitoring and
analysis of computer network traffic to collect information,
evidence and proof of any intrusion detection or vulnerabil-
ity. For this purpose, several well-known tools exist, such
as network forensic analysis tools which provide function-
alities such as traffic sniffing, Intrusion Detection Systems
(IDS), protocol analysis, and Security Event Management
(SEM) [40]–[43]. Nevertheless, one of the challenges of
network forensics is to achieve accurate and efficient packet
analysis in encrypted network traffic since it is far more chal-
lenging than the analysis of unencrypted traffic. As authors
stated in [40], [44], utilizing machine learning in packet
analysis is evolving into a complex research field that aims
to address the analysis of unknown features and encrypted
network data streams. q
y
The diversity of embedded OSs with shorter product life
cycles, as well as the numerous smartphone manufacturers
around the world, stand out as significant challenges in the
MF domain [47]. In general, MF presents various challenges
due to a multitude of reasons. For example, in [48] the authors
identify the following limitations for successfully carrying
out MF investigations: 1) data-related issues (anonymity-
enforced browsing and other anonymity services, and the
considerable volume of data acquired during an investiga-
tion) 2) forensic tools-related issues (MF research approaches
have long focused on acquisition techniques, while minor
importance was given to the other phases of MF investigative
process) 3) device and operating systems diversity 4) security
aspects (development of new and more sophisticated anti-
forensic methods from the manufacturers) 5) cloud-related
issues (current MF tools do not consider cloud aspects,
cloud investigation barriers such as access to forensics data
due to multi-jurisdictional legal frameworks, forensics data
security) and 6) process automation. It is worth noting that
MF faces significant challenges concerning the focus of the
overall MF processes. For example, it is not clear whether
investigation procedures should be model-specific for each
device or should be generic enough to form a standardized
set of guidelines applicable to forensics procedures [49]. C. MOBILE Smartphones and mobile devices may contain valuable infor-
mation for a plethora of investigation purposes. Mobile foren-
sics (MF) is a sub-branch within the digital forensics domain
relevant to the extraction of digital evidence from portable
and/or mobile devices. Mobile forensics processes could be
broken down into the following three categories: seizure,
acquisition, and examination/analysis. III. TAXONOMY OF CHALLENGES-BASED DIGITAL
FORENSICS RESEARCH and Pichan et al., the authors in [28]
also identified the challenges in cloud forensics and ana-
lyzed them on the basis of their significance. Park et al. [29]
discussed the different challenges within cloud forensic
investigations highlighting the relevance of proactive mod-
els, and discussing the integration of smart environments
to enhance the robustness of forensic investigations. The
authors in [30] provided a categorization of the cloud g
As evident from the large number of publications in lit-
erature reviews/surveys, cloud forensics is quite an explored
research topic. Despite the considerable amount of research
in cloud forensics, there still exist a number of chal-
lenges/limitations that need much attention, as discussed
by NIST [34]. In Table 3, we present a summary of the
extracted challenges in the cloud forensic review/survey
articles. From this summary, we observe that there is a
dearth of research work focusing on cloud forensic stan-
dard tools and technologies in the cloud environment. Also,
very limited works have concentrated on pointing out the
feasible solutions related to the challenges present in cloud
forensics. 25469 25469 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews TABLE 4. High level extraction of challenges in network forensics. TABLE 4. High level extraction of challenges in network forensics. TABLE 3. High level extraction of limitations in cloud forensics. TABLE 3. High level extraction of limitations in cloud forensics. D. IOT Although significant in terms of improved data availability
and operational excellence, the broad adoption of IoT devices
and IoT-related applications have brought forward new secu-
rity and forensics challenges. IoT forensics is a branch of
digital forensics dealing with IoT-related cybercrimes and
includes the investigation of connected devices, sensors and
the data stored on all possible platforms. According to the literature, several of the current limita-
tions of IoT forensics include the management of different
streams of data sources, the complicated three-tier architec-
ture of IoT, the lack of standardized systems for capturing
real-time logs and storing them in a valid uniform form,
the preparation of highly detailed reports of all information
gathered its corresponding representation, the preservation
and acquisition of evidence considering its volatility and
value of data, and the adoption of routine forensic tasks in
the IoT ecosystem [52]–[56]. Data encryption trends also
present additional challenges for IoT forensic investigators,
and arguably cryptographically protected storage systems is
one of the most significant barriers hindering efficient dig-
ital forensic analysis [54], [57], [58]. Other studies high-
light additional limitations of IoT forensics processes such
as interoperability and availability issues related to the vast
amount of connected IoT devices [54]–[56], [59], the Big
Data nature of the IoT forensics evidence (Variety, Velocity,
Volume, Value, Veracity) [55], [58], [60] and the various
security storage challenges of IoT forensics evidence, espe-
cially when related to biometric data [61]. Finally, various
regulatory-related challenges also exist in the IoT forensics
domain, particularly issues relevant to the ownership of data
in the cloud as defined by region-specific laws [54]–[56],
[58], [59]. For instance, service-level agreements stipulating
the ‘‘terms of use’’ of the cloud resources between the cloud
customer and the cloud service provider do not incorporate B. NETWORKS Another challenge is the need to perform live forensics
(mobile device should be powered on) [50]. In addition,
an important barrier for actually conducting MF investiga-
tions relates to the various networking capabilities of smart-
phones, which render the overall MF processes difficult to
manage, particularly due to the complex structure of the cloud
computing environment [51]. Finally, due to the security
measures inherent to modern mobile devices, an investigator
must actually break into the device using an exploit that will
most likely alter the device data. Clearly, the latter violates
the Association of Chief Police Officers (ACPO) principle Regarding the research and forensics-related surveys tack-
ling such issues, several reviews recall the primary method-
ologies and tools for network forensic analysis, such as the
works seen in [36], [45], yet they were conducted almost
a decade ago. Therefore, taxonomies classifying forensic
frameworks suitable for Network Forensics are crucial [40]. An interesting review focusing on the attackers perspective,
in terms of attack behaviour and plan identification, as well
as prevention mechanisms, can be found in [46]. Finally,
some protocol-oriented reviews, analyzing IEEE 802.11
protocol [43], and more recently, 5G networks [42], dis-
cuss specific vulnerabilities in their corresponding contexts. In general, the main challenges of network forensics, as iden-
tified by the authors in the aforementioned works, are classi-
fied in Table 4. 25470 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews and introduces numerous procedural issues for a forensic
investigation. In Table 5, we provide a classification of MF
approaches’ current challenges. forensic investigations’ provisions. Legislative frameworks
adopted in specific regions, such as the GDPR in Europe, also
pose significant challenges for IoT forensic investigations,
particularly data privacy provisions [53]–[56]. Finally, the
use of blockchain and its capability to enhance IoT forensic
investigations has been also discussed in [54]. In Table 6
we provide a classification of the current challenges of IoT
forensics approaches. TABLE 5. High level extraction of limitations in mobile forensics. TABLE 5. High level extraction of limitations in mobile forensics. TABLE 6. High level extraction of limitations in IoT forensics. TABLE 6. High level extraction of limitations in IoT forensics. E. FILESYSTEMS, MEMORY AND DATA
STORAGE FORENSICS A system’s memory can contain evi-
dence related to the usage of the system, including the list of
running processes, network connections, or the keys for the
driver’s encryption. Usually, such data are not stored in the
permanent storage of the system and are completely lost when
the system is turned off or unplugged from the power. In the
literature, we can find surveys devoted to the analysis of the
memory acquisition techniques [66], [67] (i.e., both hardware
and software-based), the subsequent memory analysis [68],
and the available tools [67]. The main challenges of memory
forensics derive from the fact that memory is volatile, so it has
to be acquired when the system is running and thus probably
modified by the running applications. This can lead to the
page smearing issue [68], i.e., inconsistencies between the
state of the memory as described by the page tables compared
with the actual contents of the memory. Another issue that
can occur during the memory acquisition is the incorpora-
tion of pages, which are not present in the memory due to
page swapping or demand paging [68]. Finally, although the
memory acquisition techniques should be OS and hardware
agnostic [66], each OS architecture handles the memory dif-
ferently and is equipped with distinctive tampering protection
mechanisms that hinder access to memory. aggregation and analysis, as well as their structural architec-
ture to benefit forensic procedures [69], [73]. Digital triage
is of special relevance here since reviewing many poten-
tial sources of digital evidence for specific information by
using either manual or automated analysis is mandatory to
enhance investigations [73]. Nevertheless, the authors high-
light that the legitimacy of several acquisition procedures is
constrained by the applicable legislation and that the current
state of practice requires more efficient solutions, especially
when dealing with huge amounts of data. In [74], the authors
presented a framework for database forensic investigations
enhanced by forensic experts’ opinions with the aim to over-
come the main issues that investigator’s face, such as the lack
of standardized tools and different data structures and log
structures. y
A database (DB) is the most traditional way to organise
and store data. The majority of applications and online ser-
vices deploy some type of DB to store records about their
customers, financial records, inventory, etc. E. FILESYSTEMS, MEMORY AND DATA
STORAGE FORENSICS Forensic analysis of large filesystems requires efficient meth-
ods to manage the potentially large amount of files and
data contained in them. System logs are one of the most
used information sources to leverage forensic investigations. In [62] the authors provide a review of the publicly available
datasets used in operating system log forensics research and
taxonomy of the different techniques used in the forensic
analysis of operating system logs. The taxonomy is cre-
ated based on a common investigation format that includes
event logs recovery, event correlation, event reconstruction
and visualization. Distributed filesystem forensics is even a
more challenging task, such as in the case of identifying
the malicious behaviour of the attackers by analysing cloud
logs [63]. Nevertheless, the accessibility attributes associated
with cloud logs impede the goals of investigating such infor-
mation, as well as other challenges, similar to those extracted
in Section III-A. Another challenging area is the analysis of proprietary
systems such as SCADA systems. In [64] the authors present
a survey on digital forensics that are applied to SCADA
systems. The survey describes the challenges that involve 25471 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews TABLE 7. High level extraction of challenges in file system, memory and
data storage forensics. TABLE 7. High level extraction of challenges in file system, memory and
data storage forensics. applying digital forensics to SCADA systems as well as
the range of proposed frameworks and methodologies. The
work also focuses on the research that has been carried out
to develop forensic solutions and tools that can be tailor-
made for the SCADA systems. Recent research has revealed
that malware developers have been using a broad range of
anti-forensic techniques and escape routes in-memory attacks
and system subversion, including BIOS and hypervisors. In addition, code-reuse attacks such as returned oriented
programming pose a serious remote code execution threat. To neutralise the effects of malicious code, specific tech-
niques and tools such as transparent malware tracers, system-
wide debuggers were proposed. In [65], authors present a
survey on the state-of-the-art techniques that demonstrate the
capability of thwarting the anti-forensic strategies previously
mentioned. Memory forensics refers to the forensic analysis of a sys-
tem’s memory dump. E. FILESYSTEMS, MEMORY AND DATA
STORAGE FORENSICS Besides the vast
amount of data that could be contained in a DB, a database
management system (DBMS) which allows the end-users
to administer the DB and store and access the data in a
specific format, can also provide evidence of actions in user-
level granularity. For instance, it can reveal who and when
stored/accessed specific records. Therefore, digital forensics
for DB has attracted the attention of the research commu-
nity [69]. From this perspective, several surveys focused on
database digital forensics based on the log files, metadata,
and similar types of artefacts for the case of relational and
NoSQL DB [70]–[72]. Furthermore, other authors addressed
the digital forensic opportunities on the procedure of data Considering the increasing amount of IoT technologies
and small devices that require live data analysis due to the
volatility of the data stored in them, it is crucial to develop
new strategies to enhance data acquisition procedures [75]. In the context of database forensics and data acquisition, the
challenges of big data analysis and data mining techniques
for digital forensics [76], [77], and text clustering [78] were
investigated. Moreover, a survey of techniques to perform
similarity digest search is provided in [79]. Table 7 summarises the main limitations and challenges
extracted from the literature analysed in this section. 2https://wearesocial.com/blog/2020/01/digital-2020-3-8-billion-people-
use-social-media,
https://www.cisco.com/c/en/us/solutions/collateral/
executive-perspectives/annual-internet-report/white-paper-c11-741490.
html F. BLOCKCHAIN Besides the financial domain to
which it was initially applied, through bitcoin, blockchain
technology is currently used in various other use cases such
as supply chain management, cybersecurity enhancement,
document/certificates validation, crowdfunding campaigns,
and more [80]. Additionally, because financial system set
on blockchain provide more privacy than traditional pay-
ment systems, it is common for cryptocurrencies to be used
for criminal activities [81]. This sets blockchain forensics
methodologies as a necessity [82] due to the large volume of
data that are stored in blockchain systems and the number of
processes that are managed by such systems. The main prop-
erty of blockchain-based systems is the guaranteed protection
of data integrity, which is directly related to forensic analysis. On the one side, this property makes forensic analysis more
manageable. However, on the other side, this may complicate
the process as users may be more cautious when interacting
with such systems. It has to be noted that a large portion of blockchain systems
are public, allowing access to everybody and thus making
forensic analysis a surplus process. A forensics investigator
can set up a node in a public blockchain network, sync it
with the rest of the nodes and obtain a local copy of the
ledger. Even in such cases, the structure of the information
stored in the ledger of blockchain systems is not optimal
with respect to retrieving all required data (e.g., for a specific
account or a specific smart contract), so efficient mechanisms
are required [83] to extract valuable information out of the
large volume of data stored in public ledgers [84]. In the
case of private blockchain systems, the ledger data are not
publicly available and traditional forensics approaches have
to be applied to blockchain nodes to extract data. TABLE 8. High level extraction of challenges in blockchain forensics. TABLE 8. High level extraction of challenges in blockchain forensics. F. BLOCKCHAIN Blockchain technology has been constantly integrated into
existing systems or used as the basis to rebuild systems from 25472 VOLUME 10, 2022 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews While the data stored in the ledger are of great impor-
tance, there are more data to be considered when analyzing
a blockchain node. The ledger holds all committed transac-
tions, but a blockchain node stores more information with
respect to its interactions with other nodes or clients. For
example, the IP of the client that has connected to a node to
submit a transaction or the activity of a specific node in the
network (e.g., sync requests) are not included in the ledger’s
data. On top of those, multiple security blockchain attacks
are mainly targeted against the infrastructure or the network’s
backbone and not against its content. Mining attacks, network
and long-range attacks [89], [90] target at taking control of the
blocks formation process, to maliciously alter past committed
transactions and achieve double-spending attacks. In such
cases, digital evidence from deployed nodes is the only way
to prove malicious activity. At the same time, the size of the
network in public blockchain systems makes it even harder to
retrieve the required evidence. Table 8 summarises the main
challenges extracted from the blockchain forensics literature. scratch in various domains. Besides the financial domain to
which it was initially applied, through bitcoin, blockchain
technology is currently used in various other use cases such
as supply chain management, cybersecurity enhancement,
document/certificates validation, crowdfunding campaigns,
and more [80]. Additionally, because financial system set
on blockchain provide more privacy than traditional pay-
ment systems, it is common for cryptocurrencies to be used
for criminal activities [81]. This sets blockchain forensics
methodologies as a necessity [82] due to the large volume of
data that are stored in blockchain systems and the number of
processes that are managed by such systems. The main prop-
erty of blockchain-based systems is the guaranteed protection
of data integrity, which is directly related to forensic analysis. On the one side, this property makes forensic analysis more
manageable. However, on the other side, this may complicate
the process as users may be more cautious when interacting
with such systems. scratch in various domains. G. MULTIMEDIA Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews TABLE 9. High level extraction of challenges in multimedia digital
forensics. TABLE 9. High level extraction of challenges in multimedia digital
forensics. several examples of anti-forensic methodologies [129], such
as encryption, data obfuscation (e.g., trail obfuscation), arti-
fact wiping, steganography and image tampering [130],
protected/hidden communications (e.g., tunnelling, onion
routing), malware anti-sandbox/debug, VM and in general
anti-analysis methods [131]–[134], and spoofing. As stated
in [135], anti-forensic methods exploit the dependence of
human elements on forensic tools, as well as the limita-
tions of the underlying hardware in terms of architecture
and computational power. Therefore, enhancing the train-
ing and knowledge level of investigators and more robust
forensic procedures (e.g., anti-anti forensic techniques [130])
are critical to minimise the impact of anti-forensics. In this
line, some authors argue that the use of proactive foren-
sics models could help enhancing the robustness of forensic
investigations [136]. TABLE 9. High level extraction of challenges in multimedia digital
forensics. g
[
]
Another emerging topic in digital forensics is related to
unmanned aerial vehicles (UAVs), or more commonly known
as drones [137]. The applications and versatility of these
devices are becoming more popular in a myriad of contexts,
from industrial to military applications. One of the main
challenges of drone forensics is the set of different hardware
components that are part of a drone [138], and the partic-
ular treatment that they require (i.e., with special regard to
advanced anti-forensic techniques taking place [139], as well
as the necessity of live forensics [137], [140] in this context). For instance, drones consist of sensors, flight controllers,
electronic and hardware components, on-board computers,
and radiofrequency receivers, each one linked to one or many
evidence sources in terms of, e.g., data storage (the differ-
ent memory sources present in the drone, such as memory
cards storing media, or other software), data communications
and other logs and data stored in sources related to the
drone, such as the drone controller and external cloud-based
sources [141], [142]. At the moment of writing, there are no
baseline principles, standards, nor legislation covering all the
particularities of forensic drone investigations [137], [142]. Thus, efforts towards the establishment of sound protocols,
specific forensic frameworks, as well as drone-based forensic
tools are critical [137]. G. MULTIMEDIA passive techniques to detect forgery [105], or carving on
specific file formats such as JPEG [106]. Other image foren-
sics surveys analysed topics such as hyperspectral image
[92], [107], image authentication [108], the affectation of
noise in images [109] and image steganalysis [110]–[114]. Another set of surveys focus on the specific context of
child abuse material and its detection through image and
video analysis [115]–[118]. More recently, the advent of
deep learning techniques has enhanced the capabilities of
image integrity detection and verification, outperforming tra-
ditional methods in several image-related tasks, especially in
these where anti-forensic tools were used [113], [114], [119]. In the context of video files, we can find surveys on video
steganalysis [113], [114], [120], video forgery detection
[95], [96], [98], [114], [121], [122], video forensic tools [95],
[113], [123], [124], video surveillance analysis [125], [126],
and video content authentication [127]. Finally, digital audio
forensics has also been studied in [128]. Table 9 summarises
the main limitations and challenges extracted from the multi-
media forensic literature. In [143], authors surveyed the different dimensions and
concerns which digital forensics should cover in the context
of social networks. The authors discussed several aspects
of social networks, such as privacy and security issues, the
criminal and illegal acts that can occur, and the attacks on the
underlying platform and the users. In addition, they describe
several strategies to detect such abnormal behaviours along
with the necessity to develop both pro-active and reactive
mechanisms. In terms of community detection, graph analytic
methods and tools are crucial to detect criminal networks in
different contexts, such as finance, terrorism, and other het-
erogeneous sources [144]. In [8], authors surveyed the efforts
done so far on the analysis of social network shared data
according to source identification, integrity verification and
platform provenance. Moreover, authors discussed the cur-
rent methodologies, and highlighted the current challenges G. MULTIMEDIA Due to the increasing number of ubiquitous technologies
(e.g., IoT devices, smartphones, wearables) leveraged by the
4th industrial revolution, as well as a substantial improvement
in the connectivity capabilities in smart scenarios due to 5G,
the amount of multimedia prosumers (i.e., both producers
and consumers of data) is increasing dramatically year after
year.2 Nevertheless, such multimedia content growth is a
double-edged sword. On the one hand, it is a synonym of
opportunities for the industry, companies and users. On the
other hand, it augments the possible vulnerabilities and attack
vectors of such systems, which malicious users can exploit. Even if data are by default publicly available, it is still
challenging to identify malicious activity on such platforms. It is common for deployed smart contracts to suffer from var-
ious vulnerabilities either due to poor implementation or not
properly configured blockchain networks [85]. In such cases,
users can take advantage of such vulnerabilities, mainly aim-
ing at financial profit. It is challenging to detect such activity
and identify the actors that have initiated it. Smart contracts
execution is not a straightforward process, and past execution
cannot be easily repeated in a forensic sound way [86]. Apart
from that, smart contracts may also get self-destructed by a
special OPCODE that makes following past transactions even
harder [87]. Digital forensics in the context of multimedia has received
substantial attention from the research community. There
exist numerous image forgery detection surveys exploring
the topic from a global perspective [91]–[99]. In this con-
text, pixel-based image forgery detection is one of the main
topics [100], including image splicing forgery [101], and
copy-move forgery [102]–[104], which is a well-known tech-
nique in which parts of the current images are used to
cover/hide specific characteristics. Some authors focused on Furthermore, privacy concerns have been raised concern-
ing early open public blockchain systems, and thus, there
have been multiple alternative systems that make use of var-
ious privacy-enhancing techniques such as zero-knowledge
proofs, onion routing or ring confidential transactions to pro-
tect users privacy [88]. In such cases, forensics analysis of
either network nodes or users’ wallets is required to retrieve
either logs or cryptographic keys that can be used along with
data existing on public ledgers and provide more information
about the transactions that have taken place. 25473 25473 VOLUME 10, 2022 VOLUME 10, 2022 F. H. MISCELLANEOUS This section is devoted to the digital forensics reviews that fall
beyond the domain categorisation of the previous paragraphs. As observed in most topics, anti-forensics can be under-
stood as a standalone concern in digital forensics, which
requires investigation in each context. The term anti-forensics
refers to methods and strategies that prevent forensic inves-
tigators and their tools from achieving their goals. There are 25474 25474 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews along with the need for multidisciplinary approaches to over-
come them. reproducibility and pushes the advancement in the state of
the art, which is needed to keep up with the pace of tech-
nology development [148], [149]. The next most challenging
issue is related to anti-forensics methods, which has been
discussed in several sections of the taxonomy as well as in
Section III-H. Anti-forensic strategies leveraged by malicious
actors include adversarial methods such as obfuscation or
encryption applied to, e.g., data and storage systems, as well
as hardware-related technological challenges, such as mobile
phones due to their inherent security measures, or in the case
of drones due to their specific particularities, and software,
as well as in the case of malware. In the case of tools and eval-
uation benchmarks, it is evident that the community needs
to devote more efforts towards fighting novel cybercrime,
especially in topics where, e.g., different data sources and
technologies are present. For instance, in the case of IoT and
UAVs, different data sources may necessitate different digital
forensics strategies, including tools related to device level
forensics, network forensics, and cloud forensics. Another
challenge that affects digital forensics is the lack of juris-
dictional and legal requirements for different investigation
scenarios such as ethics and data management of confidential
and personal data. This is particularly relevant nowadays
due to the widespread use of distributed systems such as
blockchain and the cloud. The latter means that software
and data may reside in different countries, and thus, specific
cross-border collaborations are required, adding another layer
of complexity to digital investigations. Moreover, this sce-
nario impedes the adoption of proactive measures due to the
difficulty of applying measures that conform to different legal
frameworks. I. CHALLENGE ANALYSIS AND AGGREGATED RESULTS The classification of challenges and limitations according to
each topic of the taxonomy has been conducted to keep a
balance between accurate descriptions of challenges and hier-
archical classification. On the one hand, we want to facilitate
identifying the gaps and limitations of each topic and pro-
vide a clear path for both new and experienced investigators
towards the corresponding literature. On the other hand, and
as stated in Section I, we provide the reader with a clear
overview of the research lines that should be strengthened
in the digital forensics ecosystem, as well as their interre-
lations according to each topic of our taxonomy. Therefore,
we used the extracted challenges of each topic and merged
the ones appearing more than once (i.e., the ones appearing
only in their corresponding topic were ignored due to their
specificity) to create a comprehensive overview of the digital
forensics challenges in Table 10. As it can be observed,
we identified several limitations of digital forensics that can
be applied in several topics or contexts and thus, indicate the
need to devote more research efforts towards them. Note that,
for instance, the last topic of the Table 10 appears to be only
affecting IoT, yet we identified this challenge in the miscella-
neous topic, and thus, we decided to include it. Nevertheless,
since several topics are analysed in such a category, we did
not represent them in Table 10. A proper understanding between all the actors involved in
the digital forensics context, including stakeholders, LEAs,
and court members, is mandatory to ensure the success-
ful prosecution of perpetrators. In this regard, one of the
highlighted challenges is to ensure that all partners have a
sufficient level of training (including technical knowledge
and standardised guidelines) and a proper understanding,
including readable reports to enable a fruitful collaboration. Moreover, while it seems procedural, the chain of custody is
still a challenge. This can be attributed to multiple reasons,
such as obvious negligence of the corresponding personnel
to properly report evidence acquisition and/or handling, cor-
rupted officers, or even gaps in the process. Nevertheless, all
of them cause severe issues in a court as a case can be missed
or misjudged. Secure and auditable means of storing and
processing the chain of custody, as proposed by LOCARD3
with the use of blockchain technology seems like a logical and
stable solution. 3https://locard.eu/ H. MISCELLANEOUS A sector that is receiving increasing attention due to its crit-
ical relevance to the proper functioning of our society is the
energy sector, and more concretely, the smart grid. In [145],
authors explore practical cybersecurity models and propose
some guidelines to enhance the protection of the smart
grid against cyber threats. Moreover, they explore software-
defined networks and their main benefits and challenges. Finally, the authors propose a conceptual forensic-driven
security monitoring framework and highlight the relevance
of forensics by design in development phases. Context-aware
scenarios such as smart cities have been also receiving
increased attention due to their complex structures, requiring
the continuous data collection, processing and interaction
between a myriad of devices [146], [147]. Digital forensics
in this particular scenario is a recent paradigm which requires
further efforts from the research community to enhance
cyber resilience and to provide efficient incident response
mechanisms [147]. overcome them. The investigation and forensic analysis cat-
egory contains the highest number of challenges. Therefore,
the topics identified in the taxonomy share similar technical
concerns in their corresponding contexts, and more multidis-
ciplinary collaboration is needed towards such direction. The
reporting and presentation category highlights one yet crit-
ical challenge since the proper reporting of an investigation
affects the outcome of the whole investigation. We further dis-
cuss about forensic readability and reporting in Section IV-C. challenges, since logging and data acquisition in specific
scenarios may pose technical challenges. This issue is
exacerbated when coupled with cross-border investigation
requirements due to data fragmentation. Moreover, once data
corresponds to multiple forensic contexts, the complexity of
performing digital investigation grows exponentially, leaving
aside the need to perform live forensics according to the par-
ticularities of the hardware. Additionally, the availability of
some devices due to their resource-constraint nature is a fur-
ther challenge. For instance, IoT botnets have high volatility,
and UAVs may implement self-defence mechanisms, even at
the physical level. In the case of the Miscellaneous category,
we included the challenges and limitations of anti-forensics,
drone forensics, smart grid, smart cities and social networks. I. CHALLENGE ANALYSIS AND AGGREGATED RESULTS A more thorough description of forensic read-
ability and its challenges is discussed later in Section IV-C. The most reported challenge is the sound data acquisition
from heterogeneous sources and its interpretation, includ-
ing different hardware and monitoring processes collecting
data and logs dynamically. Note that data acquisition and
management is a challenge affecting activities related to dig-
ital forensics. Moreover, data fragmentation, a common sce-
nario nowadays, hinders investigations further. It is important
to note that data acquisition is critical to creating bench-
marks, which help researchers and practitioners to evalu-
ate their models. The latter enables characteristics such as Data acquisition, as previously stated, is not only a chal-
lenge in terms of the existing heterogeneous data sources and
context but also in terms of size. The big data era comes with
a myriad of opportunities but also with their corresponding 25475 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews TABLE 10. Cross-domain abstraction of the challenges and limitations of digital forensics, ordered by relevance according to the amount of times they
were found in the topics of the taxonomy. For the sake of fairness, the general column Miscellaneous has been omitted. TABLE 10. Cross-domain abstraction of the challenges and limitations of digital forensics, ordered by relevance according to the amount of times they
were found in the topics of the taxonomy. For the sake of fairness, the general column Miscellaneous has been omitted. TABLE 10. Cross-domain abstraction of the challenges and limitations of digital forensics, ordered by relevance according to the amount of times they
were found in the topics of the taxonomy. For the sake of fairness, the general column Miscellaneous has been omitted. TABLE 10. Cross-domain abstraction of the challenges and limitations of digital forensics, ordered by relevance according to the amount of times they
were found in the topics of the taxonomy. For the sake of fairness, the general column Miscellaneous has been omitted. IV. DIGITAL FORENSICS METHODOLOGIES,
PRACTICES AND STANDARDS In addition to the topic-based taxonomy presented in
Section III, we collected a set of literature reviews, included
in our research methodology, that analysed forensic frame-
works and process models, and the adaptability and forensic
readiness of the actual practices. In the following sections,
we analyse the content of such reviews by extracting the chal-
lenges and identifying the main qualitative features required
to achieve forensically sound investigations. According to the outcomes depicted in Table 10, we can
observe that topics such as IoT, cloud, and mobile are
affected by the highest amount of challenges. Therefore,
we believe that researchers and practitioners should devote
more efforts to solving such topics’ challenges by leveraging
cross-domain collaborations to enhance the quality and appli-
cability of their outcomes. Similarly, other challenges which
appear in several topics could be tackled more quickly if they
were targeted with a multidisciplinary approach, with experts
from the different digital forensics topics. A. FORENSIC FRAMEWORKS AND PROCESS MODELS A digital forensics framework, also known as a digital foren-
sics process model, is a sequence of steps that, along with the
corresponding inputs, outputs and requirements, aim to sup-
port a successful forensics investigation [150], [151]. A digi-
tal forensics framework is used by forensics investigators and
other related users to ease investigations and the identification
and prosecution of perpetrators. In addition to a set of specific
steps identifying each investigation phase, the use of digital
forensic frameworks enables timely investigations, as well as
a proper reconstruction of the timeline of events and their
associated data. In this regard, one of the most critical aspects
of a digital investigation is the proper preservation of the
evidence chain of custody, since it could lead to unsolvable
inconsistencies, risking the admissibility of evidence in court. To create a visual representation of these challenges,
we believe that mapping each challenge into different cate-
gories will highlight which need to be reinforced. Therefore,
Figure 4 presents the outcomes of our taxonomy in terms
of topic challenges mapped into different categories repre-
senting different phases, from the creation of the legal basis
and framework of an investigation to the final reporting of
the outcomes. As it can be observed, the challenges most
cited in the literature are present in the evidence acquisition
and data pre-processing category. They are mainly related
to data acquisition issues and anti-forensics. Notably, these
challenges affect the forensic procedures from the beginning
(i.e., if we do not consider the standards, legislation and
procedural category), and thus, it is crucial to devote efforts to According to their phases and their granularity, there are
different investigation models suitable for different types 25476 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews FIGURE 4. Main digital forensic challenges mapped into different categories according to their application context, from the initial steps of an
investigation (left) to the final ones (right). The size of each circle denotes the times it appeared considering the topics of the taxonomy. FIGURE 4. Main digital forensic challenges mapped into different categories according to their application context, from the initial steps of an
investigation (left) to the final ones (right). The size of each circle denotes the times it appeared considering the topics of the taxonomy. of investigations. In this regard, Kohn et al. A. FORENSIC FRAMEWORKS AND PROCESS MODELS provide [152] an
integrated suitability framework that maps a set of require-
ments derived from an ongoing investigation to the most
suitable forensic procedure. Moreover, the authors also use
a graph-based approach to associate the most well-known
forensic frameworks and their interrelationships regarding
the number of phases and their content. Other well-known
frameworks include the Analytical Crime Scene Procedure
Model (ACSPM) [153], the Systematic digital forensic inves-
tigation model (SRDFIM) [154], and the advanced data
acquisition model (ADAM) [155]. In general, law enforce-
ment agencies follow variants of the ACPO (Association of
Chief Police Officers) guidelines [156]. Finally, other foren-
sic guidelines and models proposed by NIST and INTERPOL
can be found in [5], [157]. The most well-known digital
forensic frameworks are summarised in Table 11. digital investigation models and proposed some measures to
palliate them. In [177] the authors presented a chronological
review of the most well-known forensic frameworks and their
characteristics. The work presented in [178] evaluates the cur-
rent frameworks among European law enforcement agencies,
identifying and defining elements of robustness and resilience
in the context of sustainable digital investigation capacity so
that organisations can adapt and overcome deviations and
novel trends. In [175], the authors identified the need to
define specific models according to the forensic context,
such as in the case of Mobile Forensics [175]. Moreover, the
authors proposed a specific forensic framework to improve
Mobile Forensics investigations. Further reviews of the most
used forensic frameworks and their features can be found in
[179], [180]. Table 14 reports the main challenges in forensic
frameworks identified by each literature review. In parallel to forensic guidelines and frameworks, stan-
dards are crucial to ensure conformance and mutual compli-
ance across geographical and jurisdictional borders. There are
currently numerous standards and established practices pro-
vided by organisations worldwide using accepted methods. The technical details on how to forensically approach a given
investigation differ depending on the device. The analysis of
electronic evidence is typically categorised into the phases
stated in Table 12. However, the exact phases naming may
vary due to different forensic models’ usage according to each
organisation’s needs. In general, the procedures summarised in Table 11 have
a common hierarchical structure [165], [166], which can be
divided in the steps described in Table 12. 4https://caseontology.org/ A. FORENSIC FRAMEWORKS AND PROCESS MODELS Note that some of
the models may include more granular approaches to some of
the steps, which are necessary due to the investigation context
(e.g., specific devices and seizure/acquisition constraints). In the case of the chain of custody and trail of events
preservation, a forensically sound procedure needs to ensure
features such as integrity, traceability, authentication, veri-
fiability and security [167], [168]. In this regard, Table 13
provides a description of each feature. While not an official standard, the Cyber-investigation
Analysis Standard Expression (CASE)4 is a community-
driven standard that aims to develop an ontology that can effi-
ciently represent all exchanged information and roles within
the context of investigations regarding digital evidence. The
International Organization for Standardization (ISO) has
released a series of standards to assist in this effort by
providing the family of ISO 27000, focusing on informa-
tion security standardisation procedures. In what follows, In the past, several authors identified several challenges
in digital investigation processes [77], [169]–[175], mainly
related to the chain of custody preservation, the growth of
the data to be processed, and privacy and ethical issues when
collecting such data. In addition, our research methodology
identified several literature reviews which discussed the chal-
lenges and limitations of forensic frameworks. For instance,
in [176], the authors leveraged a summary of digital forensic
frameworks and tools as well as their interrelationships by
using a graph analysis methodology. In addition, they dis-
cussed some challenges and limitations of privacy-preserving 25477 25477 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews TABLE 11. Most well-known forensic models and guidelines. TABLE 11. Most well-known forensic models and guidelines. TABLE 11. Most well-known forensic models and guidelines. TABLE 14. High level extraction of challenges reported in forensic
frameworks literature reviews. TABLE 14. High level extraction of challenges reported in forensic
frameworks literature reviews. TABLE 12. Main steps in a digital forensic investigation model. TABLE 12. Main steps in a digital forensic investigation model. TABLE 13. Main features required to guarantee chain of custody
preservation. various areas such as computer forensics, image,
audio and video analysis, as well as facial iden-
tification. As a result, ASTM E2916-19 creates a
common language framework for all. TABLE 13. Main features required to guarantee chain of custody
preservation. A. FORENSIC FRAMEWORKS AND PROCESS MODELS ISO 21043-2:2018: This standard specifies many
requirements for the forensic processes in focus-
ing on recognition, recording, collection, transport
and storage of items of potential forensic value. It includes requirements for the assessment and
examination of scenes but is also applicable to activ-
ities that occur within the facility. This document
also includes quality requirements. ISO 21043-2:2018: This standard specifies many
requirements for the forensic processes in focus-
ing on recognition, recording, collection, transport
and storage of items of potential forensic value. It includes requirements for the assessment and
examination of scenes but is also applicable to activ-
ities that occur within the facility. This document
also includes quality requirements. we present the most relevant standards about digital forensics
investigations, which are summarised in Figure 5. ISO/IEC 27035: This is a three-part standard
that provides organisations with a structured and
planned approach to the management of security
incident management covering a range of incident
response phases ISO/IEC 27035: This is a three-part standard
that provides organisations with a structured and
planned approach to the management of security
incident management covering a range of incident
response phases ISO/IEC 17025:2017: In some terms, this standard
can be considered an ‘‘infrastructure’’ standard for
forensic labs. It defines the managerial and techni-
cal requirements that testing and calibration labora-
tories must conform to ensure technical competence
and guarantee that their test are calibration results
are acceptable by the corresponding suppliers and
regulatory authorities. ISO/IEC 27037:2012: This standard provides gen-
eral guidelines about the handling of the evidence
of the most common digital devices and the cir-
cumstances including devices that exist in vari-
ous forms, giving the example of an automotive
system [181]. ISO/IEC 27037:2012: This standard provides gen-
eral guidelines about the handling of the evidence
of the most common digital devices and the cir-
cumstances including devices that exist in vari-
ous forms, giving the example of an automotive
system [181]. ASTM E2916-19: The goal of this standard is
to assemble the necessary technical, scientific and
legal terms and the corresponding definitions in the
context of the examination of digital and multi-
media evidence. Therefore, the standard spans to ISO/IEC 27038:2014: Describes the digital redac-
tion of information that must not be disclosed,
taking extreme care to ensure that removed infor-
mation is permanently unrecoverable. 25478 VOLUME 10, 2022 F. A. FORENSIC FRAMEWORKS AND PROCESS MODELS Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews FIGURE 5. Applicability of standards and guidelines to the investigation process classes
and activities. FIGURE 5. Applicability of standards and guidelines to the investigation process classes
and activities. ISO/IEC 27040:2015: Provides detailed technical
guidance on how organisations can define an appro-
priate level of risk mitigation by employing a well-
proven and consistent approach to the planning,
design, documentation, and implementation of data
storage security. ISO/IEC 27043:2015: It defines the key common
principles and processes underlying the investiga-
tion of incidents and provides a framework model
for all stages of investigations. ISO/IEC 27050: This recently revised stan-
dard guides non-technical and technical person-
nel to handle evidence on electronically stored
information (ESI). ISO/IEC 27050: This recently revised stan-
dard guides non-technical and technical person-
nel to handle evidence on electronically stored
information (ESI). ISO/IEC 27041:2015: Describes other standards
and documents to provide guidance, setting the
fundamental principles ensuring that tools, tech-
niques and methods, appropriately selected for the
investigation. ISO/IEC 30121:2015: Provides a framework for
organizations to strategically prepare for a digital
investigation before an incident occurs, to maximise
the effectiveness of the investigation. ISO/IEC 30121:2015: Provides a framework for
organizations to strategically prepare for a digital
investigation before an incident occurs, to maximise
the effectiveness of the investigation. ISO/IEC 27042:2015: This standard describes how
methods and processes to be used during an investi-
gation can be designed and implemented to allow
correct evaluation of potential digital evidence,
interpretation of digital evidence, and effective
reporting of findings. ETSI is a European Standards Organization that produces
standards for ICT systems and services used worldwide,
collaborating with numerous organisations. In 2020, ETSI
published TS 103 643 V1.1.1 (2020-01) [182], a set of tech-
niques for assurance of digital material in a legal proceeding, 25479 25479 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews TABLE 15. High level extraction of challenges reported in forensic
readiness literature reviews. TABLE 15. High level extraction of challenges reported in forensic
readiness literature reviews. A. FORENSIC FRAMEWORKS AND PROCESS MODELS to provide a set of tools to assist the legitimate presentation
of digital evidence.5 In the meantime, the National Institute
of Standards and Technology (NIST) has released guide-
lines for organisations to develop forensic capability (see
also Table 11), based on the principles of forensic science
in the aspect of the application of science to the law. Still,
it should not be used on digital forensic investigations due to
subjection to different laws and regulations, as clearly stated
in their manual. The scope of NIST guidelines is incorpo-
rating forensics into the information system life cycle of an
organisation. The most relevant guidelines are 800-86 [183]
for Integrating Forensic Techniques into Incident Response
and 800-101 [184] for Mobile Device Forensics. The Scientific Working Group on Digital Evidence
(SWGDE) is an organisation engaged in the field of digital
and multimedia evidence to foster communication and coop-
eration as well as to ensure quality and consistency within the
forensic community. SWGDE has released several documents
to provide the current best practices on a large variety of
state of the art forensics subjects. Nonetheless, none of them
is targeting or addressing drone forensics’s particularities. Finally, a review of the international development of forensic
standards can be found in [185]. discussed in the literature. Nevertheless, as previously stated
in the article, one of the main challenges is that cybercrime
evolves faster than countermeasures and legislations, and
thus, investigators are always one step behind. 5https://www.swgde.org/documents/published C. FORENSIC READABILITY AND REPORTING The continuous appearance of novel ICT technologies, paired
with discovering new vulnerabilities and attacks that threaten
them, dramatically increases the amount of information col-
lected during forensic investigations. The latter refers to the
amount of data collected from devices and systems, as well
as the heterogeneous data structures required in each case and
the specific forensic methodologies developed to detect such
threats. In this context, creating interoperable and auditable
forensic procedures is a hard task, especially due to the lack
of standardised reporting mechanisms. Moreover, qualitative
aspects such as the outcomes and conclusions supported by
the forensic analysis are often not reported accurately in an
attempt to balance between technicality and comprehensibil-
ity, hindering the robustness of the findings [14], [198], [199]. Of particular relevance is the communication and readability
of such reports, especially if these are to be interpreted by
law practitioners, judges, and other stakeholders who do
not always have the necessary technical background about
the forensic tools nor the underlying technologies anal-
ysed [200], [201]. The latter issue has been extensively anal-
ysed according to different approaches, from lexical density
and complexity [202]–[208], to cognitive and psychological
features [209], [210], showcasing the need to improve the
reporting mechanisms and the possible benefits of a common,
standardised framework. In addition to such a framework, it is
crucial to develop the corresponding training procedures for
its adoption [211]. 1) PRIVACY IN EUROPE States have numerous responsibilities concerning the protec-
tion of their citizens. Although the protection of privacy (in its
various forms) is important, it represents but one of the duties
states should fulfil [217]. Other prominent duties relate to the
need to protect the life and property of citizens, to prevent
disorder, to ensure that justice occurs where individuals have
been the victim of criminal activity and to protect national
security both offline and online [218]. In modern western
societies, it is often impossible to guarantee the exercise
and protect such rights and in an absolute manner to all
individuals all of the time due to competing interests of
stakeholder groups. Respectively, privacy is only one of such
values next to, e.g., security and the need for public order. To ensure security, the state likely has to take measures that
may infringe upon the privacy of individuals [219]. This
entails the acquisition of data or the conduct of surveillance to
prevent inter alia acts of terrorism or crime. These activities
clearly interfere with and limit the privacy of citizens but
do so for desirable reasons. However, interference with such
competing interests should be balanced, and the rights and
freedoms of all groups in society should be respected to the
greatest extent [217]. Respectively, the need to balance the
privacy and security interests implies that security measures
that infringe upon individual privacy are not acceptable unless
they really are intended to meet a need that is relating to
the protection the rights and interests of others. Where such After analysing the previous literature of forensic reporting
procedures and studying the technical level of the data to be
included [214], [215], as well as analysing existing investiga-
tion models such as ISO/IEC 27043:2015 [216], we identified
a set of key points and structural features that such document
should include. In parallel, we analysed the technical level
associated with each characteristic as reported in the liter-
ature and created a reporting guideline document, which is
represented in Table 17. As it can be observed, summaries,
overview descriptions and listings should be performed in
a comprehensive, non-technical way. In the case of tool
descriptions, as well as proofs guaranteeing the outcomes,
the report should contain some technical yet understandable
descriptions. Finally, the scientific aspects and details behind
the analysis and the corresponding methodologies require
descriptions that should be provided by qualified experts. B. FORENSIC READINESS In the past, forensic investigations leveraged a post-event
approach, mainly focusing on the analysis of data related to
a past incident. In this regard, forensic readiness in terms
of pro-active techniques and protocols appeared to minimise
the cost and the impact of incidents and are widely used
nowadays [15], [186]–[188]. We can find different research approaches, such as the
review conducted in [189], in which authors discussed how
to achieve forensic readiness by collecting the opinion of
experts to elaborate a readiness framework with which
improve forensic investigations from an organizational per-
spective. In the case of [190], authors discussed forensic
readiness and several procedures to achieve it, such as fos-
tering the use of Trusted Platform Modules (TPM). Other
authors reviewed measures to achieve forensic readiness
in a holistic way [15], [191]–[194], as well as recalling
the relevance to include and expand the actual guidelines
towards incident response readiness (e.g., as in the drafts
of the ISO/IEC JTC 1/SC 27 working groups, and the
ISO/IEC 27035), training and collaboration between stake-
holders involved in forensic investigations and prosecution,
and effective reporting readability and complexity. Table 15
describes the main forensic readiness challenges identified by
the authors in the literature. It is necessary to recall that the admissibility of a piece of
evidence and the forensic validation in court is mandatory
to the proper prosecution of perpetrators and constitute the
culminating point of an investigation [212], [213]. Therefore,
several authors collected the challenges and issues related to
the acceptance of evidence in court [196], [197], [212]. More-
over, region-focused studies can be found in [213] and [197]
for the United Kingdom and Australia, respectively. Finally, in Table 16 we provide a qualitative summary of
the literature reviewed in IV according to the topics discussed
in each article. From Table 16 we can see that topics such as
privacy and ethics and the suitability of frameworks that are
being proposed to fight novel cybercrime need to be further 25480 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews TABLE 16. Qualitative analysis of the literature reviews related with digital forensic guidelines, frameworks, tools, and readiness. Notation: ✓denotes
that this topic is analysed, while ◦denotes that its only partially discussed or just named. TABLE 16. Qualitative analysis of the literature reviews related with digital forensic guidelines, frameworks, tools, and readiness. B. FORENSIC READINESS Notation: ✓denotes
that this topic is analysed, while ◦denotes that its only partially discussed or just named. TABLE 16. Qualitative analysis of the literature reviews related with digital forensic guidelines, frameworks, tools, and readiness. Notation: ✓denotes
that this topic is analysed, while ◦denotes that its only partially discussed or just named. TABLE 17. Proposed representation of the content of a forensic report
according to the inputs collected from the literature. TABLE 17. Proposed representation of the content of a forensic report
according to the inputs collected from the literature. considered as well. As seen in [195], integrating digital foren-
sic readiness as a component in data protection legislation
could improve actual practices across different sectors and
countries. In particular, this section highlights the regulatory require-
ments of working with data in Europe and in the European
Union. To facilitate digital forensic readiness, tools should
be developed and used in line with legal requirements, with
special attention to the individual’s privacy. D. DATA MANAGEMENT AND ETHICS When discussing digital forensics and respective technology
readiness, the applicable regulatory frameworks should be 25481 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews output of analogue devices or data in digital format) of poten-
tial probative value that is manipulated, generated through,
stored on or communicated by any electronic device’’ [228]. Respectively, to use such data, specific rules concerning the
gathering and use of (digital) evidence should be adhered to
as well. Electronic evidence is admissible in courts when the
following sets of rules are adhered to: 1) general rules and
principles concerning due process in criminal proceedings;
2) general rules of evidence in criminal proceedings and;
3) specific rules relating to electronic evidence in criminal
proceedings [229]. justification does not exist, infringement of individual privacy
would not be acceptable. V. DISCUSSION In Section III, we provided a topic-based taxonomy of the
digital forensics literature. In what follows, we recall the chal-
lenges identified in each category and provide some strategies
to overcome them. In European Union Art. 4 (2) of the Treaty on the Euro-
pean Union (TEU) states that national security is the sole
responsibility of each Member State. To facilitate a harmo-
nized approach to national security, the EU adopted several
Directives and other legislative pieces in connection with
criminal matters such as: 1) Charter of Fundamental Rights
of the European Union, art 7 and 8. 2) 2016/679 General
Data Protection Regulation 3) Statement of the Article 29
Working Party, Data protection and privacy aspects of cross-
border access to electronic evidence, Brussels, 29 November
2017. 4) 2016/680/EU Law Enforcement Directive [224]
5) 2014/41/EU European Investigation Order Directive
6) EU 2000 Convention on mutual assistance in criminal
matters 7) 910/2014 eIDAS Regulation [225] 8) Electronic
evidence - a basic guide for First Responders Good prac-
tice material for CERT first responders by ENISA, and
9) E-evidence package [226] 2) DATA PROTECTION IN EUROPE In consonance with the individual’s data protection inter-
est and society’s own protective endeavours toward fighting
crime and securing national security, the Council of Europe
and European Union developed a common framework to
be observed by technology developers, security agencies,
including Police, and criminal justice system. The most rel-
evant instruments of the Council of Europe relating to the
processing of data as evidence are: 1) the European Conven-
tion for the Protection of Human Rights and Fundamental
Freedoms (ECHR) in particular with reference to the protec-
tion of the rights to privacy and due process, 2) the Council
of Europe Convention on Cybercrime, as this Convention
remains the main and only international treaty which defines
the substantive elements of cybercrimes [220], 3) the Council
of Europe Convention on Mutual Assistance in Criminal
Matters, and its 1978 Protocol [221], and 4) the Electronic
Evidence Guide [222]. There are both current, and to-be adopted elements of the
applicable legal framework, but it must be underlined that as
of now, there is no comprehensive international or European
legal framework providing rules relating to evidence [230]. From these documents, five overarching principles can be
deducted concerning the acquisition and use of electronic evi-
dence. These are: data integrity, audit trail, specialist support,
appropriate training, and legality [231]. National criminal
procedure codes (referred above) contain further, specific
provisions regarding the record and applicability of digital
evidence in criminal procedures. A second protocol concerning the ‘‘Enhanced international
cooperation on cybercrime and electronic evidence’’ is also in
development [223]. A. THE ROAD AHEAD IN DIGITAL FORENSICS’ TOPICS Thus, the efforts should focus on big data
analysis and data mining techniques to extract the relevant
investigation data from the vast amount of unrelated or redun-
dant digital objects. Another issue is the case of distributed
filesystem and databases or data stores, or when the foren-
sic analysis should be conducted on the cloud. In the latter
case, besides the specialised tools and methods, it also chal-
lenges collaboration and cooperation with the cloud service
providers. Finally, most research works and tools are bound
to specific system architecture, OS, or hardware implemen-
tation, so they have the drawback of becoming cumbersome
to adjust existing solutions to new use cases and problems. In this context, more generic approaches that allow tool reuse
in different cases are necessary. y
g
As seen in Section III-G, multimedia forensics is one
of the most explored topics, according to the number of
publications. Overall, while most authors focus on image
forgery detection, anti-forensics is one of the most challeng-
ing problems. In this regard, more efforts should be devoted
to counter anti-forensic mechanisms (i.e., as part of a global
digital forensics concern) and methodologies to capture novel
criminal trends with the help of sophisticated real-time object
detection and classification systems. In addition, multi-layer
systems and ontologies should be designed to cope with mul-
tiple threats at once, paired with the appropriate benchmarks
to evaluate them. In parallel, the issues related to the vast
amount of data to be processed should be minimised by
proposing more efficient data storage and indexing mech-
anisms and introducing algorithms that can process, e.g.,
compressed data. Following such research paths and com-
bining them with the proper legislation and standardisation
mechanisms will improve the success of multimedia digital
forensics investigations. The recovery of digital evidence from portable and/or
mobile devices is the focus of mobile forensics (MF),
a sub-branch of digital forensics. Seizure, acquisition, and
examination/analysis are the three categories that mobile
forensics processes fall into. Several challenges exist con-
cerning mobile forensics, as presented in III-C. In the MF
domain, the variety of embedded OSs with shorter prod-
uct life cycles and the numerous smartphone manufacturers
worldwide present significant challenges for applying sound
forensics approaches. A. THE ROAD AHEAD IN DIGITAL FORENSICS’ TOPICS Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews security features built into modern mobile devices, an inves-
tigator must break into the device using an exploit that will
almost certainly alter the data. the internet grows exponentially, the network traffic size to be
analysed to conduct a forensics investigation rises. Methods
that can efficiently analyse voluminous traces of network
traffic are in high demand. Additionally, the heterogeneity
of network traffic protocols increases the effort required to
collect evidence from all available sources. While the widespread adoption of IoT devices and IoT-
related applications has improved data availability and oper-
ational excellence, it has also introduced new security and
forensics challenges. As presented in Section III-D, several
challenges exist concerning IoT forensics. Such challenges
include managing multiple streams of data sources, the com-
plicated three-tier architecture of IoT and the lack of stan-
dardized systems for capturing real-time logs and storing
them in a valid uniform form. The preparation of highly
detailed reports of all information gathered, its correspond-
ing representation, and the lack of standardized systems for
capturing real-time logs also serve as barriers to establishing
sound IoT-related forensics mechanisms. Data encryption
trends are also posing new challenges for IoT forensic inves-
tigators, and cryptographically protected storage systems are
arguably one of the most significant roadblocks to effec-
tive digital forensic analysis. Interoperability and availability
issues related to the vast number of connected IoT devices, the
Big Data nature of IoT forensics evidence, and IoT forensics
evidence’s various security storage challenges also represent
significant IoT-related forensics challenges. Finally, the IoT
forensics domain faces several regulatory challenges, partic-
ularly those relating to data ownership in the cloud as defined
by regional laws. Last but not least, the main challenge that network foren-
sics research faces nowadays is encrypted traffic. When dig-
ital forensic evidence acquisition happens at an intermediate
node of the communication path, it is expected for the traffic
payload to be encrypted, and methods capable of extracting
information under such conditions are required. Filesystems, Memory, and Data Storage forensics have
attracted the research community’s attention, as they are
an abundant source of digital evidence. As discussed in
Section III-E, the main challenge of these domains lies in
the fact that there exist a large number of files and data
contained in them. A. THE ROAD AHEAD IN DIGITAL FORENSICS’ TOPICS After revising the challenges collected in cloud forensics,
most of them are closely related to data management. More
concretely, data acquisition, logging, limited access to foren-
sic data, cross-border data access and exchange are vital
parameters in cloud forensics. In terms of log management,
Marty [232] proposed using log management architecture and
the guidelines for application logging in SaaS service model
using technologies such as Django, Javascript, Apache, and
MySQL. A centralised logging scheme was proposed by
Trenwith and Venter [233] to accelerate the investigation
process and provide forensic readiness. Patrascu and Patri-
ciu [234] proposed a scheme to monitor various parallel
activities in a cloud environment. In addition to the pre-
vious works, several authors have devoted efforts towards
efficient and secure evidence management in the cloud
[235]–[237], including the use of blockchain such as seen
in [238]. We believe that efficient evidence and logging col-
lection mechanisms paired with secure and verifiable man-
agement of such evidence are crucial to guarantee sound
cloud forensic investigations. To rationalize the functioning and limit the increasing num-
ber of legal provisions, Regulation 2016/95 repealed certain
acts in the field of police cooperation and judicial cooperation
in criminal matters [227]. LEAs performing digital forensics
have confidentiality case levels depending on the severity
of the crime. The forensic examiners sign a special con-
fidentiality agreement regarding data protection upon their
employment. There are policies regarding data protection, all
the case relevant data is kept only to the internal network,
which is protected with the use of all the necessary measures
(Secure Connections, encryption, controlled access at the
physical location). The forensic examination equipment is not
connected to the internet when examinations are conducted. The data in question in digital forensics is referred to as elec-
tronic evidence, defined as ‘‘any information (comprising the Network traffic forensics is a long-standing domain with
numerous research efforts and tools. The main gaps that
currently exist and on which future efforts shall be focused are
related to the volume of the traffic, the different protocols that
emerge mainly due to the IoT rise, and the fact that traffic is
encrypted in most cases. As the use of computer systems and 25482 VOLUME 10, 2022 VOLUME 10, 2022 F. 3) DECENTRALISATION AND IMMUTABILITY The wide adoption of distributed platforms, e.g. blockchain
solutions [80] and distributed storage and filesystems, imply
significant challenges for digital forensics [239], [240]. Some
of these structures have strong privacy guarantees and can
be leveraged to exfiltrate data, orchestrate malicious cam-
paigns [241]–[244], or siphon fraudulent payments [245]. Traditional logging mechanisms and access control systems
allow an investigator to assess who, when, how or even from
where are not relevant for many of these technologies. As a
result, they are continuously abused by threat actors. These
huge obstacles for digital forensics require further research
on the field and the development of more targeted tools to
extend the capabilities of digital investigators. In this regard,
while the use of distributed platforms is not exempt from
potential issues [240], they can also be potentially used to
leverage community-based intelligence against threats and to
leverage auditable forensic investigations [82], [246]–[248]. Following such an idea and in order to accelerate the response
towards sophisticated threats and international campaigns,
the community is devoting research efforts towards federated
learning models [249], [250], and other emerging topics such
as cognitive security [251], [252]. Given the continuous evolution of cybercrime and its harm-
ful capacities, preventive strategies are paramount to fight
criminal activities. The latter implies the need to reinforce
digital forensic strategies at different levels, including guide-
lines, regulations, research and training to implement forensic
readiness holistically. According to our literature analysis,
one of the key points to reinforce the actual state of practice is
the definition of interoperable and easy-to-adopt legislations
since current ones cannot cope with the increasing sophis-
tication and the ubiquitous nature of cybercrime. Therefore,
it is crucial to devote efforts towards, e.g., interoperable cross-
border models with their corresponding dissemination and
training procedures, which all practitioners may adopt to
accelerate investigations. It is also relevant to stress the neces-
sity of appropriate forensic readability and reporting. First,
effective communication between all the actors involved in a
forensic investigation is essential to maximise the guarantees
in court. Second, the proper documentation of investigations
provides valuable feedback for future investigations, enhanc-
ing forensic readiness strategies. Third, the definition of a
common reporting framework can accelerate investigations
in which sometimes speed is crucial due to, e.g., the pos-
sible volatility of evidence or to reduce harm. A. THE ROAD AHEAD IN DIGITAL FORENSICS’ TOPICS MF, in general, present a variety of
challenges such as problems with data (anonymity-enforced
browsing and other anonymity services, and the considerable
volume of data acquired during an investigation), availability
of forensic tools (MF research approaches have long focused
on acquisition techniques, while minor importance was given
to the other phases of MF investigative process) and security-
oriented concerns (development of new and more sophisti-
cated anti-forensic methods from mobile manufacturers). It is
worth noting that MF is confronted with significant chal-
lenges regarding the overall MF processes’ focus. For exam-
ple, it is unclear whether investigation procedures should be
model-specific for each device or generic enough to form a
standardized set of forensics procedures guidelines. Another
critical issue is the requirement to perform live forensics
(mobile devices should be powered on). Finally, due to the Blockchain forensics is a relatively new domain since
blockchain technology accounts for a decade. In general,
it has to be understood that the need for blockchain foren-
sics methods is expected to grow in the coming years. As discussed in Section III-F current efforts focus on the
examination of available data on public blockchain systems. One of the main challenges encountered is to provide effi-
cient methods to conduct such analysis. The data on public
ledgers continuously grows, while the storage structure dif-
fers amongst different implementations. Developing methods 25483 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews to foster a culture of forensic preparedness. Essentially, every
organisation and resource provider must understand that its
products and services are expected to suffer a successful
cyber attack. Therefore, despite the countermeasures, recov-
ery methods, and mitigation strategies, they need to imple-
ment policies and mechanisms to facilitate digital forensics. If the latter are not well-placed, while business continuity
may not be severely harmed, one may not understand why
and how the security event occurred, what needs to change,
or miss even important evidence of the threat actor. and tools that can efficiently analyse data across commonly
used blockchain platforms is required. Moreover, forensic
analysis methods for blockchain systems’ nodes will enable
more thorough investigations with more detailed results for
public and private blockchain systems. 3) DECENTRALISATION AND IMMUTABILITY To this end,
we proposed a forensic reporting content representation by
following the common denominators found in the literature in
Section III. We argue that the devotion of more efforts on this
final part of the forensic flow will enrich investigations with
valuable feedback and successful prosecution guarantees. 7https://www.justice.gov/opa/speech/deputy-attorney-general-rod-j-
rosenstein-delivers-remarks-encryption-united-states-naval A. THE ROAD AHEAD IN DIGITAL FORENSICS’ TOPICS Finally, given the ris-
ing popularity of privacy enabled blockchain systems such as
Monero or ZCash, additional effort will be required to support
forensic investigations on cases that include interactions on
such systems. 6https://formobile-project.eu/ 4) DATA PROTECTION AND ETHICS IN
CRIMINAL INVESTIGATIONS Ransomware may be regarded as the most obvious case of
exploiting cryptographic primitives for malicious acts; nev-
ertheless, this is not by any chance the only. Threat actors
and cybercriminals, for instance, use encrypted and even
covert channels to communicate, further hindering investi-
gations. The latter has sparked a huge debates as many are
promoting concepts such as responsible encryption7 with
the adoption of, e.g., weakened encryption, cryptographic
schemes such as key escrow, backdooring of cryptographic
primitives etc. [253]–[256]. While they may facilitate dig-
ital investigations, essentially, they undermine the scope of
cryptography and security, opening the door for many inter-
pretations on what lawful interception is, when it can be
performed, by whom, let alone the exploitation of the mech-
anisms by already malicious actors as the backdoor would
be already implanted. The debate is undergoing and spans 2) FORENSIC PREPAREDNESS AND STANDARDS While in Section IV we provided an overview of digital
forensics standards, unfortunately, they do not suffice current
needs. To name just two which are standing out on the tip
of the iceberg, cloud and mobile related investigations need
to have some standards on how to be performed. Addressing
the need for mobile forensics, FORMOBILE6 has initiated a
broad dialogue and is developing a draft CENELEC Work-
shop Agreement to fill in this gap. However, due to the
specificities of cloud, IoT, drones, etc., similar actions are
expected in the near future. Beyond standards and methods, there is a definite need
from industry players, developers, system administrators etc., 25484 25484 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews ransomware, malware, and threats against data availability
and veracity, affect digital forensics in different dimensions,
regardless of the topic. NIST recently published a state of
the art analysis of cloud-related challenges [34], which is
aligned with the claims collected by in the cloud-based digital
forensics literature reviews state in Section III-A. In the case
of networks, ENISA elaborated an extensive set of security
objectives and discussed them along with their corresponding
recommendation measures in the topics of electronic commu-
nications [259] as well as 5G networks [260]. NIST provides
security guidelines for managing mobile devices in their
draft SP 800-124 (rev2) [261]. The recommendations include
scenarios from organization-provided to personally-owned
devices and describes technologies and strategies that can be
used as countermeasures and mitigations. In the context of
IoT, NIST released a set of documents related to IoT device
cybersecurity, covering aspects from the design and manu-
facturing of the components to their disposal [262]. In par-
allel, ENISA also proposed a comprehensive set of security
guidelines targeting all the entities involved in the supply
chain of IoT to improve security decisions when designing,
building, deploying, and assessing IoT technologies [263]. Concerning data storage and data processing, several guide-
lines have been proposed during the past years to reduce data
breaches [264], and the proper deployment of data storage
mechanisms that enable privacy by design [265]–[267], and
forensic readiness [268]. Finally, despite the existence of such
guidelines, forensic frameworks accommodating procedures
adapted to novel types of cybercrime such as in e.g. 2) FORENSIC PREPAREDNESS AND STANDARDS social
networks [269], and the proper review and evaluation of an
investigation process, are necessary to assess the quality of
forensic investigations [270]. multiple sectors beyond digital forensics. While fostering
such approaches may greatly benefit digital forensics, the
ethical and legal implications hinder such adoption and
are received by the security community with scepticism. As discussed, anti-forensics methods are a challenge for
almost all domains of digital forensics. Nevertheless, with
the growing adoption of TPM and TEE, these challenges can
be significantly augmented. For instance, as illustrated by
Dunn et al. [257] ransomware can exploit these technologies
to render decryption key extraction impossible. Nevertheless,
it is clear that these technologies introduce significant chal-
lenges for digital investigators since they may deprive them of
access to critical information. In this regard, it is essential to
study methods for, e.g. live forensics in the presence of TPM
and TEE and to explore how the missing information can be
compensated. VI. CONCLUSION AND FINAL REMARKS VI. CONCLUSION AND FINAL REMARKS The digitisation of our daily lives is a double-edged sword
as beyond the myriad of advantages and comforts it pro-
vides, it introduces security and privacy issues. Motivated by
the lack of a general view of the digital forensics ecosys-
tem, mainly because different topics are explored in an
isolated way and aiming to answer several research ques-
tions/concerns, this manuscript seeks to fill a literature gap by
proposing a review of reviews in the field of digital forensics. Following a thorough research methodology, we identified
the main digital forensics topics. We performed a taxonomy
by documenting the current state of the art and practice and
the main challenges in each of them. Moreover, we anal-
ysed these challenges with a cross-domain perspective to
highlight their relevance according to the times they were
discussed in the literature. According to the outcomes (i.e. see
Section III-I), such analysis provided us with enough evi-
dence to prove that the digital forensics community could
benefit from closer collaborations and cross-topic research
since it appears that researchers are trying to find solutions to
the same problems in parallel, sometimes without noticing it. 5) AUTOMATION AND EXPLAINABILITY The continuous increase in reported cybercrimes apart from
the impact on the victims implies a lot of effort from investi-
gators to analyse the cases. Therefore, automation of digital
forensics inevitably becomes a need. While automated meth-
ods for collecting log files and algorithms to identify anoma-
lies or even correlating some events may exist, this does not
practically translate to automated digital forensics. Even if
one does not consider APT attacks, one must understand
that each case has particularities differentiating it from the
others. Moreover, a digital investigator has to fill in the gaps
of missing information that the attacker managed to cover,
including those that security mechanisms failed to record or
those erroneously reported. The above implies the develop-
ment of advanced machine learning and AI algorithms and
tools that will underpin future digital forensics investigations. An important part of these systems is undoubtedly under-
standing the scope of the investigation and the explainability
of the results [258], which is critical to assess the impact of
current investigations and quantify their effectiveness [14],
a critical step to ensure the implementation of the proper mea-
sures. The latter is a crucial part of AI and machine learning
modules that have to be introduced as in order for a piece of
evidence to be admissible in a court of law, one has to justify
not only how and from where it has been collected but to also
prove the relevance to the case, how it was used, and why
it is linked with the rest of the evidence. In essence, future
digital forensics systems would have to argue and reason on
the collected information in a human-readable manner. The
latter is a huge step forward compared to the existing state
where systems prioritise log events and present the analysts
with known malicious patterns in the logs, malicious binaries,
or connections that deviate specific norms. 6) FORENSIC GUIDELINES AND BEST PRACTICES One of the main strategies to reduce the impact of cybercrime
is to implement the recommendations of the security guide-
lines and directives developed by agencies such as ENISA
and NIST. The current threat landscape [6], which includes By merging the information of Table 10 and Figure 4,
we extracted the amount of cross-domain challenges that 25485 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews each topic has in each forensic phase, and reported them
in Table 18. As it can be observed, data acquisition along
with investigation and forensic analysis are the phases that
entail more challenges, according to the research community. If we analyse the data at a topic level, we can observe that
IoT has many challenges to overcome in such phases. The
same applies to Multimedia and Mobile forensics. Since we
focus on the extracted challenges as collected in our literature
review, the fact that some challenges have not been high-
lighted either at topic or forensic phase level may indicate that
researchers and practitioners have not devoted enough effort
to them, or perhaps highlights lack of discussion towards
them. Such interesting domains include value chain and
financial forensics. Like other domains, the business sector’s
ongoing digitisation means that sound value chain forensics
mechanisms will be almost necessary within any corporate
strategy for the years to come. Therefore, the potentially
unexplored issues in such cases require proactive initiatives
before they become obstacles in the near future. usually, literature reviews are mature and long term works not
likely to be published in conferences as they do not require
a fast positioning. By discussing the open issues and future
trends in digital forensics, and after observing that many of
the challenges raised years ago are still not solved, we believe
that our literature analysis reflects with high fidelity the cur-
rent state of practice and the potential challenges that may
arise in the years to come, providing a fruitful ground of
research. The inherent cross-jurisdiction nature of modern cyber-
crime paired with the abuse of cutting edge technologies man-
dates more coordinated efforts from the security and research
community. With the continuously increasing amount of data
that have to be analysed, it is straightforward that man-
ual analysis is almost at its limits. 6) FORENSIC GUIDELINES AND BEST PRACTICES The use of fine-grained
IoCs may significantly reduce the effort of the investigator. However, as already discussed, this is not always possible,
especially when non-traditional computing devices are used,
e.g. IoT, mobile, cloud. As a result, machine learning and arti-
ficial intelligence are gradually being integrated into the logic
of many tools and methods. Nevertheless, the reasoning of the
results in an understandable human manner is a cross-domain
challenge. Moreover, the standardisation of digital forensics
processes for cloud, mobile, IoT, drones, etc., is becoming a
high priority since they are an indispensable part of almost
all modern digital investigations. Finally, the consensus on
developing these standards and the coordinated efforts made
over the past few years for countering cybercrime must be
leveraged to homogenise the legislation across jurisdictions
and facilitate digital investigations. A common answer to the
problem and using the same measures would create a strong
response against cybercrime and improve response time to
security incidents and their analysis. TABLE 18. Limitations per topic according to each phase as depicted
in Figure 4. TABLE 18. Limitations per topic according to each phase as depicted
in Figure 4. Further to merely listing the state of practice and proposing
research directions according to the identified challenges,
we analysed crucial aspects of digital forensics such as stan-
dards, forensic readiness, forensic reporting, and discussed
the ethical and legal aspects of data management in Europe in
Section IV. The insights gathered from such analysis, which
were represented in the form of structured tables, qualitative
literature analysis, and a proposed representation of forensic
report content, successfully answered the research questions
presented in Table 1. ACKNOWLEDGMENT
Th
f hi
i The content of this article does not reflect the official opinion
of the European Union. Responsibility for the information
and views expressed therein lies entirely with the authors. REFERENCES W. A. Wahab, M. Shiraz, and I. Ahmad, ‘‘Network
forensics: Review, taxonomy, and open challenges,’’ J. Netw. Comput. Appl., vol. 66, pp. 214–235, May 2016. [17] H. Hunt, A. Pollock, P. Campbell, L. Estcourt, and G. Brunton, ‘‘An intro-
duction to overviews of reviews: Planning a relevant research question
and objective for an overview,’’ Systematic Rev., vol. 7, no. 1, pp. 1–9,
Dec. 2018. [41] A. Nisioti, A. Mylonas, P. D. Yoo, and V. Katos, ‘‘From intrusion detec-
tion to attacker attribution: A comprehensive survey of unsupervised
methods,’’ IEEE Commun. Surveys Tuts., vol. 20, no. 4, pp. 3369–3388,
4th Quart., 2018. [18] J. E. McKenzie and S. E. Brennan, ‘‘Overviews of systematic reviews:
Great promise, greater challenge,’’ Systematic Rev., vol. 6, no. 1, pp. 1–4,
Dec. 2017. [42] F. Sharevski, ‘‘Towards 5G cellular network forensics,’’ EURASIP J. Inf. Secur., vol. 2018, no. 1, p. 8, Dec. 2018. [19] E. Aromataris, R. Fernandez, C. M. Godfrey, C. Holly, H. Khalil, and
P. Tungpunkom, ‘‘Summarizing systematic reviews: Methodological
development, conduct and reporting of an umbrella review approach,’’
Int. J. Evidence Based Healthcare, vol. 13, no. 3, pp. 132–140, 2015. [43] D. Takahashi, Y. Xiao, Y. Zhang, P. Chatzimisios, and H.-H. Chen, ‘‘IEEE
802.11 user fingerprinting and its applications for intrusion detection,’’
Comput. Math. Appl., vol. 60, no. 2, pp. 307–318, Jul. 2010. [44] L. F. Sikos, ‘‘Packet analysis for network forensics: A comprehensive sur-
vey,’’ Forensic Sci. Int., Digit. Invest., vol. 32, Mar. 2020, Art. no. 200892. [20] M. Pollock, R. M. Fernandes, D. Pieper, A. C. Tricco, M. Gates,
A. Gates, and L. Hartling, ‘‘Preferred reporting items for overviews of
reviews (PRIOR): A protocol for development of a reporting guideline
for overviews of reviews of healthcare interventions,’’ Systematic Rev.,
vol. 8, no. 1, pp. 1–9, Dec. 2019. [45] I. R. Adeyemi, S. A. Razak, and N. A. N. Azhan, ‘‘A review of current
research in network forensic analysis,’’ Int. J. Digit. Crime Forensics,
vol. 5, no. 1, pp. 1–26, Jan. 2013. [21] D. Denyer and D. Tranfield, ‘‘Producing a systematic review,’’ in The
Sage Handbook of Organizational Research Methods. Los Angeles, CA,
USA: SAGE, 2009, pp. 671–689. [46] A. A. Ahmed and N. A. K. Zaman, ‘‘Attack intention recognition:
A review,’’ IJ Netw. Secur., vol. 19, no. 2, pp. 244–250, 2017. [47] H.-C. Chu, D.-J. Deng, and H.-C. REFERENCES Chao, ‘‘Potential cyberterrorism via a
multimedia smart phone based on a Web 2.0 application via ubiquitous
Wi-Fi access points and the corresponding digital forensics,’’ Multimedia
Syst., vol. 17, no. 4, pp. 341–349, Jul. 2011. [22] R. Pranckut˙e, ‘‘Web of science (WoS) and scopus: The titans of bibli-
ographic information in Today’s academic world,’’ Publications, vol. 9,
no. 1, p. 12, Mar. 2021. [23] J. vom Brocke, A. Simons, K. Riemer, B. Niehaves, R. Plattfaut, and
A. Cleven, ‘‘Standing on the shoulders of giants: Challenges and rec-
ommendations of literature search in information systems research,’’
Commun. Assoc. Inf. Syst., vol. 37, no. 1, p. 9, 2015. [48] K. Barmpatsalou, T. Cruz, E. Monteiro, and P. Simoes, ‘‘Current and
future trends in mobile device forensics: A survey,’’ ACM Comput. Surv.,
vol. 51, no. 3, pp. 1–31, 2018. [49] A. Farjamfar, M. T. Abdullah, R. Mahmod, and N. Izura Udzir, ‘‘A review
on mobile device’s digital forensic process models,’’ Res. J. Appl. Sci.,
Eng. Technol., vol. 8, no. 3, pp. 358–366, Jul. 2014. [24] M. E. Alex and R. Kishore, ‘‘Forensics framework for cloud computing,’’
Comput. Elect. Eng., vol. 60, pp. 193–205, May 2017. [50] K. Barmpatsalou, D. Damopoulos, G. Kambourakis, and V. Katos,
‘‘A critical review of 7 years of mobile device forensics,’’ Digit. Invest.,
vol. 10, no. 4, pp. 323–349, Dec. 2013. [25] M. Khanafseh, M. Qatawneh, and W. Almobaideen, ‘‘A survey of various
frameworks and solutions in all branches of digital forensics with a
focus on cloud forensics,’’ Int. J. Adv. Comput. Sci. Appl., vol. 10, no. 8,
pp. 610–629, 2019. [51] X. Wan, J. He, G. Liu, N. Huang, X. Zhu, B. Zhao, and Y. Mai, ‘‘Survey
of digital forensics technologies and tools for Android based intelligent
devices,’’ Int. J. Digit. Crime Forensics, vol. 7, no. 1, pp. 1–25, Jan. 2015. [26] A. Pichan, M. Lazarescu, and S. T. Soh, ‘‘Cloud forensics: Technical
challenges, solutions and comparative analysis,’’ Digit. Invest., vol. 13,
pp. 38–57, Jun. 2015. [52] J. Hou, Y. Li, J. Yu, and W. Shi, ‘‘A survey on digital forensics in Internet
of Things,’’ IEEE Internet Things J., vol. 7, no. 1, pp. 1–15, Jan. 2020. [27] G. Palmer et al., ‘‘A road map for digital forensic research,’’ in Proc. 1st
Digit. Forensic Res. Workshop, New York, NY, USA, 2001, pp. 27–30. [53] M. Stoyanova, Y. Nikoloudakis, S. Panagiotakis, E. Pallis, and
E. K. REFERENCES Secur., vol. 49, pp. 45–69,
Mar. 2015. [33] B. Manral, G. Somani, K.-K.-R. Choo, M. Conti, and M. S. Gaur,
‘‘A systematic survey on cloud forensics challenges, solutions, and future
directions,’’ ACM Comput. Surv., vol. 52, no. 6, pp. 1–38, Nov. 2020. [10] A. M. Marshall, ‘‘Quality standards and regulation: Challenges for digital
forensics,’’ Meas. Control, vol. 43, no. 8, pp. 243–247, Oct. 2010. [11] P. S. Chen, L. M. Tsai, Y.-C. Chen, and G. Yee, ‘‘Standardizing the
construction of a digital forensics laboratory,’’ in Proc. 1st Int. Work-
shop Systematic Approaches Digit. Forensic Eng. (SADFE), Nov. 2005,
pp. 40–47. [34] N. I. of Standards and Technology. (2020). Nistir 8006 NIST
Cloud Computing Forensic Science Challenges. [Online]. Available:
https://nvlpubs.nist.gov/nistpubs/ir/2020/NIST.IR.8006.pdf [35] A. Alenezi, H. F. Atlam, and G. B. Wills, ‘‘Experts reviews of a cloud
forensic readiness framework for organizations,’’ J. Cloud Comput.,
vol. 8, no. 1, Dec. 2019. [12] A. Varol and Y. U. Sönmez, ‘‘Review of evidence collection and protec-
tion phases in digital forensics process,’’ Int. J. Inf. Secur. Sci., vol. 6,
no. 4, pp. 39–46, 2017. [13] A. H. Lone and R. N. Mir, ‘‘Forensic-chain: Blockchain based digital
forensics chain of custody with PoC in hyperledger composer,’’ Digit. Invest., vol. 28, pp. 44–55, Mar. 2019. [36] E. S. Pilli, R. C. Joshi, and R. Niyogi, ‘‘Network forensic frame-
works: Survey and research challenges,’’ Digit. Invest., vol. 7, nos. 1–2,
pp. 14–27, Oct. 2010. [14] R. E. Overill and J. Collie, ‘‘Quantitative evaluation of the results of
digital forensic investigations: A review of progress,’’ Forensic Sci. Res.,
vol. 6, no. 1, pp. 13–18, Jan. 2021. [37] (2020). N. T. S. Coalition. Cybersecurity Report 2020. [Online]. Avail-
able: https://www.ntsc.org/assets/pdfs/cyber-security-report-2020.pdf [38] C. Patsakis, F. Casino, N. Lykousas, and V. Katos, ‘‘Unravelling Ariadne’s
thread: Exploring the threats of decentralised DNS,’’ IEEE Access, vol. 8,
pp. 118559–118571, 2020. [15] K. A. Z. Ariffin and F. H. Ahmad, ‘‘Indicators for maturity and readiness
for digital forensic investigation in era of industrial revolution 4.0,’’
Comput. Secur., vol. 105, Jun. 2021, Art. no. 102237. [39] N. Hoque, M. H. Bhuyan, R. C. Baishya, D. K. Bhattacharyya, and
J. K. Kalita, ‘‘Network attacks: Taxonomy, tools and systems,’’ J. Netw. Comput. Appl., vol. 40, pp. 307–324, Apr. 2014. [16] M. Fire and C. Guestrin, ‘‘Over-optimization of academic publishing
metrics: Observing Goodhart’s law in action,’’ GigaScience, vol. 8, no. 6,
Jun. 2019. [40] S. Khan, A. Gani, A. REFERENCES [1] J. I. Thornton and J. Peterson, ‘‘The general assumptions and rationale
of forensic identification,’’ Modern scientific evidence: Law Sci. expert
testimony, vol. 2, p. 13, 1997. Finally, we discussed the main takeaways of this article
and showcased several challenges that the digital forensics
community will face in the upcoming years in Section V. In this regard, we proposed some ideas to prevent and/or
overcome them while recalling the need to design efficient
and cross-domain strategies since the latter will guarantee
faster and more robust outcomes, hopefully minimising the
impact of criminal activities. [2] E. Locard, Manuel de Technique Policière: Les Constats, les Empreintes
Digitales, 2nd ed. Paris, France: Payot, 1934. [3] F. L. Wellman and H. Münsterberg, ‘‘The art of cross-examination,’’
Amer. Bar Assoc. J., vol. 10, no. 4, p. 249, 1924. [Online]. Available:
http://www.jstor.org/stable/25711556 [4] M. Pollitt, ‘‘A history of digital forensics,’’ in IFIP International Confer-
ence on Digital Forensics. Berlin, Germany: Springer, 2010, pp. 3–15. [5] (2019). I. G. C. for Innovation. Global Guidelines for Digital
Forensics
Laboratories. [Online]. Available:
https://www.interpol.i
nt/content/download/13501/file/INTERPOL_DFL_Globa%
lGuidelinesDigitalForensicsLaboratory.pdf Notably, some limitations of our approach are worth men-
tioning. Since our article is a review of reviews, we may
have missed some recent advances and challenges should
these have not yet been collected in recent surveys. More-
over, we only considered peer-reviewed journals, which may
have lessened our approach’s comprehensive and interdisci-
plinary nature. However, we opted for this methodology since [6] (2021). The
European
Union
Agency
for
Cybersecurity
(ENISA). ENISA
Threat
Landscape
2021. [Online]. Available:
https://www.enisa.europa.eu/publications/enisa-threat-landscape-2021 [7] P. Purnaye and V. Kulkarni, ‘‘A comprehensive study of cloud forensics,’’
Arch. Comput. Methods Eng., vol. 29, no. 1, pp. 1–14, 2021. 25486 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews [8] C. Pasquini, I. Amerini, and G. Boato, ‘‘Media forensics on social media
platforms: A survey,’’ EURASIP J. Inf. Secur., vol. 2021, no. 1, pp. 1–19,
Dec. 2021. [31] A. Aminnezhad, A. Dehghantanha, M. T. Abdullah, and M. Damshenas,
‘‘Cloud forensics issues and opportunities,’’ Int. J. Inf. Process. Manage.,
vol. 4, no. 4, pp. 76–85, Jun. 2013. [9] K. Nance, H. Armstrong, and C. Armstrong, ‘‘Digital forensics: Defining
an education agenda,’’ in Proc. 43rd Hawaii Int. Conf. Syst. Sci., 2010,
pp. 1–10. [32] N. H. A. Rahman, and K.-K. R. Choo, ‘‘A survey of information security
incident handling in the cloud,’’ Comput. REFERENCES Markakis, ‘‘A survey on the Internet of Things (IoT) forensics:
Challenges, approaches, and open issues,’’ IEEE Commun. Surveys Tuts.,
vol. 22, no. 2, pp. 1191–1221, 2nd Quart., 2020. [28] K. Ruan, J. Carthy, T. Kechadi, and I. Baggili, ‘‘Cloud forensics defini-
tions and critical criteria for cloud forensic capability: An overview of
survey results,’’ Digit. Invest., vol. 10, no. 1, pp. 34–43, 2013. [54] R. Kamal, E. E.-D. Hemdan, and N. El-Fishway, ‘‘A review study on
blockchain-based iot security and forensics,’’ Multimedia Tools Appl.,
vol. 80, pp. 1–32, Sep. 2021. [29] S. Park, Y. Kim, G. Park, O. Na, and H. Chang, ‘‘Research on digital
forensic readiness design in a cloud computing-based smart work envi-
ronment,’’ Sustainability, vol. 10, no. 4, p. 1203, Apr. 2018. [55] H. F. Atlam, E. El-Din Hemdan, A. Alenezi, M. O. Alassafi, and
G. B. Wills, ‘‘Internet of Things forensics: A review,’’ Internet Things,
vol. 11, Sep. 2020, Art. no. 100220. [30] S. Simou, C. Kalloniatis, S. Gritzalis, and H. Mouratidis, ‘‘A survey on
cloud forensics challenges and solutions,’’ Secur. Commun. Netw., vol. 9,
no. 18, pp. 6285–6314, Dec. 2016. VOLUME 10, 2022 25487 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Revie [56] P. Lutta, M. Sedky, M. Hassan, U. Jayawickrama, and B. B. Bas-
taki, ‘‘The complexity of Internet of Things forensics: A state-of-
the-art review,’’ Forensic Sci. Int., Digit. Invest., vol. 38, Sep. 2021,
Art. no. 301210. [80] F. Casino, T. Dasaklis, and C. Patsakis, ‘‘A systematic literature review
of blockchain-based applications: Current status, classification and open
issues,’’ Telematics Inform., vol. 36, pp. 55–81, Mar. 2019. [81] B. Shanmugam, S. Azam, K. C. Yeo, J. Jose, and K. Kannoorpatti,
‘‘A critical review of bitcoins usage by cybercriminals,’’ in Proc. Int. Conf. Comput. Commun. Informat. (ICCCI), Jan. 2017, pp. 1–7. [57] A. Sayakkara, N.-A. Le-Khac, and M. Scanlon, ‘‘A survey of electromag-
netic side-channel attacks and discussion on their case-progressing poten-
tial for digital forensics,’’ Digit. Invest., vol. 29, pp. 43–54, Jun. 2019. Comput. Commun. Informat. (ICCCI), Jan. 2017, pp. 1–7. [82] T. K. Dasaklis, F. Casino, and C. Patsakis, ‘‘Sok: Blockchain solutions for
forensics,’’ in Technology Development for Security Practitioners. Cham,
Switzerland: Springer, 2021. [58] A. E. Omolara,
A. Alabdulatif,
O. I. Abiodun,
M. Alawida,
A. Alabdulatif, W. H. Alshoura, and H. REFERENCES Arshad, ‘‘The Internet of
Things security: A survey encompassing unexplored areas and new
insights,’’ Comput. Secur., vol. 112, Jan. 2022, Art. no. 102494. [83] A. Balaskas and V. N. L. Franqueira, ‘‘Analytical tools for blockchain:
Review, taxonomy and open challenges,’’ in Proc. Int. Conf. Cyber Secur. Protection Digit. Services (Cyber Security), Jun. 2018, pp. 1–8. [59] O. Yakubu, N. C. Babu, and O. Adjei, ‘‘A review of digital forensic
challenges in the Internet of Things (IoT),’’ Int. J. Mech. Eng. Technol.,
vol. 9, no. 1, pp. 915–923, 2018. Protection Digit. Services (Cyber Security), Jun. 2018, pp. 1–8. [84] A. Turner and A. S. M. Irwin, ‘‘Bitcoin transactions: A digital discovery
of illicit activity on the blockchain,’’ J. Financial Crime, vol. 25, no. 1,
pp. 109–130, Jan. 2018. [60] N. Koroniotis, N. Moustafa, and E. Sitnikova, ‘‘Forensics and deep learn-
ing mechanisms for botnets in Internet of Things: A survey of challenges
and solutions,’’ IEEE Access, vol. 7, pp. 61764–61785, 2019. [85] H. Chen, M. Pendleton, L. Njilla, and S. Xu, ‘‘A survey on ethereum
systems security: Vulnerabilities, attacks, and defenses,’’ ACM Comput. Surv., vol. 53, no. 3, pp. 1–43, May 2021. [61] A. Ross, S. Banerjee, and A. Chowdhury, ‘‘Security in smart cities: A
brief review of digital forensic schemes for biometric data,’’ Pattern
Recognit. Lett., vol. 138, pp. 346–354, Oct. 2020. [86] I. Homoliak, S. Venugopalan, D. Reijsbergen, Q. Hum, R. Schumi, and
P. Szalachowski, ‘‘The security reference architecture for blockchains:
Toward a standardized model for studying vulnerabilities, threats, and
defenses,’’ IEEE Commun. Surveys Tuts., vol. 23, no. 1, pp. 341–390,
1st Quart., 2021. [62] H. Studiawan, F. Sohel, and C. Payne, ‘‘A survey on forensic investigation
of operating system logs,’’ Digit. Invest., vol. 29, pp. 1–20, Jun. 2019. [63] S. Khan, A. Gani, A. W. A. Wahab, M. A. Bagiwa, M. Shiraz, S. U. Khan,
R. Buyya, and A. Y. Zomaya, ‘‘Cloud log forensics: Foundations, state
of the art, and future directions,’’ ACM Comput. Surveys, vol. 49, no. 1,
pp. 1–42, Mar. 2017. [87] Z. Wang, H. Jin, W. Dai, K.-K.-R. Choo, and D. Zou, ‘‘Ethereum smart
contract security research: Survey and future research opportunities,’’
Frontiers Comput. Sci., vol. 15, no. 2, Apr. 2021, Art. no. 152802. [88] W. Koerhuis, T. Kechadi, and N.-A. Le-Khac, ‘‘Forensic analysis of
privacy-oriented cryptocurrencies,’’ Forensic Sci. Int., Digit. Invest.,
vol. 33, Jun. 2020, Art. no. 200891. [64] R. A. Awad, S. REFERENCES 12, pp. 27–40, Mar. 2015. [71] R. Chopade and V. K. Pachghare, ‘‘Ten years of critical review
on database forensics research,’’ Digit. Invest., vol. 29, pp. 180–197,
Jun. 2019. [95] S. Bourouis, R. Alroobaea, A. Alharbi, M. Andejany, and S. Rubaiee,
‘‘Recent advances in digital multimedia tampering detection for forensics
analysis,’’ Symmetry, vol. 12, no. 11, pp. 1–26, 2020. [72] W. K. Hauger and M. S. Olivier, ‘‘NOSQL databases: Forensic attribution
implications,’’ SAIEE Afr. Res. J., vol. 109, no. 2, pp. 119–132, Jun. 2018. [96] H. Kaur and N. Jindal, ‘‘Image and video forensics: A critical survey,’’
Wireless Pers. Commun., vol. 112, no. 2, pp. 1281–1302, May 2020. [97] M. D. Ansari, E. Rashid, S. Skandha, and S. K. Gupta, ‘‘A comprehensive
analysis of image forensics techniques: Challenges and future direction,’’
Recent Patents Eng., vol. 13, pp. 1–10, Dec. 2019. [73] V. Jusas, D. Birvinskas, and E. Gahramanov, ‘‘Methods and tools of dig-
ital triage in forensic context: Survey and future directions,’’ Symmetry,
vol. 9, no. 4, p. 49, Mar. 2017. [74] A. Al-Dhaqm, S. Razak, R. A. Ikuesan, V. R. Kebande, and S. Hajar
Othman, ‘‘Face validation of database forensic investigation metamodel,’’
Infrastructures, vol. 6, no. 2, p. 13, Jan. 2021. [98] R. C. Pandey, S. K. Singh, and K. K. Shukla, ‘‘Passive forensics in image
and video using noise features: A review,’’ Digit. Invest., vol. 19, pp. 1–28,
Dec. 2016. [99] S. Gupta, N. Mohan, and P. Kaushal, ‘‘Passive image forensics using
universal techniques: A review,’’ Artif. Intell. Rev., vol. 2021, pp. 1–51,
Jul. 2021. [75] I. Sutherland, J. Evans, T. Tryfonas, and A. J. C. Blyth ‘‘Acquiring volatile
operating system data tools and techniques,’’ Operating Syst. Rev., vol. 42,
pp. 65–73, Apr. 2008. [76] N. Beebe and J. Clark, ‘‘Dealing with terabyte data sets in digital investi-
gations,’’ in Proc. IFIP Int. Fed. Inf. Process., vol. 194, 2006, pp. 3–16. [100] M. A. Qureshi and M. Deriche, ‘‘A bibliography of pixel-based blind
image forgery detection techniques,’’ Signal Process., Image Commun.,
vol. 39, pp. 46–74, Nov. 2015. [77] D. Quick and K.-K.-R. Choo, ‘‘Impacts of increasing volume of digital
forensic data: A survey and future research challenges,’’ Digit. Invest.,
vol. 11, no. 4, pp. 273–294, Dec. 2014. [101] A. R. Abrahim, M. S. M. Rahim, and G. B. Sulong, ‘‘Literature review:
Detection of image splicing forgery,’’ Int. J. Appl. Eng. Res., vol. 12,
no. 22, pp. 11855–11861, 2017. REFERENCES Beztchi, J. M. Smith, B. Lyles, and S. Prowell, ‘‘Tools,
techniques, and methodologies: A survey of digital forensics for scada
systems,’’ in Proc. 4th Annu. Ind. Control Syst. Secur. Workshop, 2018,
pp. 1–8. [89] M. Saad, J. Spaulding, L. Njilla, C. Kamhoua, S. Shetty, D. Nyang, and
D. Mohaisen, ‘‘Exploring the attack surface of blockchain: A comprehen-
sive survey,’’ IEEE Commun. Surveys Tuts., vol. 22, no. 3, pp. 1977–2008,
3rd Quart., 2020. [65] M. Botacin, P. L. D. Geus, and A. Grégio, ‘‘Who watches the watchmen:
A security-focused review on current state-of-the-art techniques, tools,
and methods for systems and binary analysis on modern platforms,’’ ACM
Comput. Surv., vol. 51, no. 4, pp. 1–34, Jul. 2019. [90] E. Deirmentzoglou, G. Papakyriakopoulos, and C. Patsakis, ‘‘A survey
on long-range attacks for proof of stake protocols,’’ IEEE Access, vol. 7,
pp. 28712–28725, 2019. [66] T. Latzo, R. Palutke, and F. Freiling, ‘‘A universal taxonomy and sur-
vey of forensic memory acquisition techniques,’’ Digit. Invest., vol. 28,
pp. 56–69, Mar. 2019. pp
[91] H. Farid, ‘‘Image forgery detection,’’ IEEE Signal Process. Mag., vol. 26,
no. 2, pp. 16–25, Mar. 2009. [67] G. Osbourne, ‘‘Memory forensics: Review of acquisition and analysis
techniques,’’ Defence Sci. Technol. Organisation Edinburgh (Australia)
Cyber Electron. Warfare Div, Tech. Rep., 2013. [92] K. A. P. da Costa, J. P. Papa, L. A. Passos, D. Colombo, J. D. Ser,
K. Muhammad, and V. H. C. de Albuquerque, ‘‘A critical literature survey
and prospects on tampering and anomaly detection in image data,’’ Appl. Soft Comput., vol. 97, Dec. 2020, Art. no. 106727. [68] A. Case and G. G. Richard, ‘‘Memory forensics: The path forward,’’ Digit. Invest., vol. 20, pp. 23–33, Mar. 2017. [93] A. H. Saber, M. A. Khan, and B. G. Mejbel, ‘‘A survey on image forgery
detection using different forensic approaches,’’ Adv. Sci., Technol. Eng. Syst. J., vol. 5, no. 3, pp. 361–370, 2020. [69] A. Al-Dhaqm, S. A. Razak, D. A. Dampier, K.-K. R. Choo, K. Siddique,
R. A. Ikuesan, A. Alqarni, and V. R. Kebande, ‘‘Categorization and
organization of database forensic investigation processes,’’ IEEE Access,
vol. 8, pp. 112846–112858, 2020. [94] L. Zheng, Y. Zhang, and L. Vrizlynn, ‘‘A survey on image tampering and
its detection in real-world photos,’’ J. Vis. Commun. Image Represent.,
vol. 58, pp. 380–399, Jan. 2019. [70] O. M. Adedayo and M. S. Olivier, ‘‘Ideal log setting for database forensics
reconstruction,’’ Digit. Invest., vol. REFERENCES Zargari, Drone Forensics: The
Impact and Challenges. Cham, Switzerland: Springer, 2021, pp. 65–124. [115] V. N. L. Franqueira, J. Bryce, N. Al Mutawa, and A. Marrington, ‘‘Inves-
tigation of indecent images of children cases: Challenges and sugges-
tions collected from the trenches,’’ Digit. Invest., vol. 24, pp. 95–105,
Mar. 2018. [140] F. Adelstein, ‘‘Live forensics: Diagnosing your system without killing it
first,’’ Commun. ACM, vol. 49, no. 2, pp. 63–66, Feb. 2006. [141] A. Renduchintala, F. Jahan, R. Khanna, and A. Y. Javaid, ‘‘A comprehen-
sive micro unmanned aerial vehicle (UAV/drone) forensic framework,’’
Digit. Invest., vol. 30, pp. 52–72, Sep. 2019. [116] L. Sanchez, C. Grajeda, I. Baggili, and C. Hall, ‘‘A practitioner survey
exploring the value of forensic tools, AI, filtering, & safer presentation for
investigating child sexual abuse material (CSAM),’’ Digit. Invest., vol. 29,
pp. S124–S142, Jul. 2019. [142] E. Mantas and C. Patsakis, ‘‘Who watches the new watchmen? The
challenges for drone digital forensics investigations,’’ arXiv preprint
arXiv:2021.12640, 2021. [117] J. Cifuentes, A. L. S. Orozco, and L. J. G. Villalba, ‘‘A survey of arti-
ficial intelligence strategies for automatic detection of sexually explicit
videos,’’ Multimedia Tools Appl., vol. 39, pp. 1–18, Nov. 2021. [143] M. Keyvanpour, M. Moradi, and F. Hasanzadeh, ‘‘Digital forensics 2.0,’’
in Computational Intelligence in Digital Forensics: Forensic Investiga-
tion and Applications. Cham, Switzerland: Springer, 2014, pp. 17–46. [118] K. V. Açar, ‘‘Osint by crowdsourcing: A theoretical model for online child
abuse investigations,’’ Int. J. Cyber Criminol., vol. 12, no. 1, pp. 206–229,
2018. [144] T. Sangkaran, A. Abdullah, and N. Z. JhanJhi, ‘‘Criminal network
community detection using graphical analytic methods: A survey,’’ EAI
Endorsed Trans. Energy Web, vol. 7, no. 26, pp. 1–15, 2020. [119] E. Nowroozi, A. Dehghantanha, R. M. Parizi, and K.-K.-R. Choo, ‘‘A sur-
vey of machine learning techniques in adversarial image forensics,’’
Comput. Secur., vol. 100, Jan. 2021, Art. no. 102092. [145] G. De La T. Parra, P. Rad, and K.-K. R. Choo, ‘‘Implementation of deep
packet inspection in smart grids and industrial Internet of Things: Chal-
lenges and opportunities,’’ J. Netw. Comput. Appl., vol. 135, pp. 32–46,
Jun. 2019. [120] M. Dalal and M. Juneja, ‘‘Video steganalysis to obstruct criminal activi-
ties for digital forensics: A survey,’’ Int. J. Electron. Secur. Digit. Foren-
sics, vol. 10, no. 4, pp. 338–355, 2018. [146] E. Batista, M. A. Moncusi, P. López-Aguilar, A. Martínez-Ballesté, and
A. REFERENCES Solanas, ‘‘Sensors for context-aware smart healthcare: A security
perspective,’’ Sensors, vol. 21, no. 20, p. 6886, Oct. 2021. [121] S. Kingra, N. Aggarwal, and R. D. Singh, ‘‘Video inter-frame forgery
detection: A survey,’’ Indian J. Sci. Technol., vol. 9, no. 44, Nov. 2016. [147] G. Ahmadi-Assalemi, H. Al-Khateeb, G. Epiphaniou, and C. Maple,
‘‘Cyber resilience and incident response in smart cities: A systematic
literature review,’’ Smart Cities, vol. 3, no. 3, pp. 894–927, Aug. 2020. [122] N. A. Shelke and S. S. Kasana, ‘‘A comprehensive survey on passive
techniques for digital video forgery detection,’’ Multimedia Tools Appl.,
vol. 80, no. 4, pp. 6247–6310, Feb. 2021. [123] A. S. Shahraki, H. Sayyadi, M. H. Amri, and M. Nikmaram, ‘‘Sur-
vey: Video forensic tools,’’ J. Theor. Appl. Inf. Technol., vol. 47, no. 1,
pp. 98–107, 2013. [148] S. Garfinkel, P. Farrell, V. Roussev, and G. Dinolt, ‘‘Bringing
science to digital forensics with standardized forensic corpora,’’
Digit. Invest., vol. 6, pp. S2–S11, Sep. 2009. [Online]. Available:
https://www.sciencedirect.com/science/article/pii/S1742287609000346 [124] M. Alsmirat, R. Al-Hussien, W. Al-Sarayrah, Y. Jararweh, and M. Etier,
‘‘Digital video forensics: A comprehensive survey,’’ Int. J. Adv. Intell. Paradigms, vol. 15, no. 4, pp. 437–456, 2020. [149] C. Grajeda,
F. Breitinger,
and
I. Baggili,
‘‘Availability
of
datasets
for
digital
forensics-
and
what
is
missing,’’
Digit. Invest., vol. 22, pp. S94–S105, Aug. 2017. [Online]. Available:
https://www.sciencedirect.com/science/article/pii/S1742287617301913 [125] F. Becerra-Riera, A. Morales-González, and H. Méndez-Vázquez, ‘‘A sur-
vey on facial soft biometrics for video surveillance and forensic applica-
tions,’’ Artif. Intell. Rev., vol. 52, no. 2, pp. 1155–1187, Aug. 2019. [150] M. Köhn, M. S. Olivier, and J. H. Eloff, ‘‘Framework for a digital forensic
investigation,’’ in Proc. ISSA, 2006, pp. 1–7. [126] S. T and S. M. Thampi, ‘‘Nighttime visual refinement techniques for
surveillance video: A review,’’ Multimedia Tools Appl., vol. 78, no. 22,
pp. 32137–32158, Nov. 2019. [151] W. Halboob and R. Mahmod, ‘‘State of the art in trusted computing
forensics,’’ in Future Information Technology, Application, and Service. Dordrecht, The Netherlands: Springer, 2012, pp. 249–258. [127] R. D. Singh and N. Aggarwal, ‘‘Video content authentication techniques:
A comprehensive survey,’’ Multimedia Syst., vol. 24, no. 11, pp. 211–240,
Mar. 2018. [152] M. D. Kohn, M. M. Eloff, and J. H. P. Eloff, ‘‘Integrated digital forensic
process model,’’ Comput. Secur., vol. 38, pp. 103–115, Oct. 2013. [128] M. Zakariah, M. K. Khan, and H. REFERENCES [78] B. Almaslukh, ‘‘Forensic analysis using text clustering in the age of large
volume data: A review,’’ Int. J. Adv. Comput. Sci. Appl., vol. 10, no. 6,
pp. 71–76, 2019. [102] R. Dixit and R. Naskar, ‘‘Review, analysis and parameterisation of tech-
niques for copy–move forgery detection in digital images,’’ IET Image
Process., vol. 11, no. 9, pp. 746–759, Sep. 2017. [79] V. H. G. Moia and M. A. A. Henriques, ‘‘Similarity digest search: A
survey and comparative analysis of strategies to perform known file
filtering using approximate matching,’’ Secur. Commun. Netw., vol. 2017,
pp. 1–17, 2017. [103] S. Teerakanok and T. Uehara, ‘‘Copy-move forgery detection: A state-
of-the-art technical review and analysis,’’ IEEE Access, vol. 7,
pp. 40550–40568, 2019. 25488 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews [104] Z. Zhang, C. Wang, and X. Zhou, ‘‘A survey on passive image copy-move
forgery detection,’’ J. Inf. Process. Syst., vol. 14, no. 1, pp. 6–31, 2018. [129] K. Conlan, I. Baggili, and F. Breitinger, ‘‘Anti-forensics: Furthering dig-
ital forensic science through a new extended, granular taxonomy,’’ Digit. Invest., vol. 18, pp. S66–S75, Aug. 2016. [105] G. K. Birajdar and V. H. Mankar, ‘‘Digital image forgery detection using
passive techniques: A survey,’’ Digital Invest., vol. 10, no. 3, pp. 226–245,
Oct. 2013. [130] M. A. Qureshi and E. M. El-Alfy, ‘‘Bibliography of digital image
anti-forensics and anti-anti-forensics techniques,’’ IET Image Process.,
vol. 13, no. 11, pp. 1811–1823, Sep. 2019. [106] R. R. Ali, K. M. Mohamad, S. Jamel, and S. K. A. Khalid, ‘‘A review
of digital forensics methods for JPEG file carving,’’ J. Theor. Appl. Inf. Technol., vol. 96, no. 17, pp. 5841–5856, 2018. [131] F. Guibernau, ‘‘Catch me if you can!—Detecting sandbox evasion tech-
niques,’’ in Proc. USENIX Assoc., San Francisco, CA, USA, Jan. 2020. [107] M. J. Khan, H. S. Khan, A. Yousaf, K. Khurshid, and A. Abbas, ‘‘Modern
trends in hyperspectral image analysis: A review,’’ IEEE Access, vol. 6,
pp. 14118–14129, 2018. [132] P. Chen, C. Huygens, L. Desmet, and W. Joosen, ‘‘Advanced or not? A
comparative study of the use of anti-debugging and anti-VM techniques
in generic and targeted malware,’’ in Proc. IFIP Int. Conf. ICT Syst. Secur. Privacy Protection. Cham, Switzerland: Springer, 2016, pp. 323–336. rivacy Protection. Cham, Switzerland: Springer, 2016, pp. 323–3 [108] P. REFERENCES Korus, ‘‘Digital image integrity—A survey of protection and verifica-
tion techniques,’’ Digit. Signal Process., vol. 71, pp. 1–26, Dec. 2017. [133] A. Bulazel and B. Yener, ‘‘A survey on automated dynamic malware
analysis evasion and counter-evasion: PC, mobile, and Web,’’ in Proc. 1st
Reversing Offensive-oriented Trends Symp. (ROOTS), 2017, pp. 1–21. [109] T. Julliand, V. Nozick, and H. Talbot, ‘‘Image noise and digital
image forensics,’’ in Proc. Int. Workshop Digit. Watermarking. Cham,
Switzerland: Springer, 2015, pp. 3–17. [134] R. R. Branco, G. N. Barbosa, and P. D. Neto, ‘‘Scientific but not academ-
ical overview of malware anti-debugging, anti-disassembly and anti-VM
technologies,’’ Black Hat, vol. 1, pp. 1–27, Jul. 2012. p
g
pp
[110] S. Chutani and A. Goyal, ‘‘A review of forensic approaches to dig-
ital image steganalysis,’’ Multimedia Tools Appl., vol. 78, no. 13,
pp. 18169–18204, Jul. 2019. [135] R. Harris, ‘‘Arriving at an anti-forensics consensus: Examining how to
define and control the anti-forensics problem,’’ Digit. Invest., vol. 3,
pp. 44–49, Sep. 2006. [111] K. Karampidis, E. Kavallieratou, and G. Papadourakis, ‘‘A review of
image steganalysis techniques for digital forensics,’’ J. Inf. Secur. Appl.,
vol. 40, pp. 217–235, Jun. 2018. [136] S. Alharbi, J. Weber-Jahnke, and I. Traore, ‘‘The proactive and reactive
digital forensics investigation process: A systematic literature review,’’ in
Information Security and Assurance, T.-H. Kim, H. Adeli, R. J. Robles,
and M. Balitanas, Eds. Berlin, Heidelberg: Springer, 2011, pp. 87–100. [112] X. Luo, F. Liu, S. Lian, C. Yang, and S. Gritzalis, ‘‘On the typical statistic
features for image blind steganalysis,’’ IEEE J. Sel. Areas Commun.,
vol. 29, no. 7, pp. 1404–1422, Aug. 2011. M. Balitanas, Eds. Berlin, Heidelberg: Springer, 2011, pp. 87–10 [113] P. Yang, D. Baracchi, R. Ni, Y. Zhao, F. Argenti, and A. Piva, ‘‘A survey
of deep learning-based source image forensics,’’ J. Imag., vol. 6, no. 3,
p. 9, Mar. 2020. [137] A. Al-Dhaqm, R. A. Ikuesan, V. R. Kebande, S. Razak, and F. M. Ghab-
ban, ‘‘Research challenges and opportunities in drone forensics models,’’
Electronics, vol. 10, no. 13, p. 1519, Jun. 2021. [138] G. Horsman, ‘‘Unmanned aerial vehicles: A preliminary analysis of
forensic challenges,’’ Digit. Invest., vol. 16, pp. 1–11, Mar. 2016. [114] M. Dalal and M. Juneja, ‘‘Steganography and steganalysis (in digital
forensics): A cybersecurity guide,’’ Multimedia Tools Appl., vol. 80, no. 4,
pp. 5723–5771, Feb. 2021. [139] S. Atkinson, G. Carr, C. Shaw, and S. s_103643v010101p.pdf 173–179, 2014. [168] Z. Tian, M. Li, M. Qiu, Y. Sun, and S. Su, ‘‘Block-DEF: A secure digital
evidence framework using blockchain,’’ Inf. Sci., vol. 491, pp. 151–165,
Jul. 2019. [192] A. M. Marshall and R. Paige, ‘‘Requirements in digital forensics method
definition: Observations from a U.K. study,’’ Digit. Invest., vol. 27,
pp. 23–29, Dec. 2018. [169] R. S. Greenfield et al., Cyber Forensics: A Field Manual for Collecting,
Examining, and Preserving Evidence of Computer Crimes. Boca Raton,
FL, USA: CRC Press, 2002. [193] V. S. Harichandran, F. Breitinger, I. Baggili, and A. Marrington, ‘‘A cyber
forensics needs analysis survey: Revisiting the domain’s needs a decade
later,’’ Comput. Secur., vol. 57, pp. 1–13, Mar. 2016. [170] D. Reilly, C. Wren, and T. Berry, ‘‘Cloud computing: Forensic challenges
for law enforcement,’’ in Proc. Int. Conf. Internet Technol. Secured
Trans., Nov. 2010, pp. 1–7. [194] M. Ozel, H. I. Bulbul, H. G. Yavuzcan, and O. F. Bay, ‘‘An analytical
analysis of Turkish digital forensics,’’ Digit. Invest., vol. 25, pp. 55–69,
Jun. 2018. [171] S. L. Garfinkel, ‘‘Digital forensics research: The next 10 years,’’ Digital
Investigation, vol. 7, pp. S64–S73, Aug. 2010. [195] S. Park, N. Akatyev, Y. Jang, J. Hwang, D. Kim, W. Yu, H. Shin, C. Han,
and J. Kim, ‘‘A comparative study on data protection legislations and gov-
ernment standards to implement digital forensic readiness as mandatory
requirement,’’ Digit. Invest., vol. 24, pp. S93–S100, Mar. 2018. [172] A. Guarino, ‘‘Digital forensics as a big data challenge,’’ in ISSE Securing
Electronic Business Processes. Wiesbaden, Germany: Springer, 2013,
pp. 197–203. [196] H. Arshad, A. B. Jantan, and O. I. Abiodun, ‘‘Digital forensics: Review
of issues in scientific validation of digital evidence,’’ J. Inf. Process. Syst.,
vol. 14, no. 2, pp. 346–376, 2018. [173] G. Mohay, ‘‘Technical challenges and directions for digital forensics,’’ in
Proc. 1st Int. Workshop Systematic Approaches to Digit. Forensic Eng. (SADFE), Nov. 2005, pp. 155–161. [197] A. Butler and K.-K.-R. Choo, ‘‘IT standards and guides do not adequately
prepare IT practitioners to appear as expert witnesses: An Australian
perspective,’’ Secur. J., vol. 29, no. 2, pp. 306–325, Apr. 2016. [174] Z. Li, Q. A. Chen, R. Yang, Y. Chen, and W. Ruan, ‘‘Threat detection and
investigation with system-level provenance graphs: A survey,’’ Comput. Secur., vol. 106, Jul. 2021, Art. no. 102282. [198] A. S. Bali, G. Edmond, K. N. Ballantyne, R. I. Kemp, and K. A. s_103643v010101p.pdf [183] K. Kent, S. Chevalier, T. Grance, and H. Dang, ‘‘SP 800-86. guide to inte-
grating forensic techniques into incident response,’’ Nat. Inst. Standards
Technol., Tech. Rep., 2006. [160] B. Carrier and E. H. Spafford, ‘‘Getting physical with the investigative
process,’’ Int. J. Digit. Evidence, 2003. [161] V. Baryamureeba and F. Tushabe, ‘‘The enhanced digital investigation
process model,’’ Digit. Invest., 2004. [184] R. Ayers, S. Brothers, and W. Jansen. (May 2014). Guidelines
on Mobile Device Forensics. [Online]. Available: https://csrc.nist. gov/publications/detail/sp/800-101/rev-1/final [162] S. O. Ciardhuáin, ‘‘An extended model of cybercrime investigations,’’
International Journal of Digital Evidence, vol. 3, no. 1, pp. 1–22,
2004. [185] L. Wilson-Wilde, ‘‘The international development of forensic science
standards—A review,’’ Forensic Sci. Int., vol. 288, pp. 1–9, Jul. 2018. [163] I. O, D. Chris, and D. David, ‘‘A new approach of digital forensic model
for digital forensic investigation,’’ Int. J. Adv. Comput. Sci. Appl., vol. 2,
no. 12, pp. 175–178, 2011. [186] M. Robinson. (2015). Digital Forensics Workbook: Hands-on Activi-
ties in Digital Forensics. CreateSpace Independent Publishing Platform. [Online]. Available: https://books.google.gr/books?id=4dyHjgEACAAJ [164] (2020). European Network of Forensic Science Institutes. Foren-
sic Guidelines. [Online]. Available: http://enfsi.eu/documents/forensic-
guidelines/ [187] J. Tan, Forensic Readiness. Cambridge, MA, USA: Stake, 2001,
pp. 1–23. [188] K. Reddy and H. S. Venter, ‘‘The architecture of a digital forensic
readiness management system,’’ Comput. Secur., vol. 32, pp. 73–89,
Feb. 2013. [165] Y. Yusoff, R. Ismail, and Z. Hassan, ‘‘Common phases of computer
forensics investigation models,’’ Int. J. Comput. Sci. Inf. Technol., vol. 3,
no. 3, pp. 17–31, 2011. [189] M. Elyas, A. Ahmad, S. B. Maynard, and A. Lonie, ‘‘Digital forensic
readiness: Expert perspectives on a theoretical framework,’’ Comput. Secur., vol. 52, pp. 70–89, Jul. 2015. [166] K. Kyei, P. Zavarsky, D. Lindskog, and R. Ruhl, ‘‘A review and compar-
ative study of digital forensic investigation models,’’ in Digital Forensics
and Cyber Crime, M. Rogers and K. C. Seigfried-Spellar, Eds. Berlin,
Germany: Springer, 2013, pp. 314–327. [190] B. Endicott-Popovsky, N. Kuntze, and C. Rudolph, ‘‘Forensic readiness:
Emerging discipline for creating reliable and secure digital evidence,’’
J. Harbin Inst. Technol., vol. 22, no. 1, pp. 1–8, 2015. [167] S. Bonomi, M. Casini, and C. Ciccotelli, ‘‘B-CoC: A blockchain-based
chain of custody for evidences management in digital forensics,’’ 2018,
arXiv:1807.10359. [191] A. Mouhtaropoulos, C. T. Li, and M. Grobler, ‘‘Digital forensic readiness:
Are we there yet?’’ J. Int. Commercial Law Technol., vol. 9, no. 3,
pp. REFERENCES Malik, ‘‘Digital multimedia audio
forensics: Past, present and future,’’ Multimedia Tools Appl., vol. 77,
no. 1, pp. 1009–1040, Jan. 2018. [153] H. I. Bulbul, H. G. Yavuzcan, and M. Ozel, ‘‘Digital forensics: An
analytical crime scene procedure model (ACSPM),’’ Forensic Sci. Int.,
vol. 233, nos. 1–3, pp. 244–256, Dec. 2013. 25489 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews [154] A. Agarwal, M. Gupta, S. Gupta, and S. C. Gupta, ‘‘Systematic digital
forensic investigation model,’’ Int. J. Comput. Sci. Secur., vol. 5, no. 1,
pp. 118–131, 2011. [179] R. Montasari, ‘‘An ad hoc detailed review of digital forensic investigation
process models,’’ Int. J. Electron. Secur. Digit. Forensics, vol. 8, no. 3,
pp. 205–223, 2016. [180] R. Sabillon, J. Serra-Ruiz, V. Cavaller, and J. J. Cano, ‘‘Digital forensic
analysis of cybercrimes: Best practices and methodologies,’’ Int. J. Inf. Secur. Privacy, vol. 11, no. 2, pp. 25–37, 2017. [155] R. Adams, V. Hobbs, and G. Mann, ‘‘The advanced data acquisition
model (Adam): A process model for digital forensic practice,’’ J. Digit. Forensics, Secur. Law, vol. 8, no. 4, pp. 25–48, 2013. [181] Information Technology—Security Techniques—Guidelines for Identifi-
cation, Collection, Acquisition and Preservation of Digital Evidence,
Joint Technical Committee ISO/IEC JTC, International Organization
for Standardization, Geneva, CH, Standard ISO/IEC 27037:2012, 2012. [Online]. Available: https://www.iso.org/standard/44381.html [156] J. Williams, ‘‘ACPO good practice guide for digital evidence,’’
Metrop. Police Service, Assoc. Chief Police Officers, GB, Tech. Rep.,
2012. [157] K. Kent, S. Chevalier, T. Grance, and H. Dang, ‘‘SP 800-86. guide to inte-
grating forensic techniques into incident response,’’ Nat. Inst. Standards
Technol., Gaithersburg, MD, USA, Tech. Rep., 2006. [Online]. Available: https://www.iso.org/standard/44381.html [182] European Telecommunications Standards Institute. (2020). Techniques
for Assurance of Digital Material Used in Legal Proceedings—ETSI TS
103 643 v1.1.1 (2020-01). [Online]. Available: https://www.etsi.org/del
iver/etsi_ts/103600_103699/103643/01.01.01_60/t%
s_103643v010101p.pdf [158] W. G. Kruse II and J. G. Heiser, Computer Forensics: Incident Response
Essentials. London, U.K.: Pearson, 2001. [159] M. Reith, C. Carr, and G. Gunsch, ‘‘An examination of digital forensic
models,’’ Int. J. Digit. Evidence, vol. 1, no. 3, pp. 1–12, 2002. s_103643v010101p.pdf Martire,
‘‘Communicating
forensic
science
opinion:
An
examination
of
expert reporting practices,’’ Sci. Justice, vol. 60, no. 3, pp. 216–224,
May 2020. [175] A. Al-Dhaqm, S. A. Razak, R. A. Ikuesan, V. R. Kebande, and
K. Siddique, ‘‘A review of mobile forensic investigation process models,’’
IEEE Access, vol. 8, pp. 173359–173375, 2020. [176] M. Abulaish and N. A. H. Haldar, ‘‘Advances in digital forensics frame-
works and tools: A comparative insight and ranking,’’ Int. J. Digit. Crime
Forensics, vol. 10, no. 2, pp. 95–119, 2018. [199] L. M. Howes and N. Kemp, ‘‘Discord in the communication of forensic
science: Can the science of language help foster shared understanding?’’
J. Lang. Social Psychol., vol. 36, no. 1, pp. 96–111, Jan. 2017. [177] R. Agarwal and S. Kothari, ‘‘Review of digital forensic investigation
frameworks,’’ in Information Science and Applications (Lecture Notes
in Electrical Engineering), vol. 339. Berlin, Germany: Springer-Verlag,
2015, pp. 561–571. [200] L. M. Howes, K. P. Kirkbride, S. F. Kelty, R. Julian, and N. Kemp,
‘‘The readability of expert reports for non-scientist report-users: Reports
of forensic comparison of glass,’’ Forensic Sci. Int., vol. 236, pp. 54–66,
Mar. 2014. [178] P. Amann and J. I. James, ‘‘Designing robustness and resilience in dig-
ital investigation laboratories,’’ Digit. Invest., vol. 12, pp. S111–S120,
Mar. 2015. [201] L. M. Howes, K. P. Kirkbride, S. F. Kelty, R. Julian, and N. Kemp, ‘‘Foren-
sic scientists’ conclusions: How readable are they for non-scientist report-
users?’’ Forensic Sci. Int., vol. 231, nos. 1–3, pp. 102–112, Sep. 2013. 25490 VOLUME 10, 2022 [202] M. A. K. Halliday, ‘‘Some grammatical problems in scientific English,’’
Genre Systemic Funct. Stud., vol. 6, pp. 13–37, Jan. 1989. [226] (2019). European Commission. E-evidence—Cross-Border Access to
Electronic
Evidence. [Online]. Available:
https://ec.europa.eu/info/
policies/justice-and-fundamental-rights/crimi%nal-justice/e-evidence-
cross-border-access-electronic-evidence_en [203] S. Eggins, Introduction to Systemic Functional Linguistics. A&C Black,
2004. [204] R. Flesch, ‘‘A new readability yardstick,’’ J. Appl. Psychol., vol. 32, no. 3,
p. 221, 1948. [227] (2016). European Union. Regulation (EU) 2016/95 of the Euro-
pean Parliament and of the Council. [Online]. Available: https://eur-
lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A32016R0095 [205] R. Flesch and A. J. Gould, The Art Readable Writing, vol. 8. New York,
NY, USA: Harper, 1949. [228] (2017). E. Union. Final Report Summary—European Informatics Data
Exchange Framework for Courts and Evidence. [Online]. Available:
https://cordis.europa.eu/project/id/608185/reporting [206] J. P. Kincaid, R. P. Fishburne, Jr., R. L. Rogers, and B. S. s_103643v010101p.pdf Chissom,
‘‘Derivation of new readability formulas (automated readability index,
fog count and flesch reading ease formula) for navy enlisted personnel,’’
Naval Tech. Training Command Millington TN Res. Branch, Tech. Rep.,
1975. [229] F. Insa, ‘‘The admissibility of electronic evidence in court (A.E.E.C.):
Fighting against high-tech crime—Results of a European study,’’ J. Digit. Forensic Pract., vol. 1, no. 4, pp. 285–289, Jun. 2007. [207] R. Clerehan, R. Buchbinder, and J. Moodie, ‘‘A linguistic framework for
assessing the quality of written patient information: Its use in assessing
methotrexate information for rheumatoid arthritis,’’ Health Educ. Res.,
vol. 20, no. 3, pp. 334–344, Jun. 2005. [230] M. A. Biasiotti, J. P. M. Bonnici, J. Cannataci, and F. Turchi, Handling
and Exchanging Electronic Evidence Across Europe. vol. 39. Cham,
Switzerland: Springer, 2018. [231] Electronic Evidence—A basic Guide for First Responders, Eur. Netw. Inf. Secur. Agency (ENISA), Athens, Greece, 2015. [208] P. B. Mosenthal and I. S. Kirsch, ‘‘A new measure for assessing document
complexity: The pmose/ikirsch document readability formula,’’ J. Adoles-
cent Adult Literacy, vol. 41, no. 8, pp. 638–657, 1998. [232] R. Marty, ‘‘Cloud application logging for forensics,’’ in Proc. ACM Symp. Appl. Comput. (SAC), 2011, pp. 178–184. [233] P. Trenwith and H. Venter, ‘‘Digital forensic readiness in the cloud,’’ in
Proc. IEEE Information Security for South Africa, 2013, pp. 1–5. [209] J. L. Calderón,
E. Fleming,
M. R. Gannon,
S.-C. Chen,
J. A. Vassalotti, and K. C. Norris, ‘‘Applying an expanded set of
cognitive design principles to formatting the kidney early evaluation
program (KEEP) longitudinal survey,’’ Amer. J. Kidney Diseases, vol. 51,
no. 4, pp. S83–S92, Apr. 2008. [234] A. Patrascu and V.-V. Patriciu, ‘‘Logging system for cloud computing
forensic environments,’’ J. Control Eng. Appl. Informat., vol. 16, no. 1,
pp. 80–88, 2014. [210] M. Graves and B. Graves, ‘‘Assessing text difficulty and accessibility,’’
in Scaffolding Reading Experiences: Designs for Student Success. Nor-
wood, MA, USA: Christopher-Gordon, 2003. [235] V. Kebande and H. Venter, ‘‘A functional architecture for cloud forensic
readiness large-scale potential digital evidence analysis,’’ in Proc. Eur. Conf. Cyber Warfare Secur., 2015, p. 373. [211] J. Cosic, ‘‘Formal acceptability of digital evidence,’’ in Multimedia Foren-
sics and Security. Cham, Switzerland: Springer, 2017, pp. 327–348. [236] S. Zawoad, A. K. Dutta, and R. Hasan, ‘‘Towards building foren-
sics enabled cloud through secure logging-as-a-service,’’ IEEE Trans. Depend. Sec. Comput., vol. 13, no. 2, pp. 148–162, Mar./Apr. 2016. [212] O. Sallavaci and C. s_103643v010101p.pdf George, ‘‘Procedural aspects of the new regime for
the admissibility of expert evidence: What the digital forensic expert
needs to know,’’ Int. J. Electron. Secur. Digit. Forensics, vol. 5, nos. 3–4,
pp. 161–171, 2013. [237] M. A. M. Ahsan, A. W. B. A. Wahab, M. Y. I. B. Idris, S. Khan,
E. Bachura, and K.-K.-R. Choo, ‘‘CLASS: Cloud log assuring soundness
and secrecy scheme for cloud forensics,’’ IEEE Trans. Sustain. Comput.,
vol. 6, no. 2, pp. 184–196, Apr. 2021. [213] P. Sommer, ‘‘Certification, registration and assessment of digital forensic
experts: The U.K. experience,’’ Digit. Invest., vol. 8, no. 2, pp. 98–105,
Nov. 2011. [238] H. Tian, J. Wang, C.-C. Chang, and H. Quan, ‘‘Public auditing of log
integrity for shared cloud storage systems via blockchain,’’ Wireless
Netw., vol. 2020, pp. 378–387, May 2020. [214] D. Garrie. (2016). The Neutral Corner: Understanding a Digital Foren-
sics Report. [Online]. Available: https://www.legalexecutiveinstitute. com/understanding-digital-forensics% -report/ [239] F. Casino, E. Politou, E. Alepis, and C. Patsakis, ‘‘Immutability and
decentralized storage: An analysis of emerging threats,’’ IEEE Access,
vol. 8, pp. 4737–4744, 2020. [215] H. Bariki, M. Hashmi, and I. Baggili, ‘‘Defining a standard for report-
ing digital evidence items in computer forensic tools,’’ in Proc. Int. Conf. Digit. Forensics Cyber Crime. Berlin, Germany: Springer, 2010,
pp. 78–95. [240] V. R. Kebande, R. A. Ikuesan, and N. M. Karie, ‘‘Review of blockchain
forensics challenges,’’ in Blockchain Security in Cloud Computing. Cham, Switzerland: Springer, 2022, pp. 33–50. [216] N. M. Karie, V. R. Kebande, H. S. Venter, and K.-K.-R. Choo, ‘‘On the
importance of standardising the process of generating digital forensic
reports,’’ Forensic Sci. Int., Rep., vol. 1, Nov. 2019, Art. no. 100008. [241] S. T. Ali, P. McCorry, P. H.-J. Lee, and F. Hao, ‘‘ZombieCoin 2.0: Man-
aging next-generation botnets using bitcoin,’’ Int. J. Inf. Secur., vol. 17,
no. 4, pp. 411–422, Aug. 2018. [242] C. Patsakis and F. Casino, ‘‘Hydras and IPFS: A decentralised play-
ground for malware,’’ Int. J. Inf. Secur., vol. 18, no. 6, pp. 787–799,
Dec. 2019. [217] D. Klitou, ‘‘Privacy by design and privacy-invading technologies: Safe-
guarding privacy, liberty and security in the 21st century,’’ Legisprudence,
vol. 5, no. 3, pp. 297–329, 2011. [218] N. Daniels, ‘‘Justice, health, and healthcare,’’ Amer. J. Bioethics, vol. 1,
no. 2, pp. 2–16, Feb. 2001. [243] (2020). O. Caspi. Trickbot Bazarloader in-Depth [Online]. Available:
https://cybersecurity.att.com/blogs/labs-research/trickbot-bazarloader-%
in-depth [219] M. s_103643v010101p.pdf He has worked for the European
Commission (DG Humanitarian Aid and Civil
Protection) and the University of Piraeus Research
Centre. He has also worked in the private sector for
three years as the Supply Chain Director. He has participated in National
and European research projects and has published papers in books, peer
reviewed journals, and conference proceedings. He is currently an Assistant
Professor with the School of Social Sciences, Hellenic Open University. His research interests include in the area of supply chain management,
operational research, humanitarian logistics/disaster response, data analysis,
and blockchain technology. He has served as a guest editor, a Program
Committee Member, and a reviewer for various international journals and
conferences. pp
[259] (2020). The European Union Agency for Cybersecurity (ENISA). Guideline on Security Measures Under the EECC. [Online]. Available:
https://www.enisa.europa.eu/publications/guideline-on-security-
measures% -under-the-eecc/ [260] (2020). The European Union Agency for Cybersecurity (ENISA). 5G
supplement—To the Guideline on Security Measures Under the EECC. [Online]. Available:
https://www.enisa.europa.eu/publications/5g-
supplement-security-measure% s-under-eecc/ [261] (2020). N. I. of Standards and Technology. SP 800-124 rev. 2—Guidelines
for Managing the Security of Mobile Devices in the Enterprise. [Online]. Available:
https://csrc.nist.gov/publications/detail/sp/800-124/rev-
2/draft [262] (2020). National Institute of Standards and Technology. NIST Releases
Draft Guidance on Internet of Things Device Cybersecurity. [Online]. Available:
https://www.nist.gov/news-events/news/2020/12/nist-
releases-draft-guida% nce-internet-things-device-cybersecurity [263] The European Union Agency for Cybersecurity (ENISA). Guidelines
for securing the Internet of Things. (2020). [Online]. Available:
https://www.enisa.europa.eu/publications/guidelines-for-securing-the-
in% ternet-of-things [264] (2017). The European Union Agency for Cybersecurity (ENISA). Guide-
lines for SMES on the Security of Personal Data Processing. [Online]. Available:
https://www.enisa.europa.eu/publications/guidelines-for-
smes-on-the-sec% urity-of-personal-data-processing [265] (2020). National Institute of Standards and Technology. NIST Privacy
Framework: A Tool for Improving Privacy Through Enterprise Risk
Management. [Online]. Available: https://www.nist.gov/system/files/
documents/2020/01/16/NIST%20Privacy%% 20Framework_V1.0.pdf GEORGIOS
P. SPATHOULAS
received the
Diploma of Electrical and Computer Engineering
degree from the Aristotle University of Thessa-
loniki, in 2002, the M.Sc. degree in computer sci-
ence from The University of Edinburgh, in 2005,
and the Ph.D. degree from the Department of Dig-
ital Systems, University of Piraeus, in 2013. He is
a member of Laboratory Teaching Staff of the
Department of Computer Science and Biomedical
Informatics, University of Thessaly, since 2014,
and he teaches in both undergraduate and postgraduate study programs of the
department. He is also a Postdoctoral Researcher with the Critical Infrastruc-
tures Security and Resilience Group at the Center for Cyber and Information
Security (CCIS), Norwegian University of Science and Technology (NTNU). s_103643v010101p.pdf degree (cum laude) in com-
puter science from Rovira i Virgili University,
Tarragona, Catalonia, Spain, in 2010, 2013, and
2017, respectively. He was a Visiting Researcher
at ISCTE-IUL, Lisbon, in 2016. He has partici-
pated in several European-, Spanish-, and Catalan-
funded research projects, and he has authored
more than 50 publications in peer-reviewed international conferences and
journals. He is a Postdoctoral Researcher with the Department of Computer
Engineering and Mathematics, Rovira i Virgili University, and the Athena
Research Center, Athens, Greece. His research interests include pattern
recognition, and data management applied to different fields such as privacy
and security protection, recommender systems, smart health, supply chain,
and blockchain. He received the Best Dissertation Award from Rovira i
Virgili University. p
p
[252] K. Demertzis, P. Kikiras, N. Tziritas, S. Sanchez, and L. Iliadis, ‘‘The next
generation cognitive security operations center: Network flow forensics
using cybersecurity intelligence,’’ Big Data Cognit. Comput., vol. 2, no. 4,
p. 35, Nov. 2018. p
[253] S. Schuster,
M. van
den
Berg,
X. Larrucea,
T. Slewe,
and
P. Ide-Kostic, ‘‘Mass surveillance and technological policy options:
Improving security of private communications,’’ Comput. Standards
Interfaces, vol. 50, pp. 76–82, Feb. 2017. f
pp
[254] D. J. Bernstein, T. Lange, and R. Niederhagen, ‘‘Dual EC: A standardized
back door,’’ in The New Codebreakers. Berlin, Germany: Springer, 2016,
pp. 256–281. pp
[255] M. Smith and M. Green, ‘‘A discussion of surveillance backdoors: Effec-
tiveness, collateral damage and ethics,’’ in Proc. Int. Secur. 21st Century,
Germany’s Int. Responsibility, 2016, pp. 131–142. y
p
y
pp
[256] E. Rice, ‘‘The second amendment and the struggle over cryptography,’’
Hastings Sci. Tech. LJ, vol. 9, p. 29, Oct. 2017. g
p
[257] A. M. Dunn,
O. S. Hofmann,
B. Waters,
and
E. Witchel,
‘‘Cloaking
malware
with
the
trusted
platform
module,’’
in
Proc. 20th
USENIX
Secur. Symp. (USENIX
Security),
San Francisco, CA, USA, Aug. 2011, pp. 1–16. [Online]. Available:
https://www.usenix.org/conference/usenix-security-11/cloaking-
malware-t% rusted-platform-module [258] A. Adadi and M. Berrada, ‘‘Peeking inside the black-box: A sur-
vey on explainable artificial intelligence (XAI),’’ IEEE access, vol. 6,
pp. 52138–52160, 2018. THOMAS K. DASAKLIS received the bachelor’s
degree from the Department of Industrial Man-
agement and Technology, University of Piraeus,
and the M.Sc. degree in supply chain management
and the Ph.D. degree in emergency supply chain
management and disaster response from the Uni-
versity of Piraeus. s_103643v010101p.pdf Neocleous, ‘‘Security, liberty and the myth of balance: Towards a
critique of security politics,’’ Contemp. Political Theory, vol. 6, no. 2,
pp. 131–149, May 2007. [244] F. Casino, N. Lykousas, V. Katos, and C. Patsakis, ‘‘Unearthing malicious
campaigns and actors from the blockchain DNS ecosystem,’’ Comput. Commun., vol. 179, pp. 217–230, Nov. 2021. [220] (2004). Council of Europe. Details of treaty no. 185. [Online]. Available:
https://www.coe.int/en/web/conventions/full-list/-
/conventions/treaty/1% 85 [245] T. de Balthasar and J. Hernandez-Castro, ‘‘An analysis of bitcoin laun-
dry services,’’ in Secure IT Systems (Lecture Notes in Computer Sci-
ence), H. Lipmaa, A. Mitrokotsa, and R. Matulevicius, Eds., vol. 10674. Springer, 2017, pp. 297–312, doi: 10.1007/978-3-319-70290-2_18. [221] (1962). C. of
Europe. Details
of
Treaty
no. 030. [Online]. Available:
https://www.coe.int/en/web/conventions/full-list/-
/conventions/treaty/0% 30 [246] G. Kumar, R. Saha, C. Lal, and M. Conti, ‘‘Internet-of-forensic (IoF):
A blockchain based digital forensics framework for iot applications,’’
Future Gener. Comput. Syst., vol. 120, pp. 13–25, 2021. [222] (2013). C. of
Europe. Data
Protection
and
Cybercrime
Division,
Electronic
Evidence
Guide. [Online]. Available:
https://rm.coe.int/16803028af [247] (2019). LOCARD: Lawful Evidence Collecting and Continuity Platform
Development. [Online]. Available: https://locard.eu [223] (2018). C. of Europe. Towards a Protocol to the Budapest Con-
vention. [Online]. Available: https://rm.coe.int/t-cy-pd-pubsummary-
v6/1680795713 [248] L. Zarpala and F. Casino, ‘‘A blockchain-based forensic model for finan-
cial crime investigation: The embezzlement scenario,’’ Digit. Finance,
vol. 3, no. 3, pp. 1–32, 2021. [224] (2016). E. Union. Directive (EU) 2016/680 of the European Parliament
and of the Council. [Online]. Available: https://eur-lex.europa.eu/legal-
content/EN/TXT/?uri=CELEX%3A32016L0680 [249] T. Li, A. K. Sahu, A. Talwalkar, and V. Smith, ‘‘Federated learning:
Challenges, methods, and future directions,’’ IEEE Signal Process. Mag.,
vol. 37, no. 3, pp. 50–60, May 2020. [225] E. Union. (2014). Regulation (EU) no 910/2014, of the European
Parliament and of the Council. [Online]. Available: https://eur-
lex.europa.eu/legal-content/EN/TXT/?uri=uriserv%3AOJ.L_.201%
4.257.01.0073.01.ENG [250] Q. Yang, Y. Liu, T. Chen, and Y. Tong, ‘‘Federated machine learning:
Concept and applications,’’ ACM Trans. Intell. Syst. Technol., vol. 10,
no. 2, pp. 1–19, 2019. 25491 25491 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews [251] L. Ogiela and M. R. Ogiela, ‘‘Cognitive security paradigm for cloud
computing applications,’’ Concurrency Comput., Pract. Exper., vol. 32,
no. 8, p. e5316, Apr. 2020. FRAN CASINO (Member, IEEE) received the
B.Sc. degree in computer science, the M.Sc. degree in computer security and intelligent sys-
tems, and the Ph.D. MARIOS ANAGNOSTOPOULOS AGUSTI
SOLANAS
(Senior Member, IEEE)
received the M.Sc. degree (Hons.) in com-
puter engineering from Rovira i Virgili Univer-
sity (URV), in 2004, the Diploma degree in
advanced studies from the Polytechnic Univer-
sity of Catalonia, in 2005, and the Ph.D. degree
from the Department of Telematics Engineering,
Polytechnic University of Catalonia, in 2007. He is
currently a Professor with the Department of Com-
puter Engineering and Mathematics and the Head
of the Smart Technologies Research Group, URV. He serves as a Scientific
Coordinator for APWG.EU. His current research interests include smart
technologies, health informatics, behavior analysis, multivariate analysis,
privacy protection, and computer security. MARIOS ANAGNOSTOPOULOS received the
master’s degree in information and communica-
tion systems security and the Ph.D. degree in infor-
mation and communication systems engineering
from the University of the Aegean, Greece. He has
worked as a Postdoctoral Research Fellow in cyber
security at the Norwegian University of Science
and Technology (NTNU) and the Singapore Uni-
versity of Technology and Design (SUTD). He has
joined the Department of Electronic Systems,
Aalborg University, Copenhagen, as an Assistant Professor at the Commu-
nication, Media and Information Technologies Section, Aalborg University,
and is a member of the Cyber-Security Research Group. He is the coauthor
of more than 20 publications in peer-reviewed international conferences and
journals. His research interests include the area of networks and computer
security, and specifically DNS security, denial of service attacks, malware
analysis, and forensics. He has also served as a Program Committee Member
for international conferences and has taken part in both national and interna-
tional research programs. MAURO CONTI (Fellow, IEEE) received the
Ph.D. degree from the Sapienza University of
Rome, Italy, in 2009. After his Ph.D. degree,
he was a Postdoctoral Researcher at Vrije Uni-
versiteit Amsterdam, The Netherlands. In 2011,
he joined as an Assistant Professor at the Univer-
sity of Padua, Italy, where he became an Associate
Professor, in 2015, and a Full Professor, in 2018. He has been a Visiting Researcher with GMU,
UCLA, UCI, TU Darmstadt, UF, and FIU. He is
a Full Professor with the University of Padua. He is also affiliated with the
Delft University of Technology (TU Delft) and the University of Washington,
Seattle. His research is funded by companies, including Cisco, Intel, and
Huawei. His main research interests include security and privacy. MARIOS ANAGNOSTOPOULOS In these
areas, he has published more than 400 papers in topmost international peer-
reviewed journals and conferences. He is a Senior Member of the ACM and
a fellow of the Young Academy of Europe. He has been awarded with a
Marie Curie Fellowship by the European Commission, in 2012, and with a
Fellowship by the German DAAD, in 2013. He was the Program Chair of
TRUST 2015, ICISS 2016, WiSec 2017, ACNS 2020, and CANS 2021, and
the General Chair for SecureComm 2012, SACMAT 2013, NSS 2021, and
ACNS 2022. He is the Editor-in-Chief of IEEE TRANSACTIONS ON INFORMATION
FORENSICS AND SECURITY, the Area Editor-in-Chief of IEEE COMMUNICATIONS
SURVEYS AND TUTORIALS, and he has been an Associate Editor of several
journals, including IEEE COMMUNICATIONS SURVEYS AND TUTORIALS, IEEE
TRANSACTIONS ON DEPENDABLE AND SECURE COMPUTING, IEEE TRANSACTIONS ON
INFORMATION FORENSICS AND SECURITY, and IEEE TRANSACTIONS ON NETWORK
AND SERVICE MANAGEMENT. AMRITA GHOSAL received the Ph.D. degree in
computer science and engineering from the Indian
Institute of Engineering Science and Technology,
India, in 2015. After her Ph.D. degree, she worked
as a Postdoctoral Researcher at the Department
of Mathematics, University of Padua, Italy. She
is currently a Marie Skłodowska-Curie Fellow
with the Department of Electronic and Computer
Engineering, University of Limerick, Ireland. She
has coauthored a number of book chapters. Her
research interests include in the areas of security and privacy for mobile
and wireless networks. Particularly, she is interested in detection, prevention,
and mitigation of different DoS style attacks for smart grid, v2x, connected
vehicle, cyber-physical systems, and the IoT. In these areas, she has pub-
lished more than 35 papers in high quality journals and refereed conference
proceedings. ISTVÁN BO ¨RO ¨CZ received the Law (JD) and
postgraduate specialist Diploma degrees in infor-
mation and communication technology law from
the University of Pécs, in 2013 and 2015, respec-
tively, and the LLM degree in law and technol-
ogy from Tilburg University, in 2016. He is a
Data Protection Officer at Ion Beam Applications
SA (IBA) and a Researcher at the Research Group
on Law, Science, Technology and Society (LSTS). He is also a member of the Health and Ageing
Law Laboratory (HALL), a spinoff group within LSTS. He is involved and
provides legal assistance in several EU co-funded research projects, such as
ARC, LOCARD, PERSONA, STAR, INTREPID, MaTHiSiS, FORENSOR,
HR-Recycler, and SUCCESS or PARENT. s_103643v010101p.pdf He is the coauthor of more than 30 publications in peer reviewed journals and
conference proceedings. His research interests include related to networks
security, privacy preserving techniques, and blockchain technology. He has
also served as the Program Committee Member for international conferences
and has taken part in both national and international research programs. [266] (2019). National Institute of Standards and Technology. Recommen-
dations on Shaping technology According to GDPR Provisions—
Exploring the Notion of Data Protection by Default. [Online]. Available: https://www.enisa.europa.eu/publications/recommendations-
on-shaping-tec% hnology-according-to-gdpr-provisions-part-2
6
i
i
i
l
d
ki [267] A. Zigomitros, F. Casino, A. Solanas, and C. Patsakis, ‘‘A survey on
privacy properties for data publishing of relational data,’’ IEEE Access,
vol. 8, pp. 51071–51099, 2020. [268] (2019). The European Union Agency for Cybersecurity (ENISA). Towards a Framework for Policy Development in Cybersecurity—
Security and Privacy Considerations in Autonomous Agents. [Online]. Available:
https://www.enisa.europa.eu/publications/considerations-in-
autonomous-a% gents [269] H. Arshad, E. Omlara, I. O. Abiodun, and A. Aminu, ‘‘A semi-automated
forensic investigation model for online social networks,’’ Comput. Secur.,
vol. 97, Oct. 2020, Art. no. 101946. [270] N. Sunde and G. Horsman, ‘‘Part 2: The phase-oriented advice
and review structure (PARS) for digital forensic investigations,’’
Forensic
Sci. International:
Digit. Invest.,
vol. 36,
Mar. 2021,
Art. no. 301074. 25492 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews MARIOS ANAGNOSTOPOULOS These projects target a range of
areas, such as law enforcement, technology-induced education, human–robot
interaction, smart cities, or helping the work of data protection authorities. He is a member of the Ethical Advisory Board of the Horizon2020 Project
CUIDAR. His research interests include the notion of the privacy of the mind
along with the legal, theoretical, and practical issues of human enhancement
technologies, with special focus on cognitive enhancement. In particular,
he focuses on technologies which passively read and actively affect the
human brain and the mind both within and outside the field of health care. He is also an Editor of the World Law Dictionary, developed by TransLegal
Sweden AB. CONSTANTINOS PATSAKIS received the B.Sc. degree in mathematics from the University of
Athens, Greece, the M.Sc. degree in information
security from the Royal Holloway, University of
London, and the Ph.D. degree in cryptography
and malware from the Department of Informatics,
University of Piraeus. He has participated in sev-
eral national (Greek, Spanish, Catalan, and Irish)
and European research and development projects
(e.g.,
TACTICS,
MITIGATE,
OPERANDO,
SAURON, PRACTICIES, and YAKSHA). He worked as a Researcher
at the UNESCO Chair in data privacy and as a Research Fellow at the
Trinity College Dublin, Dublin, Ireland. His main research interests include
cryptography, malware, security, privacy, and data anonymization. Currently,
he is Assistant Professor at University of Piraeus and adjunct researcher of
Athena Research and Innovation Center. 25493 VOLUME 10, 2022
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https://openalex.org/W3035763495
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https://escholarship.org/content/qt19v4c4wp/qt19v4c4wp.pdf?t=rdkcn7
|
English
| null |
A rare presentation of hepatolithiasis in an adolescent patient: A case report
|
International journal of surgery case reports
| 2,020
|
cc-by
| 2,837
|
a
r
t i c l e
i n
f o Article
history:
Received
22
April
2020
Accepted
7
June
2020
Available
online
12
June
2020
Keywords:
Adolescent
Case
report
Hepatic
duct
Hepatolithiasis
Intrahepatic
stone Article
history:
Received
22
April
2020
Accepted
7
June
2020
Available
online
12
June
2020 INTRODUCTION:
Hepatolithiasis
(intrahepatic
stones)
is rare in adolescent
patients
and
requires complex
management
strategies
to prevent
recurrent
infections
and progression
to hepatic
fibrosis. Surgical man-
agement
is often
required. In cases
of unclear etiology,
further
work-up
is indicated
to provide
insight
into
future management. In this
report we describe
an extensive
stone
analysis. PRESENTATION
OF CASE: A 20-year-old
Caucasian
female presented
with known
hepatolithiasis
and
multiple
prior recurrent
bouts of abdominal
pain requiring
hospitalization. Magnetic
resonance
cholangiopancreatography
(MRCP)
demonstrated
an abnormal
left-sided
hepatic
biliary ductal
system
dilatation. She was treated
surgically
with a formal left hepatectomy
and preservation
of the caudate
lobe. The right ductal system
had no stones
or evidence
of inflammation,
and her bile and stones
cultures
were
negative
for organism
growth. An extensive
analysis
demonstrated
stone
composition
primarily of
cholesterol. Keywords:
Adolescent
Case
report
Hepatic
duct
Hepatolithiasis
Intrahepatic
stone DISCUSSION:
Adolescent
presentations
of hepatolithiasis
are rare and considerations
in the differential
diagnosis
include
primary sclerosing
cholangitis,
bile
acid transporter
defects, and other known
genetic
diseases. This case is unique
because
only the left half of the intrahepatic
ductal
system
had evidence of
stone
disease
and the bile was sterile. A detailed stone
analysis demonstrating
cholesterol
supersaturation
provides
additional
context though
the
etiology
remains
unclear
in this case and will require lifelong
follow-up. p
CONCLUSION:
Early-onset
hepatolithiasis
is rare and requires expert management,
and in some cases
definitive
surgical
management
with life-long
follow-up. Extensive
stone
analysis
and genetic testing
can
be performed
to help
identify
disease etiology
in unique
cases. ©
2020 The Author(s). Published
by Elsevier
Ltd on behalf
of IJS Publishing
Group Ltd. This is an open
access
article under
the CC BY license
(http://creativecommons.org/licenses/by/4.0/). A
rare
presentation
of
hepatolithiasis
in
an
adolescent
patient:
A
case
report Jonathan
Freise a, Jorge
Mena a, Kwun
Wah
Wen b, Marshall
Stoller c, Sunita
Ho c,d,
Carlos
Corvera e,∗ a School
of
Medicine,
University
of
California
San
Francisco,
San
Francisco,
CA,
USA
b Department
of
Pathology,
University
of
California
San
Francisco,
San
Francisco,
CA,
USA
c Department
of
Urology,
University
of
California
San
Francisco,
San
Francisco,
CA,
USA
d Department
of
Preventive
and
Restorative
Dental
Sciences,
Division
of
Biomaterials
and
Bioengineering,
University
of
California
San
Francisco,
S
Francisco,
CA,
USA
e D
f S
Di i i
f S
i
l O
l
U i
i
f C lif
i
S
F
i
S
F
i
CA USA a School
of
Medicine,
University
of
California
San
Francisco,
San
Francisco,
CA,
USA
b Department
of
Pathology,
University
of
California
San
Francisco,
San
Francisco,
CA,
USA
c Department
of
Urology,
University
of
California
San
Francisco,
San
Francisco,
CA,
USA
d Department
of
Preventive
and
Restorative
Dental
Sciences,
Division
of
Biomaterials
and
Bioengineering,
University
of
California
San
Francisco,
San
Francisco,
CA,
USA a School
of
Medicine,
University
of
California
San
Francisco,
San
Francisco,
CA,
USA f
y
f
f
b Department
of
Pathology,
University
of
California
San
Francisco,
San
Francisco,
CA,
USA d Department
of
Preventive
and
Restorative
Dental
Sciences,
Division
of
Biomaterials
and
Bioengineering,
University
of
California
S
Francisco,
CA,
USA ,
,
e Department
of
Surgery,
Division
of
Surgical
Oncology,
University
of
California
San
Francisco,
San
Francisco,
CA,
USA Abbreviations:
EDX,
Electron
Dispersive
X-Ray;
Micro-XCT,
Micro
X-Ray
Com-
puterized
Tomography;
MRCP,
magnetic
resonance
cholangiopancreatography;
PSC,
primary
sclerosing
cholangitis;
SEM,
Scanning
Electron
Microscopy.
∗Corresponding
author
at:
UCSF
Helen
Diller
Family
Comprehensive
Cancer
Cen-
ter,
1825
Fourth
Street,
4th
Floor,
San
Francisco,
CA,
94158,
USA.
E-mail
address:
Carlos.corvera@ucsf.edu
(C.
Corvera). CASE
REPORT
–
OPEN
ACCESS International
Journal
of
Surgery
Case
Reports
72
(2020)
343–345 https://doi.org/10.1016/j.ijscr.2020.06.017
2210-2612/©
2020
The
Author(s).
Published
by
Elsevier
Ltd
on
behalf
of
IJS
Publishing
Group
Ltd.
This
is
an
open
access
article
under
the
CC
BY
license
(http:/
creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.ijscr.2020.06.017
2210-2612/©
2020
The
Author(s).
Published
by
Elsevier
Ltd
on
behalf
of
IJS
Publishing
Group
Ltd.
This
is
an
open
access
article
under
the
CC
BY
license
(http://
creativecommons org/licenses/by/4 0/) https://doi.org/10.1016/j.ijscr.2020.06.017
2210-2612/©
2020
The
Author(s).
Published
by
Elsevier
Ltd
on
behalf
of
IJS
Publishing
Gr
creativecommons.org/licenses/by/4.0/). ed
by
Elsevier
Ltd
on
behalf
of
IJS
Publishing
Group
Ltd.
This
is
an
open
access
article
under
the
CC
BY
license
(http:// Powered by the California Digital Library
University of California Powered by the California Digital Library
University of California eScholarship.org 3.
Discussion This
case
report
illustrates
a
unique
presentation
of
hepatolithi-
asis
for
several
reasons. While
most
patients
are
diagnosed
with
hepatolithiasis
in
the
5th
or
6th
decade
of
life,
presentation
in
ado-
lescence
is
rare. Primary
considerations
in
the
differential
diagnosis
include
primary
sclerosing
cholangitis
(PSC),
recurrent
pyogenic
cholangitis,
bile
acid
transporter
defect,
and
Caroli
disease. PSC
is
a
chronic
cholestatic
liver
disease
of
unknown
etiology
character-
ized
by
inflammation
and
fibrosis
of
intra
and
extrahepatic
bile
ducts
leading
to
progressive
stricturing
and
dilatation
of
the
biliary
ducts. Given
the
extensive
stone
disease
and
diffuse
involvement
of
the
liver
specimen,
this
diagnosis
is
unlikely. In
addition,
PSC
generally
affects
the
entire
biliary
tract
and
is
not
isolated
to
one
side
of
the
liver
as
in
our
patient. Recurrent
pyogenic
cholangitis,
which
is
endemic
in
East
Asia,
remains
unlikely
given
the
patient’s
lack
of
travels
to
that
area
and
Caucasian
descent. Moreover,
her
frequent
hospitalizations
occurred
without
systemic
signs
of
infec-
tions
and
her
bile
was
sterile. A
bile
acid
transporter
defect,
such
as
ABCB4/MDR3,
can
present
with
hepatolithiasis
at
a
younger
age,
but
the
absence
of
significant
disease
in
the
right
hepatic
duct
sys-
tem
argues
against
this
as
a
cause
[6]. Caroli
disease,
a
congenital
disorder
characterized
by
multifocal,
segmental
dilatation
of
large
intrahepatic
ducts,
is
a
possibility
in
cases
of
early-onset
hepa-
tolithiasis. However,
the
patients’
history
of
MRCP
reports
did
not
strongly
favor
this
diagnosis
because
the
biliary
dilatation
appeared
more
fusiform
than
saccular,
which
would
be
an
abnormal
pre-
sentation. Moreover,
this
diagnosis
was
not
supported
by
final
histological
examination. Ultimately,
we
are
left
with
an
unknown
cause
of
this
patient’s
development
of
early-onset
hepatolithiasis. We attribute
her
problem
to
a
yet
undescribed
or
undiscovered
genetic
cause
given
the
patient’s
strong
family
history
of
stone
dis-
ease. Her
history
was
notable
for
early
gallbladder
disease
in
her An
MRCP
showed
isolated
and
dilated
biliary
ducts
from
the
cau-
date
lobe
with
eventual
resolution
of
her
symptoms. Bile
culture
samples
from
the
operative
procedure
were
negative
for
organ-
ism
growth. Pathology
demonstrated
numerous
(up
to
0.5
mm)
yellow
tan
calculi
filling
the
entire
dilated
left
biliary
ductal
sys-
tem
and
fibrosis
and
inflammation
of
the
bile
duct
walls,
as
seen
in
the
section
of
left
liver
lobe
(Fig. 1). The
hepatic
parenchyma
showed
marked
sinusoidal
dilatation,
congestion,
and
hepatic
plate
atrophy. No
tumor
or
parasite
was
found. 2.
Presentation
of
case A
20-year-old
Caucasian
female
with
a
strong
family
history
of
early
onset
gallbladder
disease
first
presented
to
our
center
with
known
hepatolithiasis
and
recurrent
bouts
of
severe
abdominal
pain
requiring
multiple
hospitalizations. She
was
first
hospital-
ized
at
the
age
of
13
for
right
upper
quadrant
abdominal
pain
found
to
have
choledocholithiasis
with
intrahepatic
and
extrahep-
atic
biliary
duct
dilatation. She
underwent
endoscopic
retrograde
cholangiopancreatography
during
her
admission
had
a
subsequent
cholecystectomy
with
no
evidence
of
cholelithiasis. In
the
following
years,
she
experienced
recurrent
bouts
of
severe
abdominal
pain
without
any
fever
or
chills
and
multiple
MRCP
studies
that
demon-
strated
abnormal
left
hepatic
biliary
ductal
system
dilatation. At
the
time
of
her
presentation
to
our
center,
she
was
noted
to
have
a
dominant
stone
impacted
at
the
origin
of
her
left
main
hepatic
duct
with
a
left
sided
dilated
ductal
system. She
was
taken
to
the
operating
room
for
a
formal
left
hepatectomy. Intraoperatively,
she
was
found
to
have
palpable
stone
disease
throughout
her
left
liver
with
a
large
stone
aggregate
at
the
origin
of
the
left
hepatic
duct. Choledochoscopy
of
the
right
sided
ductal
system
demonstrated
normal
biliary
epithelium
without
inflammation
or
stones. She
was
treated
by
formal
left
hepatectomy
and
preservation
of
her
extra-
hepatic
biliary
tract. Her
preoperative
symptom
of
abdominal
pain
completely
resolved,
however,
her
recovery
was
complicated
by
the
development
of
pruritus
two
weeks
after
discharge. specimens
and
of
the
core
of
fractured
specimens
demonstrated
the
stones
were
made
almost
entirely
of
smooth
plates
(Fig. 2). EDX
of
the
plates
revealed
presence
of
three
major
elements:
carbon,
nitrogen,
and
oxygen,
with
carbon
being
the
predomi-
nant
element,
consistent
with
a
similar
make-up
to
cholesterol
based
stones. No
calcium
was
detected
from
EDX. Stone
specimens
were
subsequently
placed
in
different
solvents:
ethanol,
DI
water,
DI
water
+
detergent,
and
trichloromethane. Dissolution
was
only
achieved
in
the
trichloromethane
solution. J.
Freise
et
al.
/ International
Journal
of
Surgery
Case
Reports
72
(2020)
343–345 344 Fig. 2. SEM
of
the
surface
of
whole
specimens
and
of
the
core
of
fractured
specimens
demonstrating
stones
composed
almost
entirely
of
smooth
plates. Fig. 1. Hemisection
of
left
liver
lobe
demonstrating
yellow
tan
intrahepatic
stones
filling
the
dilated
left
biliary
ductal
system
and
fibrosis
and
inflammation
of
the
bile
duct
walls. Fig. 1. Hemisection
of
left
liver
lobe
demonstrating
yellow
tan
intrahepatic
stones
filling
the
dilated
left
biliary
ductal
system
and
fibrosis
and
inflammation
of
the
bile
duct
walls. Fig. 2. SEM
of
the
surface
of
whole
specimens
and
of
the
core
of
fractured
specimens
demonstrating
stones
composed
almost
entirely
of
smooth
plates. 1.
Introduction symptoms
of
the
intrahepatic
type
are
more
frequent
in
younger
age
groups
[2]. Etiology
remains
unknown
in
most
cases,
although
genetic
and
environmental
factors
are
thought
to
contribute. Stones
are
classified
by
composition
into
calcium
bilirubinate
stones
and
cholesterol
stones,
with
75%
of
cases
being
calcium
bilirubinate
[3]. Treatment
goals
focus
on
resolving
recurrent
infection
and
preventing
subsequent
hepatic
fibrosis
and
progression
to
cholan-
giocarcinoma. The
most
efficacious
treatment
to
date
has
been
surgery,
including
removal
of
affected
liver
segment(s)
to
pre-
vent
recurrence
of
stone
formation
and
progressive
hepatocellular
injury. In
this
case
report,
we discuss
a
unique
presentation
of hep-
atolithiasis
at
our
academic
center
with
a
comprehensive
work-up
of
the
patient’s
stone
disease
to
better
characterize
etiology. This
work
is
reported
according
to
the
SCARE
and
PROCESS
criteria
[4,5]. Hepatolithiasis
is
defined
as
the
presence
of
gallstones
in
the
bile
ducts
proximal
to
the
confluence
of
the
right
and
left
hepatic
ducts,
irrespective
of
the
co-existence
of
stone
disease
in
the
common
bile
duct
and
gallbladder. The
prevalence
of
hepatolithiasis
is
highest
in
East
Asia
and
rare
in
Western
countries
[1]. Moreover,
inci-
dence
occurs
most
commonly
in
the
fifth
and
sixth
decades,
though j
Author(s). Published
by
Elsevier
Ltd
on
behalf
of
IJS
Publishing
Group
Ltd. This
is
an
open
access
article
under
the
CC
BY
license
(http
enses/by/4.0/). CASE
REPORT
–
OPEN
ACCESS J. Freise
et
al. / International
Journal
of
Surgery
Case
Reports
72
(2020)
343–345 Declaration
of
Competing
Interest [3]
I. Endo,
R. Matsuyama,
R. Mori,
H. Shimada,
Chapter
39
-
intrahepatic
stones,
in: W.R. Jarnagin
(Ed.),
Blumgart’s
Surgery
of
the
Liver,
Biliary
Tract
and
Pancreas,
2-Volume
Set
(Sixth
Edition),
Content
Repository
Only,
Philadelphia,
2017,
http://dx.doi.org/10.1016/B978-0-323-34062-5.00039-X, 642-655.e3. The
authors
declare
that
they
have
no
conflicts
of
interest. Sources
of
funding [4] R.A. Agha,
M.R. Borrelli,
R. Farwana,
et
al.,
The
SCARE
2018
statement:
updating
consensus
Surgical
CAse
REport
(SCARE)
guidelines,
Int. J. Surg. Lond. Engl. 60
(2018)
132–136,
http://dx.doi.org/10.1016/j.ijsu.2018.10.028. Jorge
Mena
received
the
UCSF
PROF
PATH
Pre-Doctoral
Grant
to
support
his
research. [5]
R.A. Agha,
M.R. Borrelli,
R. Farwana,
et
al.,
The
PROCESS
2018
statement:
updating
consensus
Preferred
Reporting
of
CasE
Series
in
Surgery
(PROCESS)
guidelines,
Int. J. Surg. Lond. Engl. 60
(2018)
279–282,
http://dx.doi.org/10. 1016/j.ijsu.2018.10.031. 3.
Discussion Calculi
composition
was
noted
to
be
primarily
of
cholesterol
with
some
calcium
bilirubi-
nate,
bile
salts
and
pigments
consistent
with
a
bile
stone. Given
the
unique
presentation
of
the
patient’s
disease,
a
more
extensive
analysis
was
performed
on
the
stones. Material
analysis
of
the
stones
was
performed
using
Micro
X-Ray
Computerized
Tomography
(Micro-XCT)
for
density
and
internal
structure,
and
Scanning
Electron
Microscopy
(SEM)
for
ultrastructure. Elemental
analysis
was
performed
using
Electron
Dispersive
X-Ray
(EDX)
Spectroscopy. Micro-XCT
of
stone
cross
section
(not
shown)
demonstrated
low
mineral
density
through-
out
the
specimen
and
a
hollow
core. SEM
of
the
surface
of
whole References [1]
A. Pausawasdi,
P. Watanapa,
Hepatolithiasis:
epidemiology
and
classification,
Hepatogastroenterology
44
(14)
(1997)
314–316. [1]
A. Pausawasdi,
P. Watanapa,
Hepatolithiasis:
epidemiology
and
classification,
Hepatogastroenterology
44
(14)
(1997)
314–316. [2]
S. Tazuma,
Gallstone
disease:
epidemiology,
pathogenesis,
and
classification
of
biliary
stones
(common
bile
duct
and
intrahepatic),
Best
Pract. Res. Clin. Gastroenterol. 20
(6)
(2006)
1075–1083,
http://dx.doi.org/10.1016/j.bpg.2006. 05.009. [2]
S. Tazuma,
Gallstone
disease:
epidemiology,
pathogenesis,
and
classification
of
biliary
stones
(common
bile
duct
and
intrahepatic),
Best
Pract. Res. Clin. Gastroenterol. 20
(6)
(2006)
1075–1083,
http://dx.doi.org/10.1016/j.bpg.2006. 05.009. J.
Freise
et
al.
/ International
Journal
of
Surgery
Case
Reports
72
(2020)
343–345 345 Provenance
and
peer
review Presentations
of
early-onset
hepatolithiasis
is
rare,
particularly
in
Western
countries. Definitive
surgical
management
is
required
in
complex
cases. In
cases
with
unknown
etiology,
further
work-
up
is
indicated
and
can
include
stone
analysis
and
genetic
testing. If
indicated,
life-long
follow-up
should
be
incorporated,
as
is
the
recommended
management
in
this
case. A
detailed
family
history,
genetic
testing,
and
extensive
stone
analysis
may
be
performed
to
help
characterize
disease
etiology
in
unique
presentations. Author
contribution father
and
paternal
cousin,
aunt
and
grandmother,
which
would
argue
for
a
genetic
component. Genetic
testing
of
family
members
may
help
better
characterize
a
cause
for
this
patient’s
rare
case. father
and
paternal
cousin,
aunt
and
grandmother,
which
would
argue
for
a
genetic
component. Genetic
testing
of
family
members
may
help
better
characterize
a
cause
for
this
patient’s
rare
case. Jonathan
Freise:
data
collection,
manuscript
writing,
design
of
study. The
stone
analysis,
while
not
revealing
of
a
definite
diagnosis,
might
help
point
in
the
direction
of
the
etiology. The
stones
col-
lected
from
this
patient
were
composed
primarily
of
cholesterol. Cholesterol
is
commonly
found
as
a
major
component
of
stones
in
the
extrahepatic
ducts
and
gallbladder
but
it
is
rarely
found
as
the
main
component
in
intrahepatic
stones
[7]. In
the
absence
of
precipitating
factors
such
as
infection,
cholestasis,
or
structural
abnormalities
which
were
not
apparent
in
this
patient,
defects
of
bile
metabolism
are
likely
candidates
for
relative
cholesterol
super-
saturation
and
nucleation
within
the
liver
ducts
[8]. Jorge
Mena:
performed
stone
analysis,
manuscript
writing
and
revision. Kwun
Wah
Wen:
evidence
collection,
manuscript
revision. Marshall
Stoller:
supervision
of
stone
analysis,
design
of
study. shall
Stoller:
supervision
of
stone
analysis,
design
of Sunita
Ho:
supervision
of
stone
analysis,
design
of
study,
manuscript
revision. Sunita
Ho:
supervision
of
stone
analysis,
design
of
study,
manuscript
revision. Carlos
Corvera:
managed
patient
and
performed
the
surgery,
design
of
study,
manuscript
revision,
corresponding
author. Carlos
Corvera:
managed
patient
and
performed
the
surgery,
design
of
study,
manuscript
revision,
corresponding
author. Registration
of
research
studies Our
patient
will
need
close
clinical
follow-up
as
she
remains
at
high
risk
of
developing
intrahepatic
stone
formation
in
her
remant
right
liver. A
life-time
of
interval
liver
function
tests,
and
cross-sectional
imaging
is
strongly
recommended
to
detect
early
recurrent
disease
to
prevent
progressive
indolent
liver
injury
and
ultimately
liver
failure. Indeed,
occasionally
in
patients
with
recur-
rent
pyogenic
cholangitis,
liver
transplantation
as
a
salvage
therapy
is
required
[9]. 1. Name
of
the
registry:
n/a. 2. Unique
Identifying
number
or
registration
ID:
n/a. 3. Hyperlink
to
your
specific
registration
(must
be
publicly
acces-
sible
and
will
be
checked). 3. Hyperlink
to
your
specific
registration
(must
be
publicly
acces-
sible
and
will
be
checked). 4.
Conclusion Carlos
Corvera. Ethical
approval [6]
J. Benzimra,
S. Derhy,
O. Rosmorduc,
Y. Menu,
R. Poupon,
L. Arrivé,
Hepatobiliary
anomalies
associated
with
ABCB4/MDR3
deficiency
in
adults:
a
pictorial
essay,
Insights
Imaging
4
(3)
(2013)
331–338,
http://dx.doi.org/10. 1007/s13244-013-0243-y. Ethical
approval
exempted
by
our
institution. Not
commissioned,
externally
peer-reviewed. Not
commissioned,
externally
peer-reviewed. Consent [7]
S.D. Strichartz,
M.Z. Abedin,
A.F. Ippoliti,
M. Derezin,
J.J. Roslyn,
Intrahepatic
cholesterol
stones:
a
rationale
for
dissolution
therapy,
Gastroenterology
100
(1) (1991)
228–232,
http://dx.doi.org/10.1016/0016-5085(91)90605-k. Written
informed
consent
was
obtained
from
the
patient
for
publication
of
this
case
report
and
accompanying
images. A
copy
of
the
written
consent
is
available
for
review
by
the
Editor-in-Chief
of
this
journal
on
request. ( ) (
)
p //
g/
/
(
)
[8] X. Ran,
B. Yin,
B. Ma,
Four
major
factors
contributing
to
intrahepatic
stones,
Gastroenterol. Res. Pract. (2017),
http://dx.doi.org/10.1155/2017/7213043. [9] You
M. su,
S.H. Lee,
J. Kang,
et
al.,
Natural
course
and
risk
of
cholangiocarcinoma
in
patients
with
recurrent
pyogenic
cholangitis:
a
retrospective
cohort
study,
Gut
Liver
13
(3)
(2019)
373–379,
http://dx.doi.org/10.5009/gnl18339. Open
Access
This
article
is
published
Open
Access
at
sciencedirect.com. It
is
distributed
under
the
IJSCR
Supplemental
terms
and
conditions, which
permits
unrestricted
non
commercial
use,
distribution,
and
reproduction
in
any
medium,
provided
the
original
authors
and
source
are
credited.
|
https://openalex.org/W2922476837
|
https://digibug.ugr.es/bitstream/10481/61827/1/remotesensing-11-00643-v2.pdf
|
English
| null |
Detection of Fir Trees (Abies sibirica) Damaged by the Bark Beetle in Unmanned Aerial Vehicle Images with Deep Learning
|
Remote sensing
| 2,019
|
cc-by
| 14,775
|
Received: 4 February 2019; Accepted: 12 March 2019; Published: 16 March 2019 Abstract: Invasion of the Polygraphus proximus Blandford bark beetle causes catastrophic damage
to forests with firs (Abies sibirica Ledeb) in Russia, especially in Central Siberia. Determining tree
damage stage based on the shape, texture and colour of tree crown in unmanned aerial vehicle
(UAV) images could help to assess forest health in a faster and cheaper way. However, this task is
challenging since (i) fir trees at different damage stages coexist and overlap in the canopy, (ii) the
distribution of fir trees in nature is irregular and hence distinguishing between different crowns is
hard, even for the human eye. Motivated by the latest advances in computer vision and machine
learning, this work proposes a two-stage solution: In a first stage, we built a detection strategy that
finds the regions of the input UAV image that are more likely to contain a crown, in the second
stage, we developed a new convolutional neural network (CNN) architecture that predicts the fir tree
damage stage in each candidate region. Our experiments show that the proposed approach shows
satisfactory results on UAV Red, Green, Blue (RGB) images of forest areas in the state nature reserve
“Stolby” (Krasnoyarsk, Russia). Keywords: multi-class classification; drone; aerial photography; Siberian fir; Siberia; deep-learning;
convolutional neural networks; forest health remote sensing remote sensing remote sensing Article Anastasiia Safonova 1,2,3,4,*
, Siham Tabik 3
, Domingo Alcaraz-Segura 4,5,6
, Anastasiia Safonova 1,2,3,4,*
, Siham Tabik 3
, Domingo Alcaraz-Segura 4,5,6
,
Alexey Rubtsov 1
, Yuriy Maglinets 1 and Francisco Herrera 3 1
Institute of Space and Information Technologies, Siberian Federal University, 660074 Krasnoyarsk, Russia;
alexeyruss@gmail.com (A.R.); maglinets@mail.ru (Y.M.) 1
Institute of Space and Information Technologies, Siberian Federal University, 660074 Krasnoyarsk, Russia;
alexeyruss@gmail.com (A.R.); maglinets@mail.ru (Y.M.) 2
The Earth Science Museum of M.V. Lomonosov Moscow State University, 119991 Moscow, Russia
3
Andalusian Research Institute in Data Science and Computational Intelligence, University of Granada,
18071 Granada, Spain; siham@ugr.es (S.T.); herrera@decsai.ugr.es (F.H.) p
g
g
4
Department of Botany, University of Granada, 18071 Granada, Spain; dalcaraz@ugr.es
5 5
Andalusian Center for the Assessment and Monitoring of Global Change (CAESCG), University of Almería,
04120 Almería, Spain p
6
iEcolab., Interuniversity Institute for Earth System Research in Andalusia (IISTA), University of Granada,
18006 Granada, Spain p
*
Correspondence: safonova.nastya1@gmail.com
1. Introduction Taiga and Boreal forests play an important role in the global climate through the carbon, water
and energy balance (Bonan [1]). In Russia, fir forests provide multiple provisioning, regulating and
cultural ecosystem services and are considered a national asset of the country. Despite forests are a
renewable resource, in vast areas of the world, forest degradation is too high and not compensated
by regeneration (Hansen et al. [2]). One of the main problems of Russian forest degradation is the
spatial spread acceleration of the four-eyed fir bark beetle (Polygraphus proximus Blandford) invasion
that causes fast death of fir trees (Abies sibirica Ledeb) in various forest ecosystems. The geographic
origin of P. proximus propagation comes from its natural habitat in Japan, the Korean Peninsula, Remote Sens. 2019, 11, 643; doi:10.3390/rs11060643 www.mdpi.com/journal/remotesensing 2 of 19 Remote Sens. 2019, 11, 643 Eastern China and the Russian Far East (Khabarovsk and Primorskii krai, Sakhalin and Kuril Islands)
(Kuznetsov et al. [3]. In Siberia, the first beetle occurrence was registered in 2008 (Kerchev [4]) and
within the last 10 years, massive outbreaks of P. proximus occurred on large forested areas in the
south-eastern part of the West Siberian Plain: Tomsk, Kemerovo and Novosibirsk oblasts, Altai region,
in the Altai Republic as well as in Krasnoyarsk region (Pashenova et al. and Baranchikov et al. [5,6]),
where the problem became catastrophic and got out of control. The invasion of this type of beetle
can lead not only to degradation of fir forests but also to create a threat to fir existence as a forest
type species, with the subsequent broad implications for the regional and global climate (Helbig et al.,
Ma [7,8]). Death of fir trees due to P. proximus outbreaks occurs after several stages. The beetle usually attacks
trunks of weakened trees, fallen deadwood and newly harvested wood. In case of massive outbreaks,
P. proximus also attacks healthy trees, which can resist against the beetle attack during 2–3 years. Penetration of P. proximus under tree bark results in pervasion and reproduction of ophiostomatoid
fungi (different species) and their phytopathogenic activity leads to the gradual weakening of fir
trees. As the beetle further colonizes the tree, it begins to dry out. Increase of beetles quantity in a
local forest stand leads to a massive death of fir trees. 1. Introduction Usually, fir trees die within 2–4 years from the
moment of first beetle attack, so the task of the beetle invasion monitoring is really important and
poses a challenge to develop a remote survey and early-warning system for precise estimation of forest
damage states. Advances in Earth remote sensing techniques, particularly very high resolution satellite and
airborne imagery, open the possibility to develop tools for regional mapping of consequences of forest
pest activities. Nowadays, application of unmanned aerial vehicles (UAV) tends to be more popular in
local scale forestry research because of better spatial resolution (Lehmann et al. [9]). Also, UAV data
provides the basis for development of new methods in data analysis which can be applied later at
larger scales (satellite data). Recently, application of deep learning, in particular convolutional neural
networks (CNN), in processing of colour (RGB) images of the Earth surface from various data sources
have provided high accuracy results in recognition of different plant species [10–14]. However, there
are still no published studies on the application of CNN methods to very high resolution imagery
for detection of forest health decline caused by pest invasions. The goal of this study was to test the
possibilities of neural networks as a new approach to detect bark beetle outbreaks in fir forests. In
particular, our aim was to develop and test a CNN method to automatically detect individual fir trees
disturbed by P. proximus in very high resolution imagery of mixed forests. The main contributions of this paper can be listed as follows: •
As far as we know, this is the first work in addressing the problem of forest damage detection
caused by the P. proximus beetle in very high resolution images from UAVs with deep learning. W b ilt
l b ll d d t
t f
th i
ith f
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fit •
We built a new labelled dataset of orthoimages with four categories of damage stages in fir tr g
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•
We designed a new CNN architecture to accurately classify trees in UAV images into different
damage categories of trees and compared it to the most powerful CNN models in the stat. •
We designed a new CNN architecture to accurately classify trees in UAV images into different
damage categories of trees and compared it to the most powerful CNN models in the stat. 1. Introduction •
We developed the detection model as follows. First, a new detection method selects the candidate
regions that contain trees in UAV images. Then, these candidate regions are processed by the
multiclass model to finally predict the category of tree damage. •
We developed the detection model as follows. First, a new detection method selects the candidate
regions that contain trees in UAV images. Then, these candidate regions are processed by the
multiclass model to finally predict the category of tree damage. •
We provide a complete description of the used methodology and the source code so that it can be
replicated by other researchers to detect and classify tree damage by bark beetle in other images. Our source code can be found at https://github.com/ansafo/OurCNN. The rest of the paper is organized as follows. An introduction to Deep learning and CNNs is
presented in Section 2, which includes a description of the CNN models and of the auxiliary techniques
of transfer learning and data augmentation. The related works on tree classification are provided in
Section 3. The study area and data acquisition are presented in Section 4. The proposed methodology is
presented in Section 5, which includes a definition of fir trees damage categories, pre-processing dataset 3 of 19 Remote Sens. 2019, 11, 643 and data augmentation techniques of sample patches for training CNN models, description of the
development of the classification model algorithm and creation of a data subset for independent model
verification. Section 6 presents the obtained results and data analysis. Discussion and conclusions are
provided in Section 7. 2. Introduction to Deep learning and CNNs Conceptually, this operation
is applied to increase the abstraction of the extracted features and it usually follows the
convolution layer. •
A fully connected layer, which is used as a classifier of the previously calculated high level features
to derive scores for each target class. •
A fully connected layer, which is used as a classifier of the previously calculated high level features
to derive scores for each target class. Deep CNNs are built by alternating several convolutional layers with pooling layers. At the
end of the sequence, the obtained convolutions and pooling layers are connected to one or more
fully connected layers. Softmax function is used to convert the scores of the output of the final fully
connected layer into a set of probabilities between 0 and 1, which represent the final output layer. Before starting the training process, the weights of the CNN are initialized with random values. The CNN is then trained iteratively until convergence during a number of epochs. The loss function is
used to measure how good the training process is by quantifying the distance between the prediction
and the expected value. To find the best weights that correctly assign the input to the correct answer,
the training process is reformulated into an optimization problem that aims to minimize the loss
function. This minimization can be achieved using several optimization algorithms depending on the
problem. One example is the ADAptive Moment estimation (ADAM) optimizer that computes the
best weights. 2. Introduction to Deep learning and CNNs In this Section, we make a brief introduction to deep learning and particularly to CNN models
(Section 2.1), including the two main approaches used to improve the learning of CNN: that is, transfer
learning and data augmentation (Section 2.2). Deep Neural Networks (DNNs) are a subset of machine learning algorithms that learn from a
first set of data to make predictions on new data. Unlike most machine learning algorithms, DNNs
are capable of extracting the existent patterns from data automatically, without the need of external
hand-crafted features. Their computation is organized into layers; each layer is composed of a number
of artificial neurons. To introduce non-linearity in the network, non-linear functions are introduced
at the neuron level, such as the Rectified Linear Unit (ReLU) function. Deep networks are built by
stacking a very large number of layers. They provide a powerful framework for supervised learning
when trained on a large number of labelled samples (Goodfellow et al. [15]). A DNN includes an input
layer, an output layer and hidden layers between them where the calculations take place. One of the most powerful types of DNNs are Convolutional Neural Networks (CNNs), which
have shown impressive results in image classification (given an input image, the CNN produces a
label that indicates the visual content of that image) and object detection in images (given an input
image the CNN produces a label together with a bounding box that indicates the region where the
object-class is located in the image). There are three main types of hidden layers in a CNN: •
A convolution layer, the main building block of a CNN. It is based on a fundamental operation in
image processing called convolution, which consists of filtering a 2D input image with a small
2D-matrix called filter or kernel. •
A convolution layer, the main building block of a CNN. It is based on a fundamental operation in
image processing called convolution, which consists of filtering a 2D input image with a small
2D-matrix called filter or kernel. •
A pooling layer, which reduces the input matrix in general by half. Conceptually, this operation
is applied to increase the abstraction of the extracted features and it usually follows the
convolution layer. •
A pooling layer, which reduces the input matrix in general by half. 2.1. CNNs Models In Appendix B, we provide a brief description of the CNNs that are compared against the
CNN developed in this work, that is, VGG, ResNet, Inception-V3, InceptionResNet-V2, Xception
and DensNet. Remote Sens. 2019, 11, 643 4 of 19 2.2. Transfer Learning and Data Augmentation CNNs need a large volume of data to achieve good results, however, this is not always possible
since building new labelled dataset is costly and time consuming. To overcome this limitation in
practice, CNNs are seldom trained from scratch. Instead, their weights are initialized by the pre-trained
weights on a large dataset such as, the massive ImageNet. This approach is called transfer learning. In addition, to address a new problem, not all the weights are re-trained but only the weights of the
last layers, for example, the fully connected layers. This process is called fine-tuning. Data-augmentation technique consists of creating new training examples by applying geometrical
transformations such as rotation and translation to the original samples. Its objective is artificially
increasing the volume of the dataset. In fact, it was demonstrated in several works (Tabik et al. [16]). that increasing the volume of the dataset improves the learning of CNNs and reduces overfitting. In this paper we used the transformations explained in Table 1. Table 1. The stages followed for pre-processing the input UAV imagery and data augmentation
methods used for increasing the amount of sample patches. Table 1. The stages followed for pre-processing the input UAV imagery and data augmentation
methods used for increasing the amount of sample patches. Table 1. The stages followed for pre-processing the input UAV imagery and data augmentation
methods used for increasing the amount of sample patches. Steps
The Pre-Processing Stages of Input UAV Imagery
Step 1
Selection of image patches—a set of pictures manually cropped from orthophotomosaics
using QGIS 7.2.2. Step 2
Resizing of patches to 150 × 200 RGB pixels using cubic interpolation. Step 3
Manual assignment of each patch to the appropriate tree damage category. Methods for Increasing the Amount of Data
Step 1
Change the saturation of RGB channels. Step 2
Remove Gaussian blur filter with a blur value of 5% and a width and height of a kernel 0.5. Step 3
Pixel averaging by collapsing an image with a normalized 4 × 4 pixel window filter. Step 4
Image rotation relatively to its centre with 5◦, 15◦, 50◦, 90◦, 180◦rotation angles. Step 5
Cropping the central rectangle of each image to half distance from each edge
(discarding the framing borders) and resizing back the central rectangle to 150 × 200 RGB
pixels using cubic interpolation. 3. Classification of Trees in High Resolution Imagery and Related Works [12] detected coffee crops in
high-resolution SPOT images (2.5 m/pix). They used a binary classification model and presented a
comparison of the accuracy of various CNN and OBIA methods. In addition, the authors used a large
number of methods of preparation and preliminary processing of the image, which in the final result
leads to a high accuracy of more than 95%. Very few woks have used CNNs to detect plants in UAV images. Noteworthy is the work by
Ferreira et al. [21], where weed was detected in soybean cultures using CNN based on the CaffeNet
architecture. The authors used a set of data acquired with an UAV in manual mode at a height
of 4 m above the Earth level using an RGB camera. The accuracy of the classification was 99.5%. Though, according to the authors, the accuracy may be lower in operational circumstances due to the
heterogeneity in types of soils and weeds, since testing was performed on an experimental field. In 2018,
Onishi and Ise [22] classified 7 types of trees (deciduous broad-leaved tree, deciduous coniferous tree,
evergreen broad-leaved tree, Chamaecyparis obtuse, pinus, others) on UAV images using supervised
deep learning (GoogLeNet) with an accuracy of 89%. A similar problem to ours, which is the detection of damaged trees, is revised in the next series of
works, though they mainly used classical image classification methods. In 2009, Abdullah et al. [23]
identified healthy and infested trees in satellite imagery (Sentinel-2, WorldView-2 and 3 (up to
0.5 m/pix)) using partial least squares regression (PLSR). Heurich et al. [24] presented semi-automatic
detection of dead trees following a spruce bark beetle outbreak in CIR aerial photographs using
OBIA with accuracy up to 91%. In this work, authors used several classes, as deadwood areas, vital
vegetation, non-woodland and assistant classes (shadows among dead trees and shadows among
vital trees). In 2013, Ortiz et al. [25] detected a bark beetle attack with TerraSAR-X and RapidEye
Data (up to 1.18 m/pix) using generalized linear models (GLM), maximum entropy (ME) and random
forest (RF), with accuracies up to 74%. Meddens et al. [26] used multi-temporal disturbance detection
methods to detect bark beetle-caused tree mortality and to map the subsequent forest disturbance on
Landsat images with different classes (green trees, red damage stage, grey damage stage, herbaceous
vegetation, bare soil, shadows, water). Kussul et al. 3. Classification of Trees in High Resolution Imagery and Related Works 3. Classification of Trees in High Resolution Imagery and Related Works In this section, we consider related works devoted to the problems of classification and detection
of objects (from land-covers to organisms) on Earth remote sensing (ERS) data. Scientists from around
the world are trying to solve this problem by using various ERS information with different methods
and algorithms. In general, few works have addressed the detection of damaged trees using satellite
or UAV images, in general they have only considered few health classes (e.g., healthy and dead trees)
and did not apply CNNs but classical image classification methods. For example, Deli et al. [17] developed a new CNN-based method for classifying four land-cover
classes (crops, houses, soil and forests) in land surface images. They trained the algorithm with
100 images per class. Längkvist et al. [18] proposed the classification and segmentation of satellite
orthoimagery (including five classes: vegetation, ground, road, parking, railroad, building and water)
using CNNs in a small city for a full, fast and accurate per-pixel classification. They selected the
parameters and analysed their influence on the neural network model architecture. They also found a
better performance of the CNN model compared to object-oriented methods of classification, reaching
a maximum CNN classification accuracy of 94.49%. Few papers have used neural network classifiers to recognize plant species and plant growth using
images from a digital camera and a cell phone camera. Dyrmann et al. [19] created a CNN capable of
recognizing 22 plant species on colour images with an accuracy of 86.2%. For this, the authors used
different data sets, depending on the lighting, image resolution and soil type. Razavi and Yalcin [20]
proposed a CNN architecture to classify types of plants growing in agro stations. To evaluate the
effectiveness of their approach, the results of the created CNN model were compared to Support Vector Remote Sens. 2019, 11, 643 5 of 19 Machine (SVM; RBF kernel and polynomial kernel) classifiers. The accuracy of the CNN classification
was greater than the SVM, reaching 97.47%. Few studies have also used CNN on satellite imagery. Li et al. used deep learning models [10]
to detect oil palm trees in QiuckBird images (0.6 m/pix). Guirado et al. in Reference [11] detected
Ziziphus lotus shrubs on RGB satellite images from Google Earth using TensorFlow and got better
accuracies than using object-based image analysis (OBIA). Baeta et al. 4. Study Area and Data Acquisition The study area is located in the territory of the “Stolby” State Nature Reserve, near Krasnoyarsk
city in Central Siberia of the Russian Federation. Most of the territory (80%) constitutes the
mid-mountain belt (500–800 m a.s.l.), mainly covered by mixed forests composed by seven tree species
in different proportions: conifers such as pines (Pinus sylvestris, Pinus sibirica), larch (Larix sibirica),
fir (Abies sibirica), spruces (Picea abies, Picea obovata) and parvifoliate trees such as birch (Betula pendula,
Betula pubescens) and aspen (Populus tremula) (Ryabovol [30]). Pine trees dominate among other species
and occupy 41% of the total forested area, mainly in low-mountains (200–500 m a.s.l.). Distribution of
Siberian fir expands on 25% of the Stolby forest. R
t S
2019 1
FOR PEER REVIEW
6
f 21 First appearance of P. proximus in the Stolby nature reserve was registered in 2011 and nowadays
the roughly estimated forest damage by the beetle is about 25–30% of the fir area. Remote Sens. 2019, 1, x FOR PEER REVIEW
6 of 21
First appearance of P. proximus in the Stolby nature reserve was registered in 2011 and nowadays Four plots with different rates of the P. proximus invasion were chosen for our study at Stolby
(Figure 1). the roughly estimated forest damage by the beetle is about 25–30% of the fir area. Four plots with different rates of the P. proximus invasion were chosen for our study at Stolby
(Figure 1). Figure 1. Location of the four plots in the nature reserve “Stolby,” Krasnoyarsk city (Russia), where
A, B plots are fragments from the orthophotos used to build the training dataset; and C, D plots are
fragments from the orthophotos used to build the testing dataset used for external validation or
independent testing. A set of RGB images with ultra-high spatial resolution (≈5–10 cm/pix) were obtained for the
Figure 1. Location of the four plots in the nature reserve “Stolby,” Krasnoyarsk city (Russia), where
A, B plots are fragments from the orthophotos used to build the training dataset; and C, D plots
are fragments from the orthophotos used to build the testing dataset used for external validation or
independent testing. Figure 1. 3. Classification of Trees in High Resolution Imagery and Related Works [13] classified tree species and different levels of
ash tree (Fraxinus sp.) mortality in WorldView-2 images with resolution of 1.85 m/pix using classical
methods, in particular, the linear discriminant analysis (LDA), principal component analysis (PCA),
stepwise regression and OBIA methods. Species diversity and the magnitude of ash loss were assessed. The authors needed to use a wide set of remote sensing indices to obtain good accuracies. The overall
accuracy varied between 83% for the seven tree species and 77% for the four different levels of ash
damage. In 2015, Näsi et al. [27] also identified damaged trees (with three classes: healthy, infected
and dead) using UAV images and OBIA methods. In 2017, Dash et al. [28] used a non-parametric
approach based on classification trees on UAV images to monitor forest health for disease outbreak in
mature Pinus radiata trees. In 2018, Näsi et al. [29] published a paper that most closely relates to ours. They identified of bark beetle infestation at the individual tree level (healthy, infested and dead) in
urban forests on UAV images using support vector machine (SVM), with accuracies up to 93%. As far as we know, our work is the first in addressing the detection of damaged fir trees caused
by the bark beetle using CNN methods on images acquired by UAVs (resolution below 0.1 m/pix). We considered four tree-health categories, which is higher than in previous works and allows to better
assess the stage of the infestation. We also showed that it is possible to achieve good results using a
relatively small training set of data. Remote Sens. 2019, 11, 643 6 of 19 4. Study Area and Data Acquisition Location of the four plots in the nature reserve “Stolby,” Krasnoyarsk city (Russia), where
A, B plots are fragments from the orthophotos used to build the training dataset; and C, D plots are
fragments from the orthophotos used to build the testing dataset used for external validation or
independent testing. A set of RGB images with ultra-high spatial resolution (≈5–10 cm/pix) were obtained for the
Figure 1. Location of the four plots in the nature reserve “Stolby,” Krasnoyarsk city (Russia), where
A, B plots are fragments from the orthophotos used to build the training dataset; and C, D plots
are fragments from the orthophotos used to build the testing dataset used for external validation or
independent testing. research sites during multiple flights of the DJI Phantom 3 Pro quadcopter (with standard built-in
camera) in July 2016 (plot A and C) and of the Yuneec Typhoon H hexacopter (with CGO3+ camera)
in May 2016 (plot B) and August 2018 (plot D). Imagery for research plots A and C were recorded in
cloudy weather conditions at 670 m (A) and 700 m (C) altitudes (120–150 m elevation above ground),
plot B and D was surveyed in sunny weather at 120 m height. Default camera settings (auto white
balance, ISO 100) were applied in all aerial shots. Image composites (orthophotomosaics) were
created for each plot from a set of overlapping geolocated images (300–400 partially overlapping
images per plot) using the Agisoft Photoscan Professional v1.4.0 (64-bit) software (www.agisoft.com,
Agisoft LLC, St. Petersburg, Russia). This software provides photogrammetric and 3D-model
A set of RGB images with ultra-high spatial resolution (≈5–10 cm/pix) were obtained for the
research sites during multiple flights of the DJI Phantom 3 Pro quadcopter (with standard built-in
camera) in July 2016 (plot A and C) and of the Yuneec Typhoon H hexacopter (with CGO3+ camera)
in May 2016 (plot B) and August 2018 (plot D). Imagery for research plots A and C were recorded
in cloudy weather conditions at 670 m (A) and 700 m (C) altitudes (120–150 m elevation above
ground), plot B and D was surveyed in sunny weather at 120 m height. Default camera settings
(auto white balance, ISO 100) were applied in all aerial shots. Image composites (orthophotomosaics) 7 of 19 Remote Sens. 5.1. Definition of Fir Trees Damage Categories
presents the classification model (Section 5
especially designed for detecting bark beet 5.1. Definition of Fir Trees Damage Categories
p
(
especially designed for detecting bark bee To define the categories of health status of fir trees, we followed the entomological approach by
Krivets et al. [32], which differentiates six categories according to the level P. proximus invasion into
the trunk and its influence on the canopy: I—healthy trees; II—weakened trees; III—heavily weakened
trees; IV—dying trees; V—recently died trees; VI—old deadwood. Since the first, second and third
categories can only be recognized in-situ by trunk signs that do not visibly translate into the crown,
we reclassified them into four categories (skipping the second and the third one). Hence, the final
categories in our classification were: a—completely healthy tree or recently attacked by beetles, b—tree
colonized by beetles, c—recently died tree and d—deadwood (Figure 2). 5.1. Definition of Fir Trees Damage Categories
To define the categories of health status of fir trees, we followed the entomological approach by
Krivets et al. [32], which differentiates six categories according to the level P. proximus invasion into
the trunk and its influence on the canopy: I—healthy trees; II—weakened trees; III—heavily
weakened trees; IV—dying trees; V—recently died trees; VI—old deadwood. Since the first, second
and third categories can only be recognized in-situ by trunk signs that do not visibly translate into
the crown, we reclassified them into four categories (skipping the second and the third one). Hence,
the final categories in our classification were: a—completely healthy tree or recently attacked by
beetles, b—tree colonized by beetles, c—recently died tree and d—deadwood (Figure 2). Figure 2. Damage categories of Siberian fir trees used in this study (adapted from Krivets et al. [32]):
(a) completely healthy tree or recently attacked by beetles, (b) tree colonized by beetles, (c) recently
died tree and (d) deadwood. Top figures illustrate the vertical orthoimages corresponding to the
bottom horizontal pictures. (a)
(b)
(c)
(d)
Figure 2. Damage categories of Siberian fir trees used in this study (adapted from Krivets et al. [32]):
(a) completely healthy tree or recently attacked by beetles, (b) tree colonized by beetles, (c) recently died
tree and (d) deadwood. Top figures illustrate the vertical orthoimages corresponding to the bottom
horizontal pictures. (d) (c) (b) (a) (a) (d) (b) (c) Figure 2. Damage categories of Siberian fir trees used in this study (adapted from Krivets et al. 4. Study Area and Data Acquisition 2019, 11, 643 were created for each plot from a set of overlapping geolocated images (300–400 partially overlapping
images per plot) using the Agisoft Photoscan Professional v1.4.0 (64-bit) software (www.agisoft. com, Agisoft LLC, St. Petersburg, Russia). This software provides photogrammetric and 3D-model
reconstruction tools [31]. The data processing workflow in Photoscan consisted of the following steps:
alignment of each image location, generation of dense point cloud (3D-model), creation of digital
elevation model (DEM) and eventually production of a georeferenced orthophotomosaic (orthophotos
or orthoimages from now). The workflow was run several times with varying settings in order to
achieve better stich of images over the complex mountain landscape of the study sites. As a result, we
got four orthophotos for each one of the four sites (A, B, C and D for 2016 and 2018). Remote Sens. 2019, 1, x FOR PEER REVIEW
7 of 21
elevation model (DEM) and eventually production of a georeferenced orthophotomosaic
(
h
h
h i
f
) Th
kfl
l i
i h
i
i 5. Methods
(orth
in ord As we have mentioned, our aim is to evaluate the possibilities of convolutional neural networks
as a new approach to detect bark beetle outbreaks in fir trees in very high resolution imagery in fir
and mixed forests. Thus, this section includes a definition of fir-tree damage categories (Section 5.1),
describes the data pre-processing techniques we used for training the CNN models (Section 5.2),
presents the classification model (Section 5.3) and finally presents the proposed detection technique
especially designed for detecting bark beetle outbreaks in fir forests (Section 5.4). a result, we got four orthophotos for each one of the four sites (A, B, C and D for 2016 and 2018). 5. Methods
As we have mentioned, our aim is to evaluate the possibilities of convolutional neural networks
as a new approach to detect bark beetle outbreaks in fir trees in very high resolution imagery in fir
and mixed forests. Thus, this section includes a definition of fir-tree damage categories (Section 5.1),
describes the data pre-processing techniques we used for training the CNN models (Section 5.2), 5.1. Definition of Fir Trees Damage Categories
presents the classification model (Section 5
especially designed for detecting bark bee 5.2. Dataset Preprocessing and Data Augmentation Techniques of Sample patches for Training the CNN Models 5.2. Dataset Preprocessing and Data Augmentation Techniques of Sample patches for Training the CNN Models 5.2. Dataset Preprocessing and Data Augmentation Techniques of Sample patches for Training the CNN Models Dataset Preprocessing and Data Augmentation Techniques of Sample patches for Training the CNN Models The design of the training dataset is the key to the performance of a good CNN classification
model. For labelling the training dataset, we identified 50 image patches containing 4 categories of
trees. In particular, we built two different datasets: •
First, the dataset for training, validation and testing consisted in 50 manually sampled
image-patches of single trees per each tree damage category, resulting in 200 image-patches
from the plot A and B. To train the CNN model we used 80% of this selection. The remaining 20%
of images were used for model internal validation. •
First, the dataset for training, validation and testing consisted in 50 manually sampled
image-patches of single trees per each tree damage category, resulting in 200 image-patches
from the plot A and B. To train the CNN model we used 80% of this selection. The remaining 20%
of images were used for model internal validation. •
Second, the dataset for external testing or external validation consisted in 88 patch-images
generated by our proposed detection technique from the areas C and D. •
Second, the dataset for external testing or external validation consisted in 88 patch-images
generated by our proposed detection technique from the areas C and D. Table 2. Four fir tree categories training and testing datasets. Each image has 150 × 200-pixels. Table 2. Four fir tree categories training and testing datasets. Each image has 150 × 200-pixels. Categories
of Trees
Training Dataset (Plots A and B)
Testing Dataset for
External Test
Without Data
Augmentation
With Data Augmentation
Training
Internal
Validation
Training
Internal
Validation
Test Area C
Test Area D
1
40
10
880
220
5
13
2
40
10
880
220
36
14
3
40
10
880
220
5
7
4
40
10
880
220
2
6
Total:
160
40
3520
880
48
40 The pre-processing method used for preparing the input UAV images is described in Table 1 To improve the robustness and accuracy of the CNN classification models, we increased the
volume of samples using data augmentation techniques. 5.2. Dataset Preprocessing and Data Augmentation Techniques of Sample patches for Training the CNN Models In particular, we increased the amount of
sample patches for training the CNN models using the methods presented in Table 1. The images of the training dataset were used for training the parameters of the neural network. The internal validation set is a set of examples used to tune the parameters of the neural network
and determine a stopping point for the back-propagation algorithm. The testing dataset is a
set of independent external examples used to assess or validate the performance of the final
classification model. 5.1. Definition of Fir Trees Damage Categories
presents the classification model (Section 5
especially designed for detecting bark beet [32]):
(a) completely healthy tree or recently attacked by beetles, (b) tree colonized by beetles, (c) recently
died tree and (d) deadwood. Top figures illustrate the vertical orthoimages corresponding to the
bottom horizontal pictures. Figure 2. Damage categories of Siberian fir trees used in this study (adapted from Krivets et al. [32]):
(a) completely healthy tree or recently attacked by beetles, (b) tree colonized by beetles, (c) recently died
tree and (d) deadwood. Top figures illustrate the vertical orthoimages corresponding to the bottom
horizontal pictures. 8 of 19 Remote Sens. 2019, 11, 643 5.3. Classification Model A deep neural network was developed and trained on the prepared image samples to adapt
its weights to the task of tree-damage recognition. To create a CNN and improve the quality of its
training, we manually tuned the hyperparameters in the network. Several series of experiments were
performed. For each experiment, the hyperparameters were altered and the consequent network’s
operation quality change was estimated. The loss function, which should be minimized in the CNN
(see Section 1), was established by a categorical cross-entropy loss between the input and the output
classification of the images. The ADAM’s optimization was chosen among existing optimization
algorithms since it was the most efficient one due to the possibility of initial calibration of the CNN. When exploring the effect of changing the learning rate parameter with the ADAM’s optimizer,
the highest accuracy for CNN on the test data was achieved at a value of 0.0001. The total number of
the network layers was determined by creating cascades of convolutional layers and a pooling layer. To assess the impact of the number of training epochs on the CNN accuracy, training was conducted
and compared in the range from 1 to 50 epochs (see Section 6.2). Remote Sens. 2019, 11, 643
(each includes one
included maximum p 9 of 19
blocks
ers and The overall architecture of the CNN (Table 3, Figure 3) consisted of six convolutional blocks
(each includes one convolutional layer). Additionally, the first and third convolutional blocks included
maximum pooling layers. At the top of the CNN, there are two fully connected layers and one output
layer. The ReLU activation function was used in the last four convolutional blocks and the Softmax
function for the output layer. In order to keep the overtraining of the network under control, we used
the Dropout regularization method which reduced the complexity of the model, saving the number
of its parameters low. It is very important to choose an appropriate regularization coefficient, so it
was set to 0.25 after the second, fourth and fifth layers and it was set to 0.5 before the output layer. Our network with various activation functions is presented in Table 3. the Softmax function for the output layer. In order to keep the overtraining of the network under
control, we used the Dropout regularization method which reduced the complexity of the model,
saving the number of its parameters low. 5.3. Classification Model It is very important to choose an appropriate regularization
coefficient, so it was set to 0.25 after the second, fourth and fifth layers and it was set to 0.5 before the
output layer. Our network with various activation functions is presented in Table 3. Table 3. The CNN network developed in this study, with different activation functions, where layers,
output size and networks parameters are represented. Layers
Output Size, pix
Network y
Our network with various activation functions is presented in Table 3. Layers
Output Size, pix
Network Table 3. The CNN network developed in this study, with different activation functions, where layers,
output size and networks parameters are represented. Layers
Output Size, pix
Network
Convolution
150 × 200
3 × 3, 96
Max pooling
75 × 100
2 × 2, stride 2
Convolution
75 × 100
5 × 5, 128
Dropout
75 × 100
0.25
Convolution
75 × 100
3 × 3, 128
Max pooling
38 × 50
2 × 2, stride 2
Convolution
38 × 50
3 × 3, 128
Dropout
38 × 50
0.25
Convolution
38 × 50
5 × 5, 128
Dropout
38 × 50
0.5
Convolution
38 × 50
5 × 5, 512
Global Average pooling
1 × 972,800
stride 1
Dense
1 × 972,800
ReLu
Dropout
1 × 400
0.5
Dense
1 × 100
ReLu
Dense
1 × 4
Softmax
y
p
p
Convolution
150 × 200
3 × 3, 96
Max pooling
75 × 100
2 × 2, stride 2
Convolution
75 × 100
5 × 5, 128
Dropout
75 × 100
0.25
Convolution
75 × 100
3 × 3, 128
Max pooling
38 × 50
2 × 2, stride 2
Convolution
38 × 50
3 × 3, 128
Dropout
38 × 50
0.25
Convolution
38 × 50
5 × 5, 128
Dropout
38 × 50
0.5
Convolution
38 × 50
5 × 5, 512
Global Average pooling
1 × 972,800
stride 1
Dense
1 × 972,800
ReLu
Dropout
1 × 400
0.5
Dense
1 × 100
ReLu
Dense
1 × 4
Softmax
Consequently, our CNN model has the following form, presented in Figure 3. Figure 3. The architecture of our CNN model of deep machine learning. Figure 3. The architecture of our CNN model of deep machine learning. Table 3. The CNN network developed in this study, with different activation functions, where layers,
output size and networks parameters are represented. 5.3. Classification Model Layers
Output Size, pix
Network
Convolution
150 × 200
3 × 3, 96
Max pooling
75 × 100
2 × 2, stride 2
Convolution
75 × 100
5 × 5, 128
Dropout
75 × 100
0.25
Convolution
75 × 100
3 × 3, 128
Max pooling
38 × 50
2 × 2, stride 2
Convolution
38 × 50
3 × 3, 128
Dropout
38 × 50
0.25
Convolution
38 × 50
5 × 5, 128
Dropout
38 × 50
0.5
Convolution
38 × 50
5 × 5, 512
Global Average pooling
1 × 972,800
stride 1
Dense
1 × 972,800
ReLu
Dropout
1 × 400
0.5
Dense
1 × 100
ReLu
Dense
1 × 4
Softmax
Convolution
150 × 200
3 × 3, 96
Max pooling
75 × 100
2 × 2, stride 2
Convolution
75 × 100
5 × 5, 128
Dropout
75 × 100
0.25
Convolution
75 × 100
3 × 3, 128
Max pooling
38 × 50
2 × 2, stride 2
Convolution
38 × 50
3 × 3, 128
Dropout
38 × 50
0.25
Convolution
38 × 50
5 × 5, 128
Dropout
38 × 50
0.5
Convolution
38 × 50
5 × 5, 512
Global Average pooling
1 × 972,800
stride 1
Dense
1 × 972,800
ReLu
Dropout
1 × 400
0.5
Dense
1 × 100
ReLu
Dense
1 × 4
Softmax
Consequently, our CNN model has the following form, presented in Figure 3. Figure 3. The architecture of our CNN model of deep machine learning. 5 4 P
d D t ti
P
Figure 3. The architecture of our CNN model of deep machine learning. C
tl
CNN
d l h
th f ll
i
f
t d i
Fi
3 Table 3. The CNN network developed in this study, with different activation functions, where layers,
output size and networks parameters are represented. Convolution
150 × 200
3 × 3, 96
Max pooling
75 × 100
2 × 2, stride 2
Convolution
75 × 100
5 × 5 128 Figure 3. The architecture of our CNN model of deep machine learning. Figure 3. The architecture of our CNN model of deep machine learning. Figure 3. The architecture of our CNN model of deep machine learning. Figure 3. The architecture of our CNN model of deep machine learning. . Proposed Detection Process
Consequently, our CNN model has the following form, presented in Figure 3. We developed a candidate
lik l
t
b
t
Th
5.4. Proposed Detection Process are more likely to be a tree. Then we analysed each one of these candidate regions using our CNN-
classification model. The proposed candidate selection technique is a data processing algorithm that
includes a sequence of the steps presented in the Table 4. We developed a candidate selection technique to find the potential regions in the test image
that are more likely to be a tree. Then we analysed each one of these candidate regions using our
CNN-classification model. The proposed candidate selection technique is a data processing algorithm
that includes a sequence of the steps presented in the Table 4. 10 of 19
10 of 21 Remote Sens. 2019, 11, 643
Remote Sens. 2019, 1, x FOR Table 4. The proposed candidate selection technique in the test areas cropped from test areas C and D. Steps
Data Processing Algorithm
Step 1
Conversion of RGB images to a grayscale colour palette
Step 2
Blurring of grayscale images using the Gaussian high-pass filter in order to
reduce Gaussian noise with the following function parameters: kernel size of
11 × 11 pixels and the standard deviation equal to 0
Step 3
Creation of binary images from the grayscale blurred images by application
of a threshold function with the optimal brightness threshold value of input
image pixels equal to 100
Step 4
Structuring of picture elements outlines by application of two successive
functions (erosion and dilation) with several iterations for the binary pictures
in order to distinguish individual tree crown contours and to minimize the
effect of their confluence (fusion) in one object
Step 5
Detection of image patches was implemented using a contour area calculation
function based on the Green formula (Dawkins [33]), object size for the
function was set in the range between 50 × 50 and 200 × 200 pixels
D. We developed a candidate
lik l
t
b
t
Th
5.4. Proposed Detection Process Steps
Data Processing Algorithm
Step 1
Conversion of RGB images to a grayscale colour palette
Step 2
Blurring of grayscale images using the Gaussian high-pass filter in order to reduce
Gaussian noise with the following function parameters: kernel size of 11 × 11 pixels an
the standard deviation equal to 0
Step 3
Creation of binary images from the grayscale blurred images by application of a
threshold function with the optimal brightness threshold value of input image pixels
equal to 100
Step 4
Structuring of picture elements outlines by application of two successive functions
(erosion and dilation) with several iterations for the binary pictures in order to
distinguish individual tree crown contours and to minimize the effect of their
confluence (fusion) in one object
Step 5
Detection of image patches was implemented using a contour area calculation functio
based on the Green formula (Dawkins [33]), object size for the function was set in the
range between 50 × 50 and 200 × 200 pixels The output of this process are the candidate regions indicated by red bounding boxes in Figure 4. Finally, all the bounding boxes were analysed by the classification model. The output of this process are the candidate regions indicated by red bounding boxes in Figure
4. Finally, all the bounding boxes were analysed by the classification model. The output of this process are the candidate regions indicated by red bounding boxes in Figure 4. Finally, all the bounding boxes were analysed by the classification model. The output of this process are the candidate regions indicated by red bounding boxes in Figure
4. Finally, all the bounding boxes were analysed by the classification model. Figure 4. Pre-processing consisted of the following steps: first, converting the three band image into
one grey-scale band image (PAN); second, converting grey-scale band image into blurred image;
third, converting the blurred image into a binary image based on a 100 over 256 digital value
threshold; and fourth, detecting categories of trees on RGB images. The 48 candidate patches
identified in test area C and 40 candidate patches identified in test area D are labelled with red contour
in the right panel. Figure 4. We developed a candidate
lik l
t
b
t
Th
5.4. Proposed Detection Process Pre-processing consisted of the following steps: first, converting the three band image into
one grey-scale band image (PAN); second, converting grey-scale band image into blurred image; third,
converting the blurred image into a binary image based on a 100 over 256 digital value threshold;
and fourth, detecting categories of trees on RGB images. The 48 candidate patches identified in test area
C and 40 candidate patches identified in test area D are labelled with red contour in the right panel. Figure 4. Pre-processing consisted of the following steps: first, converting the three band image into
one grey-scale band image (PAN); second, converting grey-scale band image into blurred image;
third, converting the blurred image into a binary image based on a 100 over 256 digital value
threshold; and fourth, detecting categories of trees on RGB images. The 48 candidate patches
identified in test area C and 40 candidate patches identified in test area D are labelled with red contour
i
th
i ht
l
Figure 4. Pre-processing consisted of the following steps: first, converting the three band image into
one grey-scale band image (PAN); second, converting grey-scale band image into blurred image; third,
converting the blurred image into a binary image based on a 100 over 256 digital value threshold;
and fourth, detecting categories of trees on RGB images. The 48 candidate patches identified in test area
C and 40 candidate patches identified in test area D are labelled with red contour in the right panel. in the right panel.
6. Results and Analysis 6. Results and Analysis
In this section, we will first describe the metrics used for the evaluation of the results of our
proposed model, we will then show the performance of the training and internal test process (sites A
and B) and finally we will show the results of the detection on two external test datasets of UAV
In this section, we will first describe the metrics used for the evaluation of the results of our
proposed model, we will then show the performance of the training and internal test process (sites A
and B) and finally we will show the results of the detection on two external test datasets of UAV images
(sites C and D). 6. Results and Analysis
In this section, we will first describe the metrics used for the evaluation of the results of our
proposed model, we will then show the performance of the training and internal test process (sites A
and B) and finally we will show the results of the detection on two external test datasets of UAV
In this section, we will first describe the metrics used for the evaluation of the results of our
proposed model, we will then show the performance of the training and internal test process (sites A
and B) and finally we will show the results of the detection on two external test datasets of UAV images
(sites C and D). 6.2. Evaluation of the Training Process and Comparison
6 2 Evaluation of the Training Process and Comparison To evaluate the training process of our new CNN, we used Python, Keras and TensorFlow
framework with different pre-processing data augmentation techniques. Keras is a high-level API
to ease the process of building and training deep learning models [35]. TensorFlow is open source
software library for high performance numerical computation, which runs on various heterogeneous
systems, including distributed graphics processing unit (GPU) clusters (Murray [36]). The used
hardware environment in this study was Intel Xeon E5-2630v4 CPU accelerated with NVIDIA Titan
Xp GPU as a platform for learning and testing. 6.2. Evaluation of the Training Process and Comparison
To evaluate the training process of our new CNN, we used Python, Keras and TensorFlow
framework with different pre-processing data augmentation techniques. Keras is a high-level API to
ease the process of building and training deep learning models [35]. TensorFlow is open source
software library for high performance numerical computation, which runs on various heterogeneous
systems, including distributed graphics processing unit (GPU) clusters (Murray [36]). The used
hardware environment in this study was Intel Xeon E5-2630v4 CPU accelerated with NVIDIA Titan
X
GPU
l
f
f
l
i
d
i The application of data augmentation techniques to the sample patches for training the CNN
models resulted in the creation of 4400 image-patches from the first data subset, which had 200 patches
(Section 5.2). Xp GPU as a platform for learning and testing. The application of data augmentation techniques to the sample patches for training the CNN
models resulted in the creation of 4400 image-patches from the first data subset, which had 200
h
(S
i
5 2) The maximum performance of our CNN model with augmented dataset was achieved at the
23th training epoch, providing an internal test accuracy of 99.7% and a minimum training loss lower
than 0.01. After the 23th epoch, the validation loss was stabilized and the difference between the
training and validation loss increased (Figure 5). patches (Section 5.2). The maximum performance of our CNN model with augmented dataset was achieved at the
23th training epoch, providing an internal test accuracy of 99.7% and a minimum training loss lower
than 0.01. After the 23th epoch, the validation loss was stabilized and the difference between the
training and validation loss increased (Figure 5) g
(
g
)
Figure 5. Loss and accuracy for each epoch of the CNN model training with data augmentation. Figure 5. images (sites C and D)
6.1. Evaluation Metrics 6.1. Evaluation Metrics
To evaluate and compare the results of the proposed CNN model with other CNN models, we
used the performance metrics based on the confusion matrix. The confusion matrix is four by four
To evaluate and compare the results of the proposed CNN model with other CNN models, we
used the performance metrics based on the confusion matrix. The confusion matrix is four by four
(four categories) and contains the results of multi-class classifier (one class against the rest) in terms of Remote Sens. 2019, 11, 643
Remote Sens. 2019, 1, x FOR 11 of 19
11 of 21 number of true positive (TP) predictions, true negative (TN) predictions, false positive (FP) predictions
and false negative (FN) predictions [34]. of number of true positive (TP) predictions, true negative (TN) predictions, false positive (FP)
predictions and false negative (FN) predictions [34]. The main metrics we can extract from the matrix of confusion are: accuracy (1) precision (2) The main metrics we can extract from the matrix of confusion are: accuracy (1), precision (2), recall
(3) and F-score (4). Accuracy is calculated as the total number of the correct predictions (TP + TN)
divided by the total number of examples in the test set. Precision is calculated as the number of
correct positive predictions (TP) divided by the total number of positive predictions (TP + FP). Recall
is calculated as the number of correct positive predictions (TP) divided by the number of true positives
and false negatives. F-score indicates the balance between precision and recall. The highest and best
value of all these metrics is 1.0 and the worst is 0.0. The main metrics we can extract from the matrix of confusion are: accuracy (1), precision (2),
recall (3) and F-score (4). Accuracy is calculated as the total number of the correct predictions (TP +
TN) divided by the total number of examples in the test set. Precision is calculated as the number of
correct positive predictions (TP) divided by the total number of positive predictions (TP + FP). Recall
is calculated as the number of correct positive predictions (TP) divided by the number of true
positives and false negatives. F-score indicates the balance between precision and recall. The highest
and best value of all these metrics is 1.0 and the worst is 0.0. images (sites C and D)
6.1. Evaluation Metrics ܶܲܶܰ Accuracy =
TP + TN
TP + TN + FN + FP
(1)
Precision =
TP
TP + FP
(2)
Recall =
TP
TP + FN
(3)
F_score = 2 × Precision × Recall
Precision + Recall
(4)
ܣܿܿݑݎܽܿݕൌ
ܶܲܶܰ
ܶܲܶܰܨܰܨܲ
(1)
ܲݎ݁ܿ݅ݏ݅݊ൌ
ܶܲ
ܶܲܨܲ
(2)
ܴ݈݈݁ܿܽൌ
ܶܲ
ܶܲܨܰ
(3)
ܨ_ݏܿݎ݁ൌ 2 ൈܲݎ݁ܿ݅ݏ݅݊ൈܴ݈݈݁ܿܽ
ܲݎ݁ܿ݅ݏܴ݈݈݅݊݁ܿܽ
(4) Accuracy =
TP + TN
TP + TN + FN + FP
(1)
Precision =
TP
TP + FP
(2)
Recall =
TP
TP + FN
(3)
F_score = 2 × Precision × Recall
Precision + Recall
(4)
ܣܿܿݑݎܽܿݕൌ
ܶܲܶܰ
ܶܲܶܰܨܰܨܲ
(1)
ܲݎ݁ܿ݅ݏ݅݊ൌ
ܶܲ
ܶܲܨܲ
(2)
ܴ݈݈݁ܿܽൌ
ܶܲ
ܶܲܨܰ
(3)
ܨ_ݏܿݎ݁ൌ 2 ൈܲݎ݁ܿ݅ݏ݅݊ൈܴ݈݈݁ܿܽ
ܲݎ݁ܿ݅ݏܴ݈݈݅݊݁ܿܽ
(4) (1)
(1) (2)
(2) (4)
(4) 6.2. Evaluation of the Training Process and Comparison
6 2 Evaluation of the Training Process and Comparison 6.2. Evaluation of the Training Process and Comparison
6 2 Evaluation of the Training Process and Comparison 6.3. External Test Detection Results
6.3. External Test Detection Results The confusion matrix of our CNN model in the four categories with data-augmentation are shown
is Figure 6. The confusion matrix of our CNN model in the four categories with data-augmentation are
shown is Figure 6. The confusion matrix of our CNN model in the four categories with data-augmentation are shown
is Figure 6. The confusion matrix of our CNN model in the four categories with data-augmentation are
shown is Figure 6. The confusion matrix of our CNN model in the four categories with data-augmentation are shown
is Figure 6. The confusion matrix of our CNN model in the four categories with data-augmentation are
shown is Figure 6. g
g
Figure 6. hown is Figure 6. Figure 6. Confusion matrix of the proposed CNN model with data augmentation on the candidate
regions obtained from test areas C and D. Figure 6. Confusion matrix of the proposed CNN model with data augmentation on the candidate
regions obtained from test areas C and D. Figure 6. Confusion matrix of the proposed CNN model with data augmentation on the candidate
regions obtained from test areas C and D. Figure 6. Confusion matrix of the proposed CNN model with data augmentation on the candidate
regions obtained from test areas C and D. The results based on the confusion matrix of our CNN model on each category with and without
data augmentation are shown in the Table 5
The results based on the confusion matrix of our CNN model on each category with and without
data augmentation are shown in the Table 5. Table 5. The performance of our CNN model with and without data augmentation on the test set for
each categories of trees. The performance is expressed in terms of true positives (TP), true negatives
Table 5. The performance of our CNN model with and without data augmentation on the test set for
each categories of trees. The performance is expressed in terms of true positives (TP), true negatives
(TN), false positives (FP), false negatives (FN), precision, recall and F-score. (TN), false positives (FP), false negatives (FN), precision, recall and F-score. 6.2. Evaluation of the Training Process and Comparison
6 2 Evaluation of the Training Process and Comparison Loss and accuracy for each epoch of the CNN model training with data augmentation. Figure 5. Loss and accuracy for each epoch of the CNN model training with data augmentation. Figure 5. Loss and accuracy for each epoch of the CNN model training with data augmentation. 12 of 19 12 of 19 Remote Sens. 2019, 11, 643 For comparison with our CNN model, we have also tested on the same input data the
most powerful CNN models such as Xception, VGG-16, VGG-19, ResNet-50, Inception-V3,
InceptionResNet-V2, DenseNet-121, DenseNet-169 and DenseNet-201 (Jordan [37]). A brief explanation
of each model is provided in Appendix B. We adapted the last layer of all these models to the 4 classes
of our problem. The obtained results of all these models after few training epochs are shown in
Appendix A (Table A1). As it can be seen from Table A1, all models provide high training accuracies. InceptionResNet-V2, DenseNet-121, DenseNet-169 and DenseNet-201 (Jordan [37]). A brief
explanation of each model is provided in Appendix B. We adapted the last layer of all these models
to the 4 classes of our problem. The obtained results of all these models after few training epochs are
shown in Appendix A (Table A1). As it can be seen from Table A1, all models provide high training
accuracies. 6.3. External Test Detection Results
6.3. External Test Detection Results 2019, 1, x FOR PEER REVIEW
13 of 21
ResNet50, Inception V3, InceptionResNetV2, DenseNet121, DenseNet169 and DenseNet201 are
powerful and computationally intensive models, our less computationally intensive architecture
provides much better results in the damaged tree categories problem. On the other hand, the proposed model with data-augmentation achieves high F1_score on g
The results of the detection on the input test areas C and D are shown in Figures 7 and 8. The red
boxes present the selected areas produced by our candidate selection technique (see Section 5.4). Each
red box has two numbers. The first number is the tree category estimated by the CNN model; C1, C2,
C3 and C4 refer to the tree damage categories 1, 2, 3 and 4 respectively. The second number indicates
the probability calculated by our classification CNN model. Actually, this probability indicates the
level of confidence of the model. damage class 1, 2, 3 and 4 with 92.75%, 89.86%, 89.66% and 88.89 respectively. This can be explained
by the fact that the model correctly distinguishes the colour, shape and texture of each one of the four
damage classes. The results of the detection on the input test areas C and D are shown in Figures 7 and 8. The
red boxes present the selected areas produced by our candidate selection technique (see Section 5.4). Each red box has two numbers. The first number is the tree category estimated by the CNN model;
C1, C2, C3 and C4 refer to the tree damage categories 1, 2, 3 and 4 respectively. The second number
indicates the probability calculated by our classification CNN model. Actually, this probability
indicates the level of confidence of the model. Figure 7. Results of the detection of damaged tree categories on test area C. C1, C2, C3 and C4 indicate
the estimated class by our CNN classification model together with the corresponding probability. The
symbols “+” and “–” indicate respectively correct and incorrect class estimation by our model. Figure 7. Results of the detection of damaged tree categories on test area C. C1, C2, C3 and C4 indicate
the estimated class by our CNN classification model together with the corresponding probability. The
symbols “+” and “−” indicate respectively correct and incorrect class estimation by our model. Figure 7. Results of the detection of damaged tree categories on test area C. 6.3. External Test Detection Results
6.3. External Test Detection Results Categories
of Trees
TP
TN
FP
FN
Accuracy (%)
Precision (%)
Recall (%)
F-Score (%)
Without Augmentation
1
18
44
3
14
78.48
85.71
56.25
67.92
2
30
32
15
6
74.7
66.67
83.33
74.07
3
10
52
0
6
91.18
100
65.5
76.92
4
4
58
8
0
88.57
33.33
100
50
With Augmentation
1
32
48
5
0
94.12
86.49
100
92.75
2
31
49
2
5
91.95
93.94
86.11
89.86
Categories of Trees
TP
TN
FP
FN
Accuracy (%)
Precision (%)
Recall (%)
F-Score (%)
Without Augmentation
1
18
44
3
14
78.48
85.71
56.25
67.92
2
30
32
15
6
74.7
66.67
83.33
74.07
3
10
52
0
6
91.18
100
65.5
76.92
4
4
58
8
0
88.57
33.33
100
50
With Augmentation
1
32
48
5
0
94.12
86.49
100
92.75
2
31
49
2
5
91.95
93.94
86.11
89.86
3
13
67
0
3
96.39
100
81.25
89.66
4
4
76
1
0
98.77
80
100
88.89 3
13
67
0
3
96.39
100
81.25
89.66
4
4
76
1
0
98.77
80
100
88.89
As it can be seen from Table 5, the trained CNN with data-augmentation provides the best test
accuracy, recall and F1_score. The average accuracy, recall and F-score improved by 14.5%, 20.4%
and 34.3% respectively. A comparison between the results obtained by our model and by more
As it can be seen from Table 5, the trained CNN with data-augmentation provides the best test
accuracy, recall and F1_score. The average accuracy, recall and F-score improved by 14.5%, 20.4%
and 34.3% respectively. A comparison between the results obtained by our model and by more
complex models with augmentation on the 88 candidate areas from images C and D is presented in
Appendix A (Table A2). As it can be seen from this Table, although Xception, VGG16, VGG19, ResNet50, Remote Sens. 2019, 11, 643 13 of 19 Inception V3, InceptionResNetV2, DenseNet121, DenseNet169 and DenseNet201 are powerful and
computationally intensive models, our less computationally intensive architecture provides much
better results in the damaged tree categories problem. On the other hand, the proposed model with data-augmentation achieves high F1_score on
damage class 1, 2, 3 and 4 with 92.75%, 89.86%, 89.66% and 88.89 respectively. This can be explained
by the fact that the model correctly distinguishes the colour, shape and texture of each one of the four
damage classes. Remote Sens. in
cr
7. Conclusions to detect infected trees, if a box has more than one tree and at least one of them is unhealthy and the
model considers the entire box as unhealthy tree class, this answer is considered as valid answer for
the purpose of finding unhealthy trees. On the other hand, detecting individual trees in dense forests using only RGB bands information
is a very complex task. In fact, previous studies (such as Mohan et al. [38]) that aimed to detect
individual tree crowns in dense forests had to include several sources of data (high resolution images
together with 3D LIDAR point cloud data), to achieve similar accuracies to our results. Actually, our
results demonstrate that the proposed CNN based approach has a high potential and can be further
improved in the future using more information, such as multispectral bands, NDVI or other spectral
indices and 3D LIDAR data. 7. Conclusions
Damage to fir trees caused by the attacks of bark beetles is a major problem in the Taiga and
Boreal Forests, particularly in Central Siberia of Russia. An efficient method for recognizing categories
of tree damage using remote sensing imagery could substantially help as an early warning system that
allows to eliminate fir trees colonized by the P. proximus beetle, to reduce its spread to new territories. The presented results are of great interest, both for scientific and practical purposes, since such work
in the task of classifying categories of tree damage caused by beetle attacks to fir trees using CNN
methods on UAV images has not yet been encountered. Recognizing the categories of damage to fir
trees is a difficult task due to the fact that the forest canopy is very dense and diverse. Damage to fir trees caused by the attacks of bark beetles is a major problem in the Taiga and
Boreal Forests, particularly in Central Siberia of Russia. An efficient method for recognizing
In this article, we developed a model based on CNNs to detect in RGB UAV images the damage
to fir trees caused by bark beetles. Our network consists of six convolutional blocks. The network
can recognize four damage categories in Siberian firs, from healthy to dead, with high prediction
accuracy on images from different survey data. We have shown that our model provides better
performance than the most powerful state-of-the-art CNN models, such as Xception, VGG, ResNet,
Inception, InceptionResNet, DenseNet. 6.3. External Test Detection Results
6.3. External Test Detection Results C1, C2, C3 and C4 indicate
the estimated class by our CNN classification model together with the corresponding probability. The
symbols “+” and “–” indicate respectively correct and incorrect class estimation by our model. Figure 7. Results of the detection of damaged tree categories on test area C. C1, C2, C3 and C4 indicate
the estimated class by our CNN classification model together with the corresponding probability. The
symbols “+” and “−” indicate respectively correct and incorrect class estimation by our model. As it can be seen from Figures 7 and 8, our model correctly recognizes the tree damage categories
in most candidate regions (red boxes). In the few cases where the boxes include more than one tree
crown, the model correctly predicts the existence of infected trees. Since the objective of this work is
to detect infected trees, if a box has more than one tree and at least one of them is unhealthy and the
model considers the entire box as unhealthy tree class, this answer is considered as valid answer for
the purpose of finding unhealthy trees. p
p
g
y
On the other hand, detecting individual trees in dense forests using only RGB bands information is
a very complex task. In fact, previous studies (such as Mohan et al. [38]) that aimed to detect individual
tree crowns in dense forests had to include several sources of data (high resolution images together
with 3D LIDAR point cloud data), to achieve similar accuracies to our results. Actually, our results
demonstrate that the proposed CNN based approach has a high potential and can be further improved
in the future using more information, such as multispectral bands, NDVI or other spectral indices and
3D LIDAR data. 14 of 19 Remote Sens. 2019, 11, 643 Figure 8. Results of the detection of damaged tree categories on test area D. C1, C2, C3 and C4 indicate
the estimated class by our CNN classification model together with the corresponding probability. The
symbols “+” and “–” indicate respectively correct and incorrect class estimation by our model. As it can be seen from Figures 7 and 8, our model correctly recognizes the tree damage categories
in most candidate regions (red boxes) In the few cases where the boxes include more than one tree
Figure 8. Results of the detection of damaged tree categories on test area D. 6.3. External Test Detection Results
6.3. External Test Detection Results C1, C2, C3 and C4 indicate
the estimated class by our CNN classification model together with the corresponding probability. The symbols “+” and “−” indicate respectively correct and incorrect class estimation by our model. Figure 8. Results of the detection of damaged tree categories on test area D. C1, C2, C3 and C4 indicate
the estimated class by our CNN classification model together with the corresponding probability. The
symbols “+” and “–” indicate respectively correct and incorrect class estimation by our model. As it can be seen from Figures 7 and 8, our model correctly recognizes the tree damage categories
Figure 8. Results of the detection of damaged tree categories on test area D. C1, C2, C3 and C4 indicate
the estimated class by our CNN classification model together with the corresponding probability. The symbols “+” and “−” indicate respectively correct and incorrect class estimation by our model. in
cr
7. Conclusions We showed that data augmentation substantially increased
the performance of our CNN model. Our model, trained with data augmentation, showed up to
98.77% accuracy for damage categories one, three and four. The second category may have the lowest
recognition accuracy due to the fact that we had to merge three categories traditionally assessed in the
field (weakened trees, heavily weakened trees and dying trees) into one category—trees colonized by
the beetle, since it was not possible to visually observe any differences between these three categories
in the UAV imagery. Regarding alternative models, the VGG-16 model, on average, showed higher results, among
other models considered in the paper. VGG-16 model recognized the first, second and fourth categories
with an accuracy of 85.9%, 79.76 and 94.37%, respectively. Recognition of the third category with an Remote Sens. 2019, 11, 643 15 of 19 accuracy higher than 88.89% is achieved by ResNet-50 model. The lowest accuracies were reached
by InceptionResNet-V2. In future works, we will focus on improving the segmentation of tree categories, introducing time
series of data and trying to implement the three missing classes: weakened trees, heavily weakened
trees and dying trees. It would also be interesting to explore if we can transfer our model to a new area
with the same species and to other species, by only changing the last output layer in the network. Author Contributions: A.S. and S.T. conceived and conducted the experiments, developed the CNN model and
wrote the manuscript. A.R. made a survey of the research plots and a field exploration of the territory. A.S. prepared the training and testing datasets. D.A.-S. and A.R. made changes, revised and edited the first submitted
version of the manuscript. A.S., S.T., D.A.-S., A.R., Y.M. and F.H. made changes, revised and edited the final
version of the manuscript. Funding: A.S. was supported by the grant of the Russian Science Foundation No. 16-11-00007. S.T. was
supported by the Ramón y Cajal Programme (No. RYC-2015-18136). S.T. and F.H. received funding from
the Spanish Ministry of Science and Technology under the project TIN2017-89517-P. D.A.-S. received support
from project ECOPOTENTIAL, which received funding from the European Union Horizon 2020 Research and
Innovation Programme under grant agreement No. 641762, from the European LIFE Project ADAPTAMED
LIFE14 CCA/ES/000612 and from project 80NSSC18K0446 of the NASA’s Group on Earth Observations Work
Programme 2016. A.R. was supported by the grant of the Russian Science Foundation No. 18-74-10048. Y. in
cr
7. Conclusions M. was
supported by the grant of Russian Foundation for Basic Research No. 18-47-242002, Government of Krasnoyarsk
Territory and Krasnoyarsk Regional Fund of Science. Acknowledgments: We are very grateful to the reviewers for their valuable comments that helped to improve
the paper. We appreciate the support of a vice-director of the “Stolby” State Nature Reserve, Anastasia Knorre. We also thank two Ph.D. students Egor Trukhanov and Anton Perunov from Siberian Federal University for their
help in data acquisition (aerial photography from UAV) on two research plots in 2016 and raw imagery processing. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript and in the
decision to publish the results. Table A1. Performance comparison of the training by proposed CNN model with other models. Table A1. Performance comparison of the training by proposed CNN model with other models. Model
Without Augmentation
With Augmentation
Loss
Accuracy
Loss
Accuracy
Our CNN model
0.05
1
0.001
1
Xception
0.3
0.89
0.002
1
VGG-16
0.03
1
0.03
1
VGG-19
0.07
1
0.01
0.99
ResNet-50 + 4
0.21
0.94
0.01
1
Inception-V3 + 4
0.34
0.89
0.003
1
InceptionResNet-V2 + 4
0.13
0.94
0.04
0.99
DenseNet-121 + 4
0.17
0.94
0.02
0.99
DenseNet-169 + 4
0.08
1
0.004
0.99
DenseNet-201 + 4
0.12
0.94
0.01
1
Table A2. The results of testing alternative models for each class that were trained on data set
with augmentation. Table A2. The results of testing alternative models for each class that were trained on data set
with augmentation. Abbreviations The following abbreviations or terms are used in this manuscript: CNN
Convolutional neural networks
UAV
Unmanned aerial vehicle
P. proximus
Polygraphus proximus
RGB
Red, green, blue
ERS
Earth remote sensing
SVM
Support vector machine
RBF
Radial basis function
EPSG
Google Earth in European Petroleum Survey Group
OBIA
Object based image analysis
LDA
Linear discriminant analysis
PCA
Principal component analysis
RSI
Remote sensing indices
DJI
Dajiang Innovation Technology
QGIS
Quantum Geographic Information System
ADAM
ADAptive Moment estimation
ReLU
Rectified linear unit
TP
True positives
TN
True negatives
FP
False positives
FN
False negatives
API
Application programming interface
GPU
Graphics processing unit
CPU
Central processing unit 16 of 19 Remote Sens. 2019, 11, 643 Table A1. Performance comparison of the training by proposed CNN model with other models. Categories of Trees
TP
TN
FP
FN
Acc (%)
Precision (%)
Recall (%)
F-Score (%)
Xception
1
30
26
19
2
72.73
61.22
93.75
74.07
2
15
41
8
21
65.88
65.22
41.67
50.85
3
7
49
1
9
84.85
87.5
53.75
58.33
4
4
52
4
0
93.33
50
100
66.67
VGG-16
1
24
43
3
8
85.90
88.89
75
81.36
2
33
34
14
3
79.76
70.21
91.67
79.52
3
6
61
0
10
87.01
100
37.5
54.55
4
4
63
4
0
94.37
50
100
66.67
VGG-19
1
26
38
6
6
84.21
81.25
81.25
81.25
2
29
35
11
7
78.05
72.5
80.56
76.32
3
5
59
0
11
85.33
100
31.25
47.62
4
4
60
7
0
90.14
36.36
100
53.33
ResNet-50
1
32
32
16
0
80
66.67
100
80
2
20
44
1
16
79.01
95.24
55.56
70.18
3
9
55
1
7
88.89
90
56.25
69.23
4
3
61
6
1
90.14
33.33
75
46.15
Inception-V3
1
30
25
21
2
70.51
58.82
93.75
72.29
2
16
39
8
20
66.27
66.67
44.44
53.33
3
6
49
1
10
83.33
85.71
37.50
52.17
4
3
52
3
1
93.22
50
75
60
InceptionResNet-V2
1
23
27
14
9
68.49
62.16
71.88
66.67
2
18
32
11
18
63.29
62.07
50
55.38
3
6
44
6
10
75.76
50
37.5
42.86
4
3
47
7
1
86.21
30
75
42.86
DenseNet-121
1
26
33
10
6
78.67
72.22
81.25
76.47
2
24
35
8
12
74.68
75
66.67
70.59
3
6
53
1
10
84.29
85.71
37.50
52.17
4
3
56
10
1
84.29
23.08
75
35.29
DenseNet-169
1
32
26
21
0
73.42
60.38
100
75.29
2
15
43
5
21
69.05
75
41.67
53.57
3
8
50
1
8
86.57
88.89
50
64
4
3
55
3
1
93.55
50
75
60
Dense Net-201
1
32
22
22
0
71.05
59.26
100
74.42
2
15
39
7
21
65.85
68.18
41.67
51.72
3
4
50
1
12
80.6
80
25
38.1
4
3
51
4
1
91.53
42.86
75
54.55 17 of 19 17 of 19 Remote Sens. 2019, 11, 643 Appendix B. Table A1. Performance comparison of the training by proposed CNN model with other models. Brief Description of the CNNs That Are Compared against the CNN Developed in
This Work, That Is, VGG, ResNet, Inception-V3, InceptionResNet-V2, Xception and DenseNet Visual Geometry Group (VGG) is a neural network architecture that has four variants, VGG-11, VGG-13,
VGG-16 and VGG-19, where the number 13, 16 and 19 indicate the number of layers in the network. VGG-16 was
designed by the University of Oxford to recognize objects in images. VGG-16 network is the 1st runner-up on the
ImageNet Large Scale Visual Recognition Challenge (ILSVRC) comparison in 2014 with an obtained accuracy of
93.3% [39]. A distinctive feature of the architecture is a small convolution kernel of 3 × 3 pixels. The neural network
architecture consists of two parts. The first part consists of alternating convolution cascades and a pooling layer:
two convolution-convolution-pooling cascades and then three convolution-convolution-convolution-pooling
cascades. On the pooling layer, Max Pooling is selected with a 2 × 2 square core. This part highlights the
characteristic features in the image. The second part is responsible for the classification of the object in the image
according to the features selected at the previous stage and includes three fully connected layers. Thus, the VGG-16
network receives images with a size of 224 × 224 pixels in three colour channels (red, green and blue) and the
output represents the probability of belonging to a particular class in one hot encoding format. In our experiments,
we compared our CNN to the following types of networks: VGG-16 and VGG-19. p
g yp
ResNet was developed by Microsoft using residual learning for recognition, localization and detection of
objects in images. It was the winner of the ILSVRC 2015 competition with 150 layers and the winner of Microsoft
Common Objects in Context 2015 (MS COCO) detection and segmentation [40]. The authors of ResNet used
the two-layer traversal approach and applied it on a large scale, which is considered as a small classifier in the
network. The network also uses a bottleneck layer, which allows the reduction of the number of features in each
layer, using a 1 × 1 pixel convolution with a lower yield of features and then a 3 × 3 pixel convolution layer and
again a 1 × 1 pixel convolution layer with more features. The bottleneck layer reduces computational resources
while preserves features combinations. The output layer is the pooling layer with the Softmax function. Table A1. Performance comparison of the training by proposed CNN model with other models. In our
experiments, we compared our CNN to ResNet-50. p
p
Inception-V3 is Google’s convolutional neural network for recognizing objects in an image. In December
2015, the third version of Inception was introduced with the inclusion of Batch-normalized Inception. Batch-normalization calculates the mean and standard deviation of all feature distribution maps in the output
layer and then normalizes their responses with these values [20]. The last layer of the Inception network is the
pooling layer with the Softmax function. To evaluate Inception-V3 on our data, we had to resize the input images
from 150 × 200 to 299 × 299 pixels using cubic interpolation. p
g
p
InceptionResNet-V2 is the union of two neural networks: Inception and ResNet. This allowed the authors to
improve the accuracy of image classification. The idea of residual blocks was preserved from the Inception network
and a combination of convolutional blocks from the ResNet network. Despite the fact that InceptionResNet-V2
demonstrates high accuracy compared to other existing models, it has two drawbacks: low speed and a high
amount of memory used. Xception was implemented in Keras by François Chollet and it is a modification of the Inception network
(Chollet [41]). The network architecture is similar to ResNet-34 but the model and code is simpler than in Inception. A distinctive feature of the network architecture is SeparableConv, which is a modifiable, separable convolution
that is located at the top of the network architecture. In the network there are residual (or shortcut/skip)
connections, taken from the network ResNet. In addition, the network contains residual connections, which
significantly increase the accuracy of classifying objects in images. g
y
y
y
g
j
g
DenseNet is the result of the development of the ResNet network and is based on its residual blocks [21]. The basic idea is that connections have all the possible combinations within each block, which represents a
gradient of more paths and the network becomes more resistant to learning. In our experiments, we compared
our CNN to the following types of networks: DenseNet-121, DenseNet-169 and DenseNet-201. 4.
Kerchev, I. Ecology of four-eyed fir bark beetle Polygraphus proximus Blandford (Coleoptera, Curcu
Scolytinae) in the west Siberian region of invasion. Rus. J. Biol. Invasions 2014, 5, 176–185. [CrossRef 5.
Pashenova, N.V.; Kononov, A.V.; Ustyantsev, K.V.; Blinov, A.G.; Pertsovaya, A.A.; Baranchikov, Y.N.
Ophiostomatoid fungi associated with the four-eyed fir bark beetle on the territory of russia. Rus. J.
Biol. Invasions 2018, 9, 63–74. [CrossRef] References Li, W.; Fu, H.; Yu, L.; Cracknell, A. Deep learning based oil palm tree detection and counting for
high-resolution remote sensing images. Remote Sens. 2016, 9, 22. [CrossRef] 11. Guirado, E.; Tabik, S.; Alcaraz-Segura, D.; Cabello, J.; Herrera, F. Deep-learning versus OBIA for scattered
shrub detection with google earth imagery: Ziziphus lotus as case study. Remote Sens. 2017, 9, 1220. [CrossRef] 12. Baeta, R.; Nogueira, K.; Menotti, D.; dos Santos, J.A. Learning deep features on multiple scales for coffee
crop recognition. In Proceedings of the 2017 30th SIBGRAPI Conference on Graphics, Patterns and Images
(SIBGRAPI), Niteroi, Brazil, 17–20 October 2017; pp. 262–268. 13. Kussul, N.; Lavreniuk, M.; Skakun, S.; Shelestov, A. Deep learning classification of land cover and crop types
using remote sensing data. IEEE Geosci. Remote Sens. Lett. 2017, 14, 1–5. [CrossRef] 14. Waser, L.T.; Küchler, M.; Jütte, K.; Stampfer, T. Evaluating the potential of worldview—2 data to classify tree
species and different levels of ash mortality. Remote Sens. 2014, 6, 4515–4545. [CrossRef] 15. Goodfellow, I.; Bengio, Y. Aaron Courville Deep Learning. MIT Press. Available online: https://mitpress. mit.edu/books/deep-learning (accessed on 4 March 2019). 16. Tabik, S.; Peralta, D.; Herrera-Poyatos, A.; Herrera, F. A snapshot of image pre-processing for convolutional
neural networks: Case study of MNIST. Int. J. Comput. Intell. Syst. 2017, 10, 555–568. [CrossRef] 17. Deli, Z.; Bingqi, C.; Yunong, Y. Farmland scene classification based on convolutional neural network. In Proceedings of the 2016 International Conference on Cyberworlds (CW), Chongqing, China, 28–30
September 2016; pp. 159–162. p
pp
18. Längkvist, M.; Kiselev, A.; Alirezaie, M.; Loutfi, A. Classification and segmentation of satellite orthoimagery
using convolutional neural networks. Remote Sens. 2016, 8, 329. [CrossRef] 19. Dyrmann, M.; Karstoft, H.; Midtiby, H. Plant species classification using deep convolutional neural network. Biosyst. Eng. 2016, 151, 72–80. [CrossRef] 20. Razavi, S.; Yalcin, H. Using convolutional neural networks for plant classification. In Proceedings of the 25th
Signal Processing and Communications Applications Conference (SIU), Antalya, Turkey, 15–18 May 2017;
pp. 1–4. 21. Dos Santos Ferreira, A.; Matte Freitas, D.; da Silva, G.; Pistori, H.; Folhes, M.T. Weed detection in soybean
crops using ConvNets. Comput. Electron. Agric. 2017, 143, 314–324. [CrossRef] 22. Onishi, M.; Ise, T. Automatic classification of trees using a UAV onboard camera and deep learning. arXiv
2018, arXiv:1804.10390. 23. Abdullah, H.; Darvishzadeh, R.; Skidmore, A.K.; Groen, T.A.; Heurich, M. References 1. Bonan, G.B. Forests and climate change: Forcings, feedbacks, and the climate benefits of forests. Science 2008,
320, 1444–1449. [CrossRef] 1. Bonan, G.B. Forests and climate change: Forcings, feedbacks, and the climate benefits of forests. Science 2008,
320, 1444–1449. [CrossRef] 2. Hansen, M.C.; Potapov, P.V.; Moore, R.; Hancher, M.; Turubanova, S.A.; Tyukavina, A.; Thau, D.;
Stehman, S.V.; Goetz, S.J.; Loveland, T.R.; et al. High-resolution global maps of 21st-century forest cover
change. Science 2013, 342, 850–853. [CrossRef] 2. Hansen, M.C.; Potapov, P.V.; Moore, R.; Hancher, M.; Turubanova, S.A.; Tyukavina, A.; Thau, D.;
Stehman, S.V.; Goetz, S.J.; Loveland, T.R.; et al. High-resolution global maps of 21st-century forest cover
change. Science 2013, 342, 850–853. [CrossRef] 3. Kuznetsov, V.; Sinev, S.; Yu, C.; Lvovsky, A. Key to Insects of the Russian Far East (in 6 Volumes). Volume
5. Trichoptera and Lepidoptera. Part 3. Available online: https://www.rfbr.ru/rffi/ru/books/o_66092
(accessed on 4 March 2019). 3. Kuznetsov, V.; Sinev, S.; Yu, C.; Lvovsky, A. Key to Insects of the Russian Far East (in 6 Volumes). Volume
5. Trichoptera and Lepidoptera. Part 3. Available online: https://www.rfbr.ru/rffi/ru/books/o_66092
(accessed on 4 March 2019). 5. Pashenova, N.V.; Kononov, A.V.; Ustyantsev, K.V.; Blinov, A.G.; Pertsovaya, A.A.; Baranchikov, Y.N. Ophiostomatoid fungi associated with the four-eyed fir bark beetle on the territory of russia. Rus. J. Biol. Invasions 2018, 9, 63–74. [CrossRef] 5. Pashenova, N.V.; Kononov, A.V.; Ustyantsev, K.V.; Blinov, A.G.; Pertsovaya, A.A.; Baranchikov, Y.N. Ophiostomatoid fungi associated with the four-eyed fir bark beetle on the territory of russia. Rus. J. Biol. Invasions 2018, 9, 63–74. [CrossRef] 18 of 19 18 of 19 Remote Sens. 2019, 11, 643 6. Baranchikov, Y.; Akulov, E.; Astapenko, S. Bark beetle Polygraphus proximus: A new aggressive far eastern
invader on Abies species in Siberia and European Russia. In Proceedings of the 21st U.S. Department of
Agriculture Interagency Research Forum on Invasive Species, Annapolis, MD, USA, 12–15 January 2010. 7. Helbig, M.; Wischnewski, K.; Kljun, N.; Chasmer, L.E.; Quinton, W.L.; Detto, M.; Sonnentag, O. Regional
atmospheric cooling and wetting effect of permafrost thaw-induced boreal forest loss. Glob. Chang. Biol. 2016, 22, 4048–4066. [CrossRef] 8. Ma, Z. The Effects of Climate Stability on Northern Temperate Forests. Ph.D. Thesis, Aarhus University,
Aarhus, Denmark, March 2016. 9. Lehmann, J.R.K.; Nieberding, F.; Prinz, T.; Knoth, C. Analysis of unmanned aerial system-based CIR images
in forestry—A new perspective to monitor pest infestation levels. Forests 2015, 6, 594–612. [CrossRef] 10. References European spruce bark beetle
(Ips typographus, L.) green attack affects foliar reflectance and biochemical properties. Int. J. Appl. Earth
Obs. Geoinf. 2018, 64, 199–209. [CrossRef] 24. Heurich, M.; Ochs, T.; Andresen, T.; Schneider, T. Object-orientated image analysis for the semi-automatic
detection of dead trees following a spruce bark beetle (Ips typographus) outbreak. Eur. J. For. Res. 2009, 129,
313–324. [CrossRef] 25. Ortiz, S.M.; Breidenbach, J.; Kändler, G. Early detection of bark beetle green attack using terraSAR-X and
rapideye data. Remote Sens. 2013, 5, 1912–1931. [CrossRef] 26. Meddens, A.J.H.; Hicke, J.A.; Vierling, L.A.; Hudak, A.T. Evaluating methods to detect bark beetle-caused
tree mortality using single-date and multi-date Landsat imagery. Remote Sens. Environ. 2013, 132, 49–58. [CrossRef] 27. Näsi, R.; Honkavaara, E.; Lyytikäinen-Saarenmaa, P.; Blomqvist, M.; Litkey, P.; Hakala, T.; Viljanen, N.;
Kantola, T.; Tanhuanpää, T.; Holopainen, M. Using UAV-based photogrammetry and hyperspectral imaging
for mapping bark beetle damage at tree-level. Remote Sens. 2015, 7, 15467–15493. [CrossRef] 19 of 19 19 of 19 Remote Sens. 2019, 11, 643 28. Dash, J.P.; Watt, M.S.; Pearse, G.D.; Heaphy, M.; Dungey, H.S. Assessing very high resolution UAV imagery
for monitoring forest health during a simulated disease outbreak. ISPRS J. Photogramm. Remote Sens. 2017,
131, 1–14. [CrossRef] 29. Näsi, R.; Honkavaara, E.; Blomqvist, M.; Lyytikäinen-Saarenmaa, P.; Hakala, T.; Viljanen, N.; Kantola, T.;
Holopainen, M. Remote sensing of bark beetle damage in urban forests at individual tree level using a novel
hyperspectral camera from UAV and aircraft. Urban For. Urban Green. 2018, 30, 72–83. [CrossRef] 30. Ryabovol, S.V. The Vegetetion of Krasnoyarsk. Modern Problems of Science and Education. Available online:
https://www.science-education.ru/en/article/view?id=7582 (accessed on 1 March 2019). 31. Agisoft PhotoScan User Manual—Professional Edition, Version 1.4. 127p. Available online: https://www. agisoft.com/pdf/photoscan-pro_1_4_en.pdf (accessed on 15 March 2019). 32. Krivets, S.A.; Kerchev, I.A.; Bisirova, E.M.; Pashenova, N.V.; Demidko, D.A.; Petko, V.M.; Baranchikov, Y.N. Four-Eyed Fir Bark Beetle in Siberian Forests (Distribution, Biology, Ecology, Detection and Survey of Damaged
Stands; UMIUM: Krasnoyarsk, Russia, 2015. 33. Dawkins, P. Calculus III–Green’s Theorem. Available online: http://tutorial.math.lamar.edu/Classes/
CalcIII/GreensTheorem.aspx (accessed on 23 November 2018). 34. Basic Evaluation Measures from the Confusion Matrix. Classifier Evaluation with Imbalanced Datasets 2015. Available online: https://classeval.wordpress.com/introduction/basic-evaluation-measures/ (accessed on
15 March 2019). 35. Keras Documentation. Available online: https://keras.io/ (accessed on 23 November 2018). 36. Murray, C. Deep Learning CNN’s in Tensorflow with GPUs. Available online: https://hackernoon.com
deep-learning-cnns-in-tensorflow-with-gpus-cba6efe0acc2 (accessed on 23 November 2018). 37. Jordan, J. Common Architectures in Convolutional Neural Networks. References Available online: https://www. jeremyjordan.me/convnet-architectures/ (accessed on 2 August 2018). 38. Mohan, M.; Silva, C.A.; Klauberg, C.; Jat, P.; Catts, G.; Cardil, A.; Hudak, A.T.; Dia, M. Individual tree
detection from unmanned aerial vehicle (UAV) derived canopy height model in an open canopy mixed
conifer forest. Forests 2017, 8, 340. [CrossRef] 39. ImageNet Large Scale Visual Recognition Competition 2014 (ILSVRC2014). Available online: http://www. image-net.org/challenges/LSVRC/2014/results (accessed on 28 February 2019). 40. COCO–Common Objects in Context. Available online: http://cocodataset.org/#home (accessed on
28 February 2019). 41. Chollet, F. Xception: Deep learning with depthwise separable convolutions. arXiv, 2016, arXiv:1610.02357. 41. Chollet, F. Xception: Deep learning with depthwise separable convolutions. arXiv, 2016, arXiv:1610.02357. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Widespread Use of Non-productive Alternative Splice Sites in Saccharomyces cerevisiae
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PLOS genetics
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cc-by
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Introduction encodes an integral transmembrane protein, for which the use of
an alternative 59-splice site changes the number of passes through
the membrane and ultimately the location of the C-terminal end of
Src1p [16,17]. Alternative 39-splice site selection has been shown
to regulate expression of the APE2 gene according to a
temperature-dependent secondary structure in the transcript
[18]. A few other alternative 39-splice sites have been described,
and the use of some of these sites produces transcripts that are
degraded by NMD [19]. Recent work analyzing alternative
splicing across fungal species has shown that S. cerevisiae has lost
some of the alternative splicing events through gene duplication
and sub-functionalization of the duplicated genes, which are
otherwise produced by alternative splicing in other species [20]. Nonsense-mediated mRNA decay (NMD) is an RNA degrada-
tion system that degrades RNAs containing premature termination
codons [1,2]. In mammalian cells and higher eukaryotes, NMD
can be used to regulate gene expression, for instance by reducing
the level of alternatively spliced isoforms containing premature
termination codons [3,4,5,6,7,8]. This interplay between alterna-
tive splicing and NMD is involved in the autoregulation of SR
proteins [3,4,5]. In addition to its function in regulating non-
productively spliced isoforms, NMD is also used in a variety of
eukaryotes to degrade unspliced pre-mRNAs that have escaped
the splicing machinery [9,10,11,12,13]. Thus, NMD is widely
involved in the proofreading of splicing efficiency and accuracy. Using RNA-Seq analysis of strains mutated for NMD factors,
we identify here a large number of alternative splice sites in S. cerevisiae. However, we show that splicing at these sites is generally
non-productive because it introduces premature termination
codons (PTC), leading to degradation of the transcripts by
NMD. Non-productive splicing can be increased during environ-
mental stress to contribute to a global regulatory mechanism that
down-regulates transcripts levels in response to environmental
cues. These results show that non-productive splice sites are widely
used in S.cerevisiae, but that transcripts spliced at these sites are The yeast Saccharomyces cerevisiae has long been used as a model
system to investigate the mechanisms of pre-mRNA splicing, as
many components of the splicing machinery were identified
through genetic screens in S. cerevisiae [14], and most splicing
factors are highly conserved from yeast to mammalian cells [15]. Despite the presence of c.a. 330 intron-containing genes in S. Abstract * E-mail: guillom@chem.ucla.edu ¤ Current address: Applied BioCode Inc., Santa Fe Springs, California, United States of America. ¤ Current address: Applied BioCode Inc., Santa Fe Springs, California, United States of America. ¤ Current address: Applied BioCode Inc., Santa Fe Springs, California, United States of Am Tadashi Kawashima1¤, Stephen Douglass2, Jason Gabunilas1, Matteo Pellegrini3,
Guillaume F. Chanfreau1* 1 Department of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, Los Angeles, California, United States of America, 2 Bioinformatics
Interdepartmental Program, UCLA, Los Angeles, California, United States of America, 3 Department of Molecular, Cellular and Developmental Biology, UCLA, Los Angeles,
California, United States of America April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org Abstract Saccharomyces cerevisiae has been used as a model system to investigate the mechanisms of pre-mRNA splicing but only a
few examples of alternative splice site usage have been described in this organism. Using RNA-Seq analysis of nonsense-
mediated mRNA decay (NMD) mutant strains, we show that many S. cerevisiae intron-containing genes exhibit usage of
alternative splice sites, but many transcripts generated by splicing at these sites are non-functional because they introduce
premature termination codons, leading to degradation by NMD. Analysis of splicing mutants combined with NMD
inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified
novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 39 splice sites and for Prp18p in
suppressing the usage of a non-canonical AUG 39-splice site in GCR1. The use of non-productive alternative splice sites can
be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during
stress. These results show that alternative splicing is frequent in S. cerevisiae but masked by RNA degradation and that the
use of alternative splice sites in this organism is mostly aimed at controlling transcript levels rather than increasing
proteome diversity. Editor: Hiten D. Madhani, University of California San Francisco, United States of America Editor: Hiten D. Madhani, University of California San Francisco, United States of America Editor: Hiten D. Madhani, University of California San Francisco, United States of America Received October 8, 2013; Accepted February 4, 2014; Published April 10, 2014 Received October 8, 2013; Accepted February 4, 2014; Published April 10, 2014 Received October 8, 2013; Accepted February 4, 2014; Published April 10, 2014 Copyright: 2014 Kawashima et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Supported by grant GM061518 to GFC. TK was supported by USPHS National Research Service Award GM07104 and by a UCLA Graduate Division
Dissertation Year Fellowship. JG was supported by a Ruth L. Kirschstein National Research Service Award GM007185. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. RNA-Seq reveals the accumulation of a large number of
non-productive splice variants in NMD mutants We previously showed that NMD degrades unspliced transcripts
arising from a large fraction of intron-containing genes in S. cerevisiae, due to suboptimal splice sites [12,13], or upon splicing
factor inactivation [21]. In addition, recent data showed that
transcripts generated by the use of alternative 39-splice sites can be
degraded by NMD [19]. To gain further insights into the function
of NMD in the proofreading of spliced isoforms, we performed
RNA sequencing of mRNAs from wild-type and isogenic upf1D,
upf2D and upf3D strains defective for NMD. To identify transcripts
spliced at alternative splice sites, we performed gapped alignment
analysis of the RNA sequences (Table S1) using BLAT [22]. This
analysis revealed numerous occurrences of spliced transcripts
arising from previously unknown splice sites, in both WT and the
NMD mutants. We will refer to these new splicing events as
alternative splicing events, even if these are found in wild-type
cells, and to the annotated splicing events as the normal or
canonical splicing events. Alternative splicing events were detected
more frequently in RNA samples obtained from the NMD
mutants (Fig. 1A and 1B; Table S2), consistent with the fact that
most of these alternative splicing events result in the introduction
of a PTC, either by inducing a translational frameshift or by
inserting an intronic PTC-containing sequence (Table S2). After
adjusting for sequencing depth, upf1D, upf2D and upf3D showed a
1.67, 1.72, and 1.90-fold enrichment in alternative splicing events
and 1.59, 1.70, and 1.79-fold enrichment in PTC-generating
alternative splicing events, respectively, versus wild-type (Table
S2). NMD mutants showed an approximately 1.7-fold increase in
unspliced mRNAs compared to the wild-type (Table S3) when
considering reads that map to intronic and exon-intron regions,
confirming our previous results from tiling arrays showing the
involvement of NMD in eliminating unspliced transcripts genome-
wide [12]. This enrichment for unspliced RNAs in NMD mutants
is probably underestimated. Although there were 4-fold more
reads that mapped only to intronic regions in NMD mutants
compared to wild-type (Table S3), we observed an unanticipated
high number of reads that mapped to exon-intron junctions in the
wild-type strain (Table S3), which lowered the overall enrichment
for unspliced RNAs in NMD mutants. The consensus sequences derived from the alternative splicing
events identified in wild-type and all three mutants exhibited
differences from the consensus sequences derived from the
canonical (normal) splicing events (Fig. 1E). Non-productive Alternative Splicing in Yeast There was limited overlap in the alternative splicing events
identified in the three UPF mutants (Fig. 1C), suggesting that the
depth of our sequencing analysis was not sufficient to saturate
identification of all alternative splicing events, particularly those
occurring at low frequencies. The list of intron containing genes
(ICG) for which we did not find the use of alternative splice sites is
provided in Table S4, and the list of genes for which alternative
splicing events were detected is shown in Table S5. 97 out of 304
intron containing genes analyzed did not exhibit alternative
splicing (Table S4). Whether this reflects the absence of competing
alternative sites or the lack of depth of our sequencing analysis
remains to be determined. Author Summary Accurate gene expression requires the transfer of gene
information from DNA to RNA. When DNA is transcribed
into RNA, part of the RNA needs to be removed (spliced) to
generate a proper copy of the genetic information. This
process needs to be very accurate to preserve the genetic
information that will be transferred into proteins. Our
study shows that in baker’s yeast, the splicing process does
not always produce the correctly spliced products, as RNA
splicing events frequently utilize incorrect splice sites. However, these deficient RNA molecules are eliminated
from cells by a quality control mechanism to preserve the
integrity of the genetic information. However, incorrect
splicing is not useless, as it can be used to regulate the
quantity of RNA that is generated. To investigate if alternative splicing events are due to rare
events or to splicing errors that occur randomly during transcript
expression, we examined the abundance of ICG mRNAs that
exhibited alternative splicing events and that of ICG mRNAs for
which no alternative splicing events were detected (Fig. 1D). This
analysis showed that some low abundance transcripts exhibited
alternative splicing, while some high abundance transcripts did not
(Fig. 1D). In addition, the median abundance of genes that showed
alternative splicing was 117 RPKM, while the median abundance
for genes with no alternative splicing events detected was 136
RPKM. Thus, even if the most highly expressed ICG (.2200
RPKM) all exhibited alternative splicing (Fig. 1D), genes with no
alternative splicing were in general expressed at higher level than
genes for which alternative splicing events were detected, showing
no clear correlation between transcript abundance and the
detection of alternative splicing events. We conclude that the
detection of alternative splicing events in our RNA-Seq analysis is
not an indirect consequence of the higher number of reads for
highly-expressed transcripts. eliminated by RNA quality control mechanisms. Thus, while
alternative splicing is frequently utilized in higher eukaryotes to
generate proteome diversity, it is mainly used in S.cerevisiae as a
means to regulate transcript levels. Introduction cerevisiae, the prevalence of alternative splicing in this organism
remains largely unexplored, as only a few examples of alternative
splice site selection have been documented. The SRC1 gene April 2014 | Volume 10 | Issue 4 | e1004249 1 PLOS Genetics | www.plosgenetics.org RNA-Seq reveals the accumulation of a large number of
non-productive splice variants in NMD mutants Alternative 59-splice
sites showed a relaxation of the conserved sequences, especially at
positions 4 and 6 compared to the consensus obtained from the
canonical splicing events. The 39-splice sites used in alternative
splicing events also showed a decrease in conservation of the
polypyrimidine sequence preceding the conserved YAG, as well as
a weaker conservation of the pyrimidine preceding the conserved
AG dinucleotide (Fig. 1E). Thus, alternative splice sites identified
by RNA sequencing showed a relaxed conservation, suggesting
that these might correspond to lower efficiency splice sites, and
possibly to regulated splicing events. Finally, we identified a
number of alternative splicing events in either wild-type or NMD
mutants that do not introduce a PTC and would potentially result
in
the
production
of
proteins
that
differ
from
the
SGD
annotations. The list of these potential alternative proteins is
presented in Table S6. However, we did not investigate these
alternative protein forms further because most of the RNAs that
would result in the production of these proteins were found in low
abundance compared to those resulting in the production of the
annotated proteins. Strategy for validation of alternative splicing events gy
p
g
The previous RNA-Seq analysis revealed the potential wide-
spread usage of alternative splice sites (SS). Figure 2 depicts
specific mRNAs that were chosen for validation and further
characterization. These transcripts were classified into three
classes: those with 1) alternative 59-SS; 2) alternative 39-SS; and
3) a combination of both. Transcripts from class 1 included
RPL22B as well as the previously reported SRC1 [16]. Class 2
transcripts included genes encoding the RNA Polymerase III April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 2 Non-productive Alternative Splicing in Yeast Figure 1. Bioinformatics analysis of alternative splice site usage in wild-type and NMD mutants. A. Venn diagram showing the overlap of
alternative splice site usage between the wild-type and three NMD mutants pooled for all unique non-canonical splicing events (both PTC-generating
and non-PTC-generating). B. Venn diagram showing the overlap of alternative splicing events between the wild-type and three NMD mutants pooled
for all unique non-canonical splicing events resulting in a potential PTC. C. Venn diagram showing the overlap of alternative splicing events between
the upf1D, upf2D, and upf3D strains for PTC-generating splicing events. D. Distributions of intron-containing gene transcripts showing alternative
splicing events (red) or no alternative splicing events (blue) according to their overall abundance in RPKM. Transcripts for which the abundance was
higher than 2,300 RPKM were grouped in the final bin. E. Sequence logo analysis of 59- and 39- splice sites for all normal and alternative splicing
events detected by RNA-Seq in wild-type and NMD mutant strains. doi:10.1371/journal.pgen.1004249.g001 Figure 1. Bioinformatics analysis of alternative splice site usage in wild-type and NMD mutants. A. Venn Figure 1. Bioinformatics analysis of alternative splice site usage in wild-type and NMD mutants. A. Venn diagram showing the overlap of
alternative splice site usage between the wild-type and three NMD mutants pooled for all unique non-canonical splicing events (both PTC-generating
and non-PTC-generating). B. Venn diagram showing the overlap of alternative splicing events between the wild-type and three NMD mutants pooled
for all unique non-canonical splicing events resulting in a potential PTC. C. Venn diagram showing the overlap of alternative splicing events between
the upf1D, upf2D, and upf3D strains for PTC-generating splicing events. D. Distributions of intron-containing gene transcripts showing alternative
splicing events (red) or no alternative splicing events (blue) according to their overall abundance in RPKM. Strategy for validation of alternative splicing events Transcripts for which the abundance was
higher than 2,300 RPKM were grouped in the final bin. E. Sequence logo analysis of 59- and 39- splice sites for all normal and alternative splicing
events detected by RNA-Seq in wild-type and NMD mutant strains. doi:10.1371/journal.pgen.1004249.g001 labeling with an appropriate 32P-labelled ladder (data not shown)
to confirm the sizes of all RT-PCR products and correlate the data
back to gels obtained with Cy3-labeled primers. In addition to the
wild-type and NMD-deficient upf1D strains, we analyzed the
phenotypes of a number of S. cerevisiae splicing mutants. Knockout
mutants of genes encoding Mud1p and Nam8p were chosen for
their association with the U1 snRNP and role in 59-SS selection
[23,24,25,26]. The HUB1 knockout was also included, as Hub1p
was recently implicated in 59-SS selection for SRC1 [17]. Prp17p
and Prp18p were selected for their involvement in the second step
of splicing and potential effects on 39-SS selection [27,28]. Finally,
Isy1p was also included as a potential splicing fidelity factor [29]. The splicing profiles were analyzed for each of the genes transcription factor TFC3 with a downstream alternative 39-SS, and
the adenosine deaminase TAN1 with two alternative 39-SS flanking
the normal 39-SS. For the third class, we examined genes encoding
the glycosylphosphatidylinositol biosynthetic enzyme GPI15 and the
transcriptional regulator GCR1. GPI15 exhibited the use of an
alternative 59-SS with the normal 39-SS, as well as the normal 59-SS
with an alternative 39-SS (Fig. 2). GCR1 showed a more complex
splicing pattern with multiple combinations of 59 and 39-SS (Fig. 2). transcription factor TFC3 with a downstream alternative 39-SS, and
the adenosine deaminase TAN1 with two alternative 39-SS flanking
the normal 39-SS. For the third class, we examined genes encoding
the glycosylphosphatidylinositol biosynthetic enzyme GPI15 and the
transcriptional regulator GCR1. GPI15 exhibited the use of an
alternative 59-SS with the normal 39-SS, as well as the normal 59-SS
with an alternative 39-SS (Fig. 2). GCR1 showed a more complex
splicing pattern with multiple combinations of 59 and 39-SS (Fig. 2). We analyzed alternative splicing events by RT-PCR using Cy3-
end labeled primers, which allowed for relative comparison of the
abundance of spliced and unspliced species, regardless of their size. Non-productive Alternative Splicing in Yeast Non-productive Alternative Splicing in Yeast mentioned above in each of these mutant strains by fractionation
of the RT-PCR products on polyacrylamide gels (Fig. 3). For the
splicing mutants for which the splicing pattern differed from the
wild-type, additional RT-PCR experiments were performed in
triplicate from three independent cultures and quantitated, as
shown in Supporting Figures S1, S2, S3, S4 and S6. previous studies have demonstrated the roles of various splicing
factors [16,17,30]. RT-PCR analysis of SRC1 splice variants
confirmed the use of these two alternative 59-SS (Fig. 3). Wild-type
samples showed a 60/40 ratio of SRC1-S/SRC1-L (Fig. S1),
consistent with previous reports [16,17,30]. Samples from the
upf1D mutant showed a pattern similar to wild-type (Fig. 3; Fig. S1), indicating that both variants are stable and not targeted by
NMD. This result is consistent with our RNA-Seq analysis, which
showed high sequence counts for both forms in all strains. Samples
from the nam8D strain showed a slight increase in the level of
unspliced transcripts (Fig. 3) due to reduced splicing efficiency
[31]. The prp17D and prp18D mutants both showed a slight RT-PCR analysis confirms the involvement of Prp17p and
Hub1p in SRC1 alternative splicing RT-PCR analysis confirms the involvement of Prp17p and
Hub1p in SRC1 alternative splicing As a first step in validating our RT-PCR strategy, we focused on
SRC1, which exhibits two possible 59-SS (Fig. 2) and for which SRC1, which exhibits two possible 59-SS (Fig. 2) and for which
[31]. The prp17D and prp18D mutants both showed a slight
Figure 2. Spliced species produced from the SRC1, RPL22B, TAN1, TFC3, GPI15 and GCR1 genes. Species labeled with an asterisk are subject
to NMD. Species labeled with two asterisks are predicted to be subject to NMD but were not observed to do so in subsequent experiments. The
alternative 39-SS of SRC1 is located 4 nt upstream from the annotated 39-SS. The alternative 39-SS of RPL22B is located 64 nt downstream from the
annotated 39-SS. The alternative 39-SS of TAN1 are located 6 nt upstream and 7 nt downstream from the annotated 39-SS. The alternative 39-SS of
TFC3 is located 17 nt downstream from the annotated 39-SS. The alternative 59 and 39-SS of GPI15 are located 36 nt downstream and 14 nt upstream,
respectively, from the annotated 59 and 39-SS. The alternative 39-SS of GCR1 are located 5 nt upstream (GUAUGG); 51 nt downstream (GUAUGG) and
627 nt downstream from the annotated 59SS. The alternative 39-SS of GCR1 are located 40 nt upstream (AUG) and 17 nt downstream (CAG) from the
annotated 39-SS. doi:10.1371/journal.pgen.1004249.g002 Figure 2. Spliced species produced from the SRC1, RPL22B, TAN1, TFC3, GPI15 and GCR1 genes. Species labeled with an asterisk are subject
to NMD. Species labeled with two asterisks are predicted to be subject to NMD but were not observed to do so in subsequent experiments. The
alternative 39-SS of SRC1 is located 4 nt upstream from the annotated 39-SS. The alternative 39-SS of RPL22B is located 64 nt downstream from the
annotated 39-SS. The alternative 39-SS of TAN1 are located 6 nt upstream and 7 nt downstream from the annotated 39-SS. The alternative 39-SS of
TFC3 is located 17 nt downstream from the annotated 39-SS. The alternative 59 and 39-SS of GPI15 are located 36 nt downstream and 14 nt upstream,
respectively, from the annotated 59 and 39-SS. The alternative 39-SS of GCR1 are located 5 nt upstream (GUAUGG); 51 nt downstream (GUAUGG) and
627 nt downstream from the annotated 59SS. The alternative 39-SS of GCR1 are located 40 nt upstream (AUG) and 17 nt downstream (CAG) from the
annotated 39-SS. Strategy for validation of alternative splicing events Because we lacked an adequate size marker for Cy3 detection, the
same RT-PCR analyses were initially performed with 32P-end We analyzed alternative splicing events by RT-PCR using Cy3-
end labeled primers, which allowed for relative comparison of the
abundance of spliced and unspliced species, regardless of their size. Because we lacked an adequate size marker for Cy3 detection, the
same RT-PCR analyses were initially performed with 32P-end April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 3 PLOS Genetics | www.plosgenetics.org RT-PCR analysis confirms the involvement of Prp17p and
Hub1p in SRC1 alternative splicing doi:10.1371/journal.pgen.1004249.g002 April 2014 | Volume 10 | Issue 4 | e1004249 4 Non-productive Alternative Splicing in Yeast mutants on SRC1 splicing patterns and showed that our RT-PCR
strategy is effective in analyzing the impact of specific splicing
factors on splice site usage.. increase in the usage of SRC1-L 59-splice site relative to the SRC-S
59-splice site (1.4 and 1.3 fold, respectively), as suggested previously
for the prp17D mutant at the protein level [17], and the prp18D
mutant also exhibited an increase in unspliced precursors
accumulation, consistent with previous results for other transcripts
[21]. The isy1D mutant strain exhibited a clear accumulation of
unspliced pre-mRNAs (Fig. 3), in agreement with the documented
role of Isy1p in maintaining the proper conformation needed for
the 1st step of splicing [29]. Hub1p inactivation resulted in a 3-fold
reduction in the amount of SRC1-S, coinciding with an increase in
SRC1-L (Fig. S1, Fig. 3), consistent with previous reports [17,30]. This reduction was also observed in the context of the upf1D
mutant (Fig. S1, Fig. 3). Thus, the results described above
confirmed the previously described effects of various splicing Alternative splicing patterns of GPI15 and GCR1 reveal
the production of alternative non-functional protein
products and the use of a non-canonical AUG 39-splice
site repressed by Prp18p p
y
p
p
GPI15 in an interesting case where the two alternatively spliced
products identified by our RNA-Seq analysis are not targeted by
NMD. The use of an alternative GUACGU 59-splice site results in
the deletion of 30 nucleotides from the 39 end of exon 1 (Fig. 2),
which maintains the open-reading frame but generates a truncated
protein. However, the protein product resulting from translation of
this alternatively spliced product is likely to be non-functional, as
this truncation removes a stretch of 10 amino acids at positions
187–197 in the most highly conserved region of this protein [35]. This transcript can be detected in samples from the wild-type and
the splicing factor mutants, and does not vary in intensity in the
context of upf1D, indicating that it is not targeted by NMD (AS 59,
Fig. 3). In contrast, the alternatively spliced transcript generated by
use of a downstream CAG 39-SS results in a PTC. However, this
PTC-containing transcript would exhibit a short 85 nt 39-UTR,
which might render it insensitive to NMD as suggested by the faux
39 UTR model [36,37]. Indeed, the abundance of this transcript
was not increased in the upf1D mutant (Fig. 3). In addition, this
transcript is expected to yield a non-functional protein due to C-
terminal truncation and deletion of amino acids within the most
conserved region of the protein [35]. Analysis of the pattern of
selection of these two alternatively spliced transcripts in the various
splicing mutants did not reveal any major effect of these mutants
(Fig. 3) in contrast to the effects described above for RPL22B,
TAN1 or TFC3. However, there was a slight increase in the use of
the downstream alternative 39-SS in the prp17Dupf1D strain,
consistent with the role of Prp17p in favoring the upstream 39-SS,
as described above for TAN1 and TFC3. TAN1 exhibits a more complex alternative 39-SS pattern, where
a canonical UAG 39-SS is flanked by two alternative 39 AAG
sequences (Fig. 2). The use of either of these sites would generate
PTC-containing transcripts. The upstream AAG (AS 39 #1) is
only 6 nt away from the canonical 39-SS. The retention of 6 nt of
intronic sequence would maintain the proper reading frame but
would result in a PTC because the UAG sequence of the normal
39-splice site corresponds to an in-frame stop codon [32]. components Nam8p and Mud1p
RPL22B showed the presence of an RPL22B showed the presence of an alternative 59-SS in the
intronic sequence, which unlike SRC1, yields a PTC-containing
transcript potentially targeted to NMD (Fig. 2). This alternatively
spliced transcript was almost 10-fold more abundant in the upf1D
mutant (Fig. 3; Fig. S2), further suggesting that it is targeted by
NMD. We also detected a large accumulation of unspliced species Figure 3. RT-PCR analysis of alternatively spliced products for SRC1, RPL22B, TAN1, TFC3, GPI15 and GCR1 in wild-type, NMD and
various splicing mutants. The unspliced (US) species is also shown on top. The middle portions of the gel where no species were visible have been
removed. In all cases, RT-PCR was performed with a Cy3-labeled primer. The labeling of the different alternatively spliced forms is according to the
nomenclature shown in Figure 2. doi:10.1371/journal.pgen.1004249.g003 Figure 3. RT-PCR analysis of alternatively spliced products for SRC1, RPL22B, TAN1, TFC3, GPI15 and GCR1 in wild-type, NMD and
various splicing mutants. The unspliced (US) species is also shown on top. The middle portions of the gel where no species were visible have been
removed. In all cases, RT-PCR was performed with a Cy3-labeled primer. The labeling of the different alternatively spliced forms is according to the
nomenclature shown in Figure 2. doi:10.1371/journal.pgen.1004249.g003 April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 5 Non-productive Alternative Splicing in Yeast in the upf1D mutant, indicating inefficient recognition of this
splicing substrate. This may be the result of both the normal
(GUACGU) and alternative (GUUUGU) 59-SS having non-
consensus sequences (see below). Interestingly, the abundance of
the alternatively spliced product was found to decrease by two to
three folds when Nam8p or Mud1p were inactivated in the context
of the upf1D deletion (Fig. 3; Fig. S2). The deletion of either one of
these two factors might hinder the ability of the U1 snRNP to bind
to the alternative suboptimal 59 GUUUGU splice site of RPL22B,
resulting in decreased usage. This is consistent with the known
roles of Mud1p and Nam8p in the first step of splicing [25], and
suggest their direct involvement in modulating 59-SS selection of
RPL22B. By contrast, no major changes were observed in the
prp17D, prp18D, isy1D, hub1D mutants, either alone or in
combination with the upf1D deletion (Fig. 3), showing the
specificity of the effects detected with Nam8p and Mud1p. components Nam8p and Mud1p
RPL22B showed the presence of an Thus,
RPL22B exhibits two competing suboptimal 59-SS, one of which is
highly sensitive to perturbations in the U1 snRNP. The functional
significance of the alternative 59-SS of RPL22B in regulating
transcript levels is investigated further below. most proximal to the branch point sequence (AS 39 #1; Fig. 3; Fig. S4). This product is barely detectable in the wild-type strain but
can be observed in the prp18D strain (Fig. 3), and inactivation of
Prp18p in the context of the upf1D deletion resulted in a 3-fold
increase in the abundance of this species (Fig. S4). The effect of
Prp18p on this 39-SS might be due to the identity of the sequences
immediately following the 39-SS, which have been shown to
influence 39-SS selection in the absence of a functional Prp18p
[33]. Isy1p inactivation resulted in an increase of unspliced species
in a similar fashion to SRC1 discussed above; however there was
no effect of Isy1p, Hub1p, Mud1p and Nam8p on alternative 39-
SS selection of TAN1 (Fig. 3), showing the specificity of the effects
observed with Prp17p and Prp18p. Finally, unspliced TAN1
transcripts were generally not affected by NMD, except in the
context of a mud1D mutant strain (Fig. 3). This observation is
consistent with a recent report showing that TAN1 unspliced
transcripts are retained in the nucleus by the RES complex, and
are subject to NMD only when the RES complex is inactivated
[34]. Overall, analysis of TFC3 and TAN1 alternative 39-SS
patterns show that Prp17p and Prp18p have antagonistic roles in
the selection of upstream and downstream 39-SS of TAN1, and
highlight the importance of Prp17p in enhancing the use of 39-SS
located closer to the branchpoint. A novel role for Prp17p in promoting the use of
branchpoint proximal alternative 39-splice sites Gapped sequence alignment showed that TFC3 exhibits an
alternative CAG 39-SS 17 nt downstream of the annotated AAG
(Fig. 2). This product can be detected in samples from the wild-
type and splicing mutants, but is 4.5-fold more abundant in the
context of the upf1D deletion, showing that a large fraction of this
product is degraded by NMD (Fig. 3; Fig. S3). This non-
productive isoform amounts to 27% of all spliced products (Fig. S3), showing that a significant fraction of splicing generates NMD-
targeted,
non-productive
transcripts. We
observed
a
slight
accumulation (1.7 fold) of the downstream alternative 39-splice
product in the prp17D mutant (Fig. 3; Fig. S3), showing that this
second step splicing factor contributes to reducing the use of this
alternative 39-SS. As expected, inactivation of the first step splicing
factors Mud1p or Nam8p had no effect on the pattern of 39-SS
selected (Fig. 3). Alternatively spliced species of RPL22B and GCR1 increase
during stress conditions Significantly, amino acid starvation did not change the levels of the
alternatively spliced species of TAN1 and TFC3 that are normally
subject to NMD (Fig. S11). This observation provides further
evidence that the increase in the amount of alternatively spliced
RPL22B transcript observed during amino acid starvation is due to
a switch in splice site selection and not to an inhibition in NMD,
since the level of alternatively spliced species of TAN1 and TFC3
that are normally degraded by NMD is unaffected in the same
conditions. In addition to this major spliced product, we also detected a series
of alternatively spliced products degraded by NMD (as denoted by
asterisks in Fig. 2 and 3). Splicing from the annotated GUAUGA 59
splice site combined with the upstream CAG 39 splice site resulted in
a PTC-containing transcript labeled as *A in Figs. 2 & 3. This
transcript is degraded by NMD, as higher amounts are observed in all
the strains containing a upf1D deletion, and it is the most abundant of
all GCR1 alternatively spliced products subject to NMD (Fig. 3; Fig. S6). Another product is generated from combining the upstream
GUAUGG 59-SS with the most downstream UAG 39-SS (*C in
Fig. 2). This splicing event results in a PTC, as it introduces a
translational frameshift, which is not detected until the 43rd amino
acid is translated. The corresponding transcript accumulates at low
abundance in all samples and appears to be targeted by NMD, as its
abundance increases slightly in all upf1D strains. In addition, the use
of this most downstream 39-SS increases almost 4-fold in the
prp17Dupf1D strain when compared to the upf1D control (Fig. 3; Fig. S6). Because the 39-SS used to generate this transcript corresponds to
the most downstream one, this observation provides another example
of the importance of Prp17p in favoring the selection of upstream 39-
SS, as shown above for TFC3, TAN1 and to a lesser extent GPI15. Another PTC-containing transcript that is degraded by NMD
results from splicing of the downstream intronic GUAUGG 59-SS
with the CAG 39-SS, (labeled *B in Fig. 2 and 3). This product is
faint, but detectable in all cases of NMD deactivation, except in
combination with nam8D or mud1D, most likely because this 59-SS
has a higher sensitivity to U1 snRNP perturbations, as described
above for RPL22B. Alternatively spliced species of RPL22B and GCR1 increase
during stress conditions g
The previous results validated our prediction that transcripts
generated from the use of alternative non-productive splice sites
are degraded by NMD and revealed the role of specific splicing
factors
in
governing
the
choice
between
alternative
sites. Strikingly, the sequence of some of these non-productive splice
sites was found to be conserved across closely related yeast species
(Fig. S8, RPL22B and Fig. S9, TAN1). Because the level of
sequence conservation in intronic sequences is usually very low
(Fig. S8, S9), these peaks in sequence conservations for intronic
alternative splice sites might reflect their functional importance. We hypothesized that the use of some of these alternative splice
sites which lead to degradation by NMD might be favored under
certain conditions to down-regulate gene expression. To test this
hypothesis, we monitored changes in the splicing patterns of
RPL22B, TAN1, and TFC3 under stress conditions such as amino
acid starvation, heat shock, LiCl-mediated hyperosmotic stress,
and rapamycin treatment, as these have been reported to elicit
diverse responses in the expression of intron containing genes
[43,44]. In addition, various stresses cause down-regulation in
ribosomal protein gene expression (many of which contain
introns), presumably to relieve the cell of massive energy
requirements of ribosome biogenesis and focus those resources
into regulations that are the most appropriate in response to the
current stress condition [45,46,47]. After 10 minutes of amino acid
depletion, RPL22B showed an increase in unspliced species as well
as well as a 4.5-fold increase in the level of the alternatively spliced
product when compared to the SDC or YPD media controls
(Fig. 4A; Fig. S10). In the upf1D strain shifted to amino acid
starvation conditions, the levels of the alternatively spliced product
increased compared to the wild-type strain grown in the same
conditions, as would be expected when NMD transcripts are no
longer degraded (Fig. 4A lanes 2 and 4). The fact that the level of
the alternatively spliced transcript is 2.5-fold higher in the upf1D
sample than in the wild-type sample under amino acid starvation
conditions (Fig. S10) argues that the increase in the abundance of
these species in the wild-type strain in these conditions is not due
to NMD inhibition in these conditions, but that a change in splice
site selection occurs that favors the use of the alternative splice site. Non-productive Alternative Splicing in Yeast Alternatively spliced species of RPL22B and GCR1 increase
during stress conditions Non-productive Alternative Splicing in Yeast 23 nt upstream of the other alternative 39-SS (Fig. 2). The use of
all of these sites was confirmed by RT-PCR, cloning and Sanger
sequencing (see below and Fig. S5). The fact that some alternative
splice sites escaped identification by mRNA sequencing indicates
that a greater depth of coverage has the potential to identify even
more alternative splice sites. Prp22p during the late stages of splicing [42], it is possible that the
absence of Prp18p might indirectly hinder the function of Prp22p
in proofreading 39-SS selection, and that the use of this unusual 39-
SS might be the consequence of a reduced Prp22p function in the
absence of Prp18p. To test this hypothesis, we analyzed GCR1
splicing in a prp22-1 mutant. RT-PCR analysis showed that the
spliced product generated from the use of the AUG 39-SS did not
accumulate in a prp22-1 splicing mutant (Fig. S7). Thus, the
accumulation of species resulting from the use of this unusual 39-
SS in the prp18Dupf1D samples is not an indirect consequence of
hindered Prp22p function. The discovery of the splicing at this
unusual 39-SS sequence reveals the importance of Prp18p in
ensuring proper 39-SS selection for GCR1 and in repressing the use
of non-canonical 39-SS sequences. Based on GCR1 annotation, the canonical spliced mRNA would
use the GUAUGA 59-SS along with the most downstream UAG
39-SS (Fig. 2). This product (labeled as S(annot.) in Fig. 2 and 3),
however was detected at very low levels (Fig. 3). The major spliced
product observed resulted from the use of the most upstream
GUAUGG 59-SS and of an upstream CAG 39-SS (labeled ‘‘S’’ in
Fig. 2 and 3). This splicing event does not introduce a PTC and
results in a protein that is very similar to the translation product of
the annotated spliced transcript S(annot.). The annotated amino
acid sequence of GCR1 from position 2 to 4 is VCT. In the major
spliced product S, this sequence is replaced by QTSVDST. Thus,
most of the protein is identical, except for a few N-terminal amino
acids which are not expected to affect Gcr1p function, as all GCR1
mutations with phenotypic effects have been mapped to a region
downstream of this short sequence stretch [38,39,40]. Based on the
relative abundances of S and S(annot.), it is clear that S, and not
S(annot.) is the main spliced product for the GCR1 gene. Alternative splicing patterns of GPI15 and GCR1 reveal
the production of alternative non-functional protein
products and the use of a non-canonical AUG 39-splice
site repressed by Prp18p The
downstream AAG (AS 39 #2) is 7 nt downstream of the normal
39-SS, resulting in a frameshift-induced PTC. RT-PCR analysis of
the wild-type and upf1D strains confirmed the RNA-Seq data by
showing that these two alternative splice products are detected at
extremely low levels, unless NMD is inhibited (Fig. 3; Fig. S4). In
samples from the upf1D strain, the two alternatively spliced
products accumulate to similar amounts, and both species are
detected at lower levels than the normal spliced product (20% of
all
spliced
products;
Fig. S4),
possibly because
these
two
suboptimal AAG sites do not compete well with the consensus
canonical UAG site. Strikingly, the usage of these alternative 39
splice sites was dramatically altered when Prp17p or Prp18p were
inactivated. Inactivating Prp17p resulted in an increase in the use
of the downstream alternative 39-SS (AS 39#2), while the
upstream alternative 39-SS (AS 39#1) was no longer used (Fig. 3;
Fig. S4), showing a role of Prp17p in enhancing the use of
upstream, branchpoint proximal 39-SS. By contrast, Prp18p
inactivation resulted in increased usage of the alternative 39-SS GCR1 showed the most complex splicing pattern of all
transcripts analyzed. Gapped alignments identified an intronic
GUAUGG alternative 59-SS as well as an upstream CAG
alternative 39-SS (Fig. 2). In addition to these alternative splice
sites identified by RNA-Seq, RT-PCR revealed the use of an
additional GUAUGG alternative 59-SS staggered 5-nt upstream of
the normal 59-SS and of a non-canonical AUG alternative 39-SS April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 6 Non-productive Alternative Splicing in Yeast Non-productive Alternative Splicing in Yeast This analysis showed that cells treated in heat shock
conditions resulted in much weaker signal than in the samples
obtained from cells grown at 25uC, consistent with a general
down-regulation
of
ribosomal
protein
genes
under
stress
[43,44,45,46,47]. Upon NMD inactivation, we observed a rescue
of transcript levels, which mostly corresponded to unspliced RNAs
and to some alternatively spliced transcripts (Fig. 5B). However,
changing the alternative 59-SS to a consensus sequence in the
context of NMD inactivation was sufficient to recover a large
amount of spliced transcripts (Fig. 5B, lane 11, lower panel). To
investigate if this effect was specific to heat shock or is also
observed during other stresses, we analyzed the expression of wild-
type and mutated forms of RPL22B during amino acid starvation
(Fig. 5C). The results observed during amino acid starvation were
similar to those described during heat shock, with a large increase
in the level of spliced transcripts upon changing the alternative 59-
splice site to the consensus sequence. We also observed an increase
in the use of the alternative 59-SS under amino acid starvation
(Fig. S13). Interestingly, shifting the Upf1p-inactivated strain with
the alternative 59-splice site consensus sequence from SDC to
amino acid starvation conditions resulted in only a minor increase We next investigated the effect of a 20 minute heat shock at 42uC
on splicing patterns. Under these conditions and in the wild-type
strain, RPL22B showed an increase in unspliced as well as a decrease
in the relative amount of the normal spliced product (Fig. 4A lane 5
vs. 7). More importantly, the NMD defective strain upf1D showed
an even larger increase in unspliced pre-mRNAs, as well a large
accumulation of the alternatively spliced product that coincides with
a decreased amount of canonical spliced product (Fig. 4A lane 6 vs. 8). In these conditions, the alternatively spliced product now
corresponds to more than half of all spliced species (Fig. S10). Under
heat shock, this alternatively spliced product is 4-fold more
abundant in the upf1D strain than in the wild-type strain. These
higher levels upon NMD inactivation show that the increased
accumulation of these species under heat shock is not due to a
decrease in NMD efficiency. Rather, this result shows that the use of
the alternative splice site is being favored in heat shock conditions. Non-productive Alternative Splicing in Yeast By contrast, TFC3 and TAN1 exhibited an accumulation of
unspliced species, but decreased levels of both the canonical and
alternatively spliced species (Fig. S11), consistent with a general
inhibition of pre-mRNA splicing under heat shock [48,49]. Thus
the pattern of alternatively spliced species of TFC3 and TAN1 that
are subject to NMD is very different from that of RPL22B, further
proving that the accumulation of the alternatively spliced RPL22B
transcript under heat shock conditions described above is not due to
a general stabilization of spliced forms degraded by NMD. Like heat shock, rapamycin treatment was shown to result in an
inhibition of ribosomal proteins mRNA splicing based on
microarray experiments [43]. Within 20 minutes of rapamycin
treatment, RPL22B indeed showed trends similar to those
observed in heat shock, but to a lesser degree, with an increase
of unspliced species and of alternatively spliced RPL22B species
(Fig. 4A), but no effect on the alternatively spliced TAN1 and TFC3
transcripts (Fig. S11). Hyperosmotic shock (300 mM LiCl expo-
sure for 10 min) only resulted in minimal effects; there were no
changes observed for TFC3 and TAN1 targets under these stress
conditions (Fig. S11), and RPL22B showed only a slight increase in
unspliced but the levels of spliced transcripts remained similar. Thus, RPL22B exhibits regulated use of its alternative 59-splice
site,
mostly
under
amino
acid
starvation
and
heat
shock
conditions, while other transcripts such as TFC3 and TAN1 did
not exhibit any change in their alternative splicing profiles. Because GCR1 exhibited a very complex splicing pattern,
especially in the absence of Prp18p, and because heat shock
conditions resulted in the most dramatic changes in splicing for
RPL22B, we next investigated the effect of heat shock on GCR1
splicing in the wild-type, upf1D, prp18D and prp18Dupf1D mutants
(Fig. 4B). Under heat-shock, we detected a general inhibition of
splicing, consistent with the data described above. However, we
also observed an increase of the abundance of the A* form relative
to the normal spliced product S, indicative of a switch from the
normal GUAUGG site to the GUAUGA site. The absence of
Prp18p resulted in an increase of the use of the non-canonical
AUG site (*D species) in heat shock conditions, and this product
now constituted one third of all spliced species. Non-productive Alternative Splicing in Yeast Thus we conclude
that GCR1, like RPL22B, exhibits a switch in splice site selection
during heat shock, and that Prp18p limits splicing at this non-
canonical AUG site under stress conditions. Non-productive Alternative Splicing in Yeast mutations of this alternative 59-SS. The suboptimal GUUUGU
alternative 59-splice site was either deleted or mutated to the
consensus GUAUGU sequence at the endogenous chromosomal
locus (CS, consensus mutation and D, deletion, Fig. 5A). Changing
the alternative 59-SS to the consensus GUAUGU sequence
resulted in detectable amounts of alternatively spliced products
at 25uC, even in a functional NMD background (Fig. 5B),
suggesting that the suboptimal GUUUGU sequence contributes to
the low usage of this alternative site in normal conditions. Inactivation of Upf1p in this context showed that 70% of all
spliced species were now being produced by splicing from the
alternative
consensus
site
(Fig. 5B;
lane
5;
Fig. S12
for
quantitation), and that splicing efficiency was improved, as shown
by a decrease in unspliced species. By contrast, deleting the
alternative splice site resulted in higher amount of unspliced
transcripts, especially in the upf1D background. Thus, deleting the
alternative 59-splice site of RPL22B is not sufficient to improve
splicing at the normal splice site, possibly because of the
suboptimal sequence of the normal RPL22B 59-splice site. In
addition to RT-PCR, the same strains were analyzed by northern
blot (Fig. 5B, bottom panel), which yielded results similar to those
obtained by RT-PCR. These results show that increasing the
strength of the alternative 59-SS of RPL22B is sufficient to enhance
the overall splicing efficiency of this transcript, while deleting this
site results in an overall increase of unspliced RNAs. Under heat
shock
and
NMD
inactivation,
this
effect
was
even
more
prominent, as mutation of the alternative splice site to the
consensus resulted in the alternatively spliced product being the
major spliced species (Fig. 5B, lane 11). Thus, under heat shock
conditions, RPL22B transcripts bearing the consensus alternative
splice site mutation are now spliced almost exclusively at this site. Analysis of the mutant with a deletion of the alternative 59-SS
under heat shock conditions showed that the use of the normal 59-
SS is not increased at elevated temperatures when the competing
alternative 59-SS has been eliminated (Fig. 5A and 5B lane 12). This mutant shows a larger accumulation of unspliced RPL22B
transcript, hinting that the normal process of spliceosome assembly
is perturbed on this transcript during heat shock, possibly due to
the suboptimal 59-SS. To obtain a more quantitative assessment of
transcript levels, rather than just assessing the ratio between the
different spliced forms, we analyzed the same samples by northern
blot. Alternatively spliced species of RPL22B and GCR1 increase
during stress conditions Analysis of other mutants did not reveal any
major influence on the pattern of 59- or 39-SS selection. Like
SRC1, GCR1 exhibits two staggered 59 splice sites. However, unlike
for SRC1, Hub1p has no influence on their selection (Fig. 3). A final set of NMD targets are produced by the use of the two
most upstream 59-SS with a highly unusual alternative AUG 39-SS
in the intronic sequence (labeled *D and *E in Figs. 2 &3). Interestingly, these products were only detected in the absence of
Prp18p, suggesting that this factor is essential in preventing the use
of this non-canonical 39-SS. The use of this highly unusual AUG
39 splice site was unambiguously confirmed through sequencing
and RT-PCR analysis of RNAs derived from prp18Dupf1D samples
(Fig. S5). The ATPase Prp22p has been implicated in the fidelity of
39-SS selection [41]. Because Prp18p functions upstream from April 2014 | Volume 10 | Issue 4 | e1004249 7 PLOS Genetics | www.plosgenetics.org Non-productive Alternative Splicing in Yeast The alternative, suboptimal 59-splice site of RPL22B
contributes to the global down-regulation of RPL22B in
stress conditions The fact that the usage of the alternative splice site of
RPL22B is reduced in this strain while transcript levels are higher
overall argues against the hypothesis that alternative splice site
usage is the result of splicing errors occurring at low frequencies, as
if this were the case, one would expect higher levels of alternatively
spliced RPL22B transcripts upon its overexpression in the strain in
which the natural RPL22B promoter was swapped for the GAL
promoter. Under heat shock conditions, the use of the alternative
splice site was reduced 8.1 fold in the upf1D strain expressing
RPL22B under the control of the GAL promoter compared to the
upf1D strain expressing RPL22B from its natural promoter and
grown in galactose medium (Fig. 6, lanes 10 and 12). Thus,
alternative splicing regulation of RPL22B upon heat shock is
tightly linked to the identity of the RPL22B promoter, as switching
the identity of the promoter is sufficient to favor the use of the
normal 59-splice site. The mechanism by which the identity of the strain in normal conditions (75%; Fig. S13). In conclusion, these
results show that low splicing efficiency due to the suboptimal normal
and alternative 59 splice sites of RPL22B, combined with NMD
degradation of the unspliced and alternatively spliced forms contribute
to the general decrease in RPL22B levels as a means to rapidly halt
production of this ribosomal protein under various stress conditions. The alternative, suboptimal 59-splice site of RPL22B
contributes to the global down-regulation of RPL22B in
stress conditions To further analyze the importance of the alternative 59-splice
site of RPL22B on its splicing patterns and expression during
normal and stress conditions, we investigated the effect of April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 8 Non-productive Alternative Splicing in Yeast Figure 4. RT-PCR analysis of alternatively spliced products under stress conditions. A. Analysis of RPL22B in various stress conditions. Shown are the RT-PCR products obtained from the wild-type or upf1D mutant strain after growth in the following conditions: SDC, synthetic define
complete medium at 30uC; -AA, 10 minutes in SDC medium at 30uC lacking amino acid (-AA); 25uC, log phase at 25uC in YPD; H.S, 20 minutes at 42uC
in YPD; YPD: log phase at 30uC in YPD; LiCl, incubation with 300 mM LiCl in YPD at 30uC for 10 minutes; RAP control, see Materials and Methods; RAP,
treatment with Rapamycin for 20 minutes. B. RT-PCR analysis of GCR1 alternative splicing in heat-shock conditions. Labeling of the different species is
similar to that of Figures 2 and 3. doi:10.1371/journal.pgen.1004249.g004 Figure 4. RT-PCR analysis of alternatively spliced products under stress conditions. A. Analysis of RPL22B in various stress conditions. Shown are the RT-PCR products obtained from the wild-type or upf1D mutant strain after growth in the following conditions: SDC, synthetic define
complete medium at 30uC; -AA, 10 minutes in SDC medium at 30uC lacking amino acid (-AA); 25uC, log phase at 25uC in YPD; H.S, 20 minutes at 42uC
in YPD; YPD: log phase at 30uC in YPD; LiCl, incubation with 300 mM LiCl in YPD at 30uC for 10 minutes; RAP control, see Materials and Methods; RAP,
treatment with Rapamycin for 20 minutes. B. RT-PCR analysis of GCR1 alternative splicing in heat-shock conditions. Labeling of the different species is
similar to that of Figures 2 and 3. doi:10.1371/journal.pgen.1004249.g004 replacement of the normal RPL22B promoter by the GAL
promoter resulted in an increase in overall RPL22B transcript
levels, but also in a decrease in the use of the alternative 59-SS
(Fig. 6). Usage of the alternative 59-splice site of RPL22B is
influenced by promoter identity Ribosomal protein genes are known to be transcriptionally
regulated in stress conditions. To investigate the use of RPL22B 59-
SS selection independently from transcriptional inhibition under
heat shock, we replaced the natural RPL22B promoter with a
galactose-inducible promoter. The wild-type and upf1D strains
containing the natural RPL22B promoter showed no detectable
difference in RPL22B splicing patterns or expression when grown
in galactose containing medium (YPGal) compared to glucose-
containing medium (YPD) at 25uC (Fig. 6 lanes 1–4), either by
RT-PCR (top panel) or northern blot (bottom panel). Strikingly, April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 9 Non-productive Alternative Splicing in Yeast Figure 5. Effects of mutations of the RPL22B alternative 59 splice site on RPL22B splicing and expression in normal and stress
conditions. A. Organization of the RPL22B precursor, with the normal and alternative 59-splice sites. Shown are the mutations to the consensus
sequence (CS) GUAUGU or the deletion that entirely removes the GUUUGU sequence. B. Analysis of the effect of these mutations on RPL22B splicing
and expression at normal temperatures (25uC) or after a 20 min heat shock at 42uC. N, natural 59-splice site (GUUUGU); CS, consensus sequence
(GUAUGU); D = deletion of the alternative 59-splice site. Top panel: RT-PCR analysis. Bottom panel: northern blot analysis. US, *AS-59, and S indicate
the location of the products corresponding to the unspliced, alternatively spliced and normal spliced products, respectively. For the northern blot,
SCR1 was used as a loading control. C. Analysis of the effect of the RPL22B alternative splice site consensus mutation on RPL22B expression during
amino acid starvation. Shown is a northern blot of RNA samples extracted from the indicated strains grown at 30uC in normal synthetic define
complete (SDC) medium with amino acid (+) or in SDC medium lacking amino acid (2) for 10 minutes. Strains contained either the natural GUUUGU
sequence at the alternative 59-splice site of RPL22B, or the consensus GUaUGU sequence. The nucleotide mutated is highlighted in lower case. Labeling of the different species is similar to that of panel B. SCR1 was used as a loading control. doi:10.1371/journal.pgen.1004249.g005
Non productive Alternative Splicing in Yeast Figure 5. Effects of mutations of the RPL22B alternative 59 splice site on RPL22B splicing and expression in normal and stress
conditions. A. Organization of the RPL22B precursor, with the normal and alternative 59-splice sites. Usage of the alternative 59-splice site of RPL22B is
influenced by promoter identity Shown are the mutations to the consensus
sequence (CS) GUAUGU or the deletion that entirely removes the GUUUGU sequence. B. Analysis of the effect of these mutations on RPL22B splicing
and expression at normal temperatures (25uC) or after a 20 min heat shock at 42uC. N, natural 59-splice site (GUUUGU); CS, consensus sequence
(GUAUGU); D = deletion of the alternative 59-splice site. Top panel: RT-PCR analysis. Bottom panel: northern blot analysis. US, *AS-59, and S indicate
the location of the products corresponding to the unspliced, alternatively spliced and normal spliced products, respectively. For the northern blot,
SCR1 was used as a loading control. C. Analysis of the effect of the RPL22B alternative splice site consensus mutation on RPL22B expression during
amino acid starvation. Shown is a northern blot of RNA samples extracted from the indicated strains grown at 30uC in normal synthetic define
complete (SDC) medium with amino acid (+) or in SDC medium lacking amino acid (2) for 10 minutes. Strains contained either the natural GUUUGU
sequence at the alternative 59-splice site of RPL22B, or the consensus GUaUGU sequence. The nucleotide mutated is highlighted in lower case. Labeling of the different species is similar to that of panel B. SCR1 was used as a loading control. doi:10.1371/journal.pgen.1004249.g005 Discussion promoter influences alternative splice site selection is unclear, but
could be linked to the influence of the promoter on the speed of
transcription. Nevertheless, we can conclude from these results
that transcriptional down-regulation and the increased use of the
alternative 59-SS provide synergistic mechanisms to limit the
expression of RPL22B during stress, consistent with the global
down-regulation of ribosome biogenesis during stress conditions. promoter influences alternative splice site selection is unclear, but
could be linked to the influence of the promoter on the speed of
transcription. Nevertheless, we can conclude from these results
that transcriptional down-regulation and the increased use of the
alternative 59-SS provide synergistic mechanisms to limit the
expression of RPL22B during stress, consistent with the global
down-regulation of ribosome biogenesis during stress conditions. Contribution of splicing factors to alternative splice site
selection and splice site fidelity Contribution of splicing factors to alternative splice site
selection and splice site fidelity y
The analysis of double mutants in which splicing factor
mutations were combined with NMD inactivation revealed some
important and unexpected functions for these factors on alterna-
tive splice site selection. We found that the Nam8p and Mud1p
components are important for the selection of some, but not all of
the alternative 59-splice sites described here. In the case of
RPL22B, this requirement was likely due to the fact that the
alternative 59-SS possesses a suboptimal splicing sequence, and
therefore exhibits a weaker affinity for U1 binding, and a stronger
requirement for Mud1p and Nam8p that impact the efficiency of
U1 snRNP assembly on the alternative splice site. Strikingly, we
identified a new role for Prp17p in favoring the use of upstream,
branchpoint-proximal 39-SS. In all cases that we have analyzed,
Prp17p inactivation resulted in an increase in the use of the
downstream 39-SS. The mechanistic basis for this novel function
that we describe here for Prp17p in promoting branchpoint
proximal 39-SS is not fully understood. Because 39-SS close to the
branchpoint are often the first ones that are being used, this novel
function for Prp17p could be linked to promoting the ability of the
spliceosome to scan and recognize 39-SS close to the branchpoint,
or to unwind secondary structures that mask branchpoint-
proximal 39-SS. The absence of Prp17p would result in a higher
rate of misrecognition of 39-SS and in the use of more distal 39-SS. In addition, we found that the absence of Prp18p resulted in the
selection of a non-canonical AUG 39-SS in GCR1, and that this
atypical 39-SS was utilized to a greater extent during heat-shock,
revealing a unique function for Prp18p in suppressing usage of a Figure 6. Replacement of RPL22B promoter by the GAL promoter results in a decrease in alternative 59-splice site usage. Shown are
the products generated when growing the indicated strains (wild-type or upf1D that contained the natural RPL22B promoter or the GAL promoter
upstream RPL22B) in glucose (YPD) or galactose (YPGal)-containing media. Top panel, RT-PCR analysis. US, *AS 59, and S indicate the location of the
products corresponding to the unspliced, alternatively spliced and normal spliced products, respectively. Bottom Panel, Northern blot analysis. The
labeling of the different species is similar to that of the top panel. SCR1 was used as a loading control. doi:10.1371/journal.pgen.1004249.g006 Figure 6. A significant fraction of splicing events in S. cerevisiae
generates non-functional RNA or protein products In this study we show that the ensemble of transcripts generated
by splicing from the S. cerevisiae genome is highly complex. Most of
the splicing events that we have characterized in this study are April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 10 Non-productive Alternative Splicing in Yeast non-productive, either because they result in transcripts that are
targeted by NMD, or because the protein products generated
from these transcripts are predicted to be non-functional (e.g. GPI15). The large number of additional splice sites identified, and
their relaxed conservation (Fig. 1D) imply that the rules
governing splice selection are intrinsically more flexible than
previously thought. This is further illustrated by the finding that a
non-canonical AUG sequence in GCR1 can be used as a 39-SS in
the absence of Prp18p (Fig. 3). In some cases, non-productive
alternatively spliced transcripts accumulate only at low levels (e.g. GCR1, GPI15, Fig. 3). However, for other genes such as TFC3,
the alternatively spliced non-productive transcripts represent a
significant fraction (close to 30%) of all RNAs generated from this
locus. Thus, non-productive splicing can significantly limit the
expression of these genes. This was further demonstrated by
mutagenesis of the non-productive splice site of RPL22B, as
changing this site to a consensus sequence was sufficient to
increase the splicing efficiency and the expression of this gene
(Fig. 5B). Thus, the presence of alternative and sometimes sub-
optimal splice sites that compete with the normal splice site
contributes to an overall decrease in the amount of productively-
spliced transcripts. Because the overlap in the alternative splicing
events detected in all three NMD-deficient strains was limited
(Fig. 1C), and because we detected by RT-PCR some alternative
splicing events that escaped detection by RNA-Seq (e.g. GCR1), it
is likely that we have not exhaustively identified the ensemble of
splice sites that can be used by S. cerevisiae, and that additional
splice sites will be identified by deeper sequencing or systematic
RT-PCR analysis. Spliceosome errors or bona-fide regulations? The widespread occurrence of non-productive splice site usage
described in this study begs the question of whether the use of
these splice sites is the result of mistakes by the spliceosome, which
occur at low frequency (as one might suggest based on their
weaker consensus sequences) or whether they correspond to sites
that have been selected throughout evolution for regulatory
purposes. The sequence of some of these intronic, non-productive
splice sites is conserved across closely related yeast species (Fig. S8
and S9), which, given the low conservation of intronic sequences in
general, argues that this might reflect some degree of functional
relevance. In addition, there is no obvious correlation between
transcript levels and the occurrence of alternative splicing events
(Fig. 1D), which argues against the suggestion that most of the
alternative splicing events that we have mapped arise from low
fidelity splicing events or errors that occur randomly, and which
would be expected to be more frequently detected in highly
abundant transcripts. Also, replacement of the RPL22B gene
promoter results in higher transcript levels but reduces the usage of
the alternative 59-splice site of RPL22B (Fig. 6), providing another
independent argument to suggest that the level of usage of
alternative splice sites is not solely a reflection of overall transcript
abundance. Finally, we show that the use of some of these
alternative splice sites can be up-regulated during stress conditions
(RPL22B, GCR1), and that this increased use participates in the
down-regulation of RPL22B in stress conditions. Thus, this
phylogenetically conserved, alternative, non-productive 59-SS of
RPL22B is functionally important because it contributes to the
down-regulation of RPL22B during stress. This is shown by the
fact that changing this sequence to a consensus sequence results in
a significant increase in transcript levels upon NMD inactivation
during stress (Fig. 5). The transcriptional down-regulation of
ribosomal proteins during stress has been documented previously
[45]. We show here that the promoter of the RPL22B gene is
essential not only because it drives transcriptional repression
during stress, but also because it controls the switch in 59-SS
selection that contributes to the overall repression of RPL22B
during heat-shock. Thus, a combination of transcriptional and
post-transcriptional
regulations,
through
splicing
inhibition
[43,44], degradation of unspliced RNAs by NMD [12,47] and
use of non-productive splice sites (this study) contributes to the
repression of ribosomal protein production during stress. Spliceosome errors or bona-fide regulations? While
several non-RPG transcripts analyzed in these stress conditions did
not shown any changes, GCR1 did exhibit a change in the use of
alternative splice sites during stress (Fig. 4B). This result raises the
possibility that other intron-containing genes may be regulated
similarly by alternative splicing as a function of different
environmental growth conditions. Overall our study has revealed
that the pattern of splicing events in the model eukaryote S. cerevisiae is highly complex, but masked by NMD-mediated
degradation. Given the recent report that another single cell
eukaryote, S.pombe shows alternative splicing patterns conserved in
higher eukaryotes [50], these observations suggest that alternative
splicing provides an important contribution to genetic regulations
and adaptations to environmental changes in unicellular eukary-
H
i
h
f S
i i
h
f l
i
li Contribution of splicing factors to alternative splice site
selection and splice site fidelity Replacement of RPL22B promoter by the GAL promoter results in a decrease in alternative 59-splice site usage. Shown are
the products generated when growing the indicated strains (wild-type or upf1D that contained the natural RPL22B promoter or the GAL promoter
upstream RPL22B) in glucose (YPD) or galactose (YPGal)-containing media. Top panel, RT-PCR analysis. US, *AS 59, and S indicate the location of the
products corresponding to the unspliced, alternatively spliced and normal spliced products, respectively. Bottom Panel, Northern blot analysis. The
labeling of the different species is similar to that of the top panel. SCR1 was used as a loading control. doi:10.1371/journal.pgen.1004249.g006 April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 11 Non-productive Alternative Splicing in Yeast sites have evolved towards fine tuning transcript levels, rather than
generating proteome diversity as shown in higher eukaryotes. non-canonical 39-SS. This function for Prp18p is independent
from Prp22p’s function in proofreading 39-SS [41], but might
complement its role to ensure the overall proper fidelity of 39-SS
selection. While we have demonstrated this function for GCR1
only, a full genomic analysis of 39-SS usage in the absence of
Prp18p might reveal further examples of non-canonical 39-SS
being used. Yeast culture and RNA analysis y
Yeast strains were grown at 25uC in YPD medium, unless
indicated otherwise in the figures. For heat shock treatment,
strains were pre-grown in YPD at 25uC, spun down in 50 mL
Falcon tubes, resuspended in pre-warmed YPD medium and heat
shocked for 20 min before harvesting. For LiCl treatment, yeast
strains were grown to mid-log phase in YPD rich media at 30uC,
harvested by centrifugation in 50 mL Falcon tubes, washed once
with pre-warmed 50 mL of YPD+300 mM LiCl before being
resuspended
in
pre-warmed
YPD
with
300 mM
LiCl
for
10 minutes. For Rapamycin treatment, cells were grown to mid-
log phase in rich media (at 30uC), and rapamycin from a stock
solution of 1 mg/mL in 90% ethanol, 10% Tween-20 was added
to a final concentration of 200 ng/mL and cells were incubated for
20 minutes. The same volume of 90% ethanol, 10% Tween-20
solution used for the rapamycin treatment was added to the
negative control. Sample preparation and RNA sequencing was
performed by Illumina. RT-PCR analysis and northern blot was
performed as described [21]. Mapping reads High throughput sequencing data have been deposited in the
GEO database (accession GSE55213). All sequence files were
aligned against the 2008 SGD assembly of the Saccharomyces
cerevisiae
genome. The
novoalign
software
package
(www. novocraft.com) and the BLAT alignment tool [22] were used to
align 75 base pair reads in two steps. In the first step, sequences
were aligned with novoalign allowing for up to four mismatches
and no gaps. In the second step, sequences that failed to align in
the first step were aligned with BLAT allowing three mismatches
and gaps up to 20000 nucleotides in length. A sequence was kept
for further analysis if it mapped with equal score to at most two
genomic locations and did not contain a gap smaller than ten
nucleotides. Intronic sequences counts Intronic sequence expression representative of unspliced RNAs
was quantified for each ICG by summing reads that aligned to
introns and exon-intron boundaries. Values between samples were
normalized by total mapped reads to account for lane effects. p-
values were computed by modeling each ICG wild-type count as a
poisson random variable and calculating the probability of
observing each mutant count if it were drawn from the same
distribution. Supporting Information Figure S1
Quantification of the SRC1-L and SRC1-S isoforms
in wild-type, upf1D and splicing mutants. Shown is the percentage
of the SRC1-L and SRC1-S transcripts in various strains. Values
shown are the average and standard deviations obtained from RT-
PCR experiments of three independent cultures for each strain. (TIF) Figure S2
Quantification of the usage of the normal and
alternative 59-splice sites of RPL22B in wild-type, upf1D and
splicing mutants. Shown is the percentage of transcripts spliced at
the normal 59-splice site (red) and at the alternative 59-splice site
(blue). Values shown are the average and standard deviations
obtained from RT-PCR experiments of three independent
cultures for each strain. (TIF) Figure S9
Conservation of the intronic alternative 39-SS in
TAN1. A. Screen capture of the web browser showing the RNA-
Seq reads mapped for TAN1 that use the intronic alternative 39-SS
in black, and the sequence conservation in closely related yeast
species as blue peaks. B. Zoomed in view of the conservation of the
sequence of the alternative intronic AAG 39-SS. (PDF) Figure S3
Quantification of the usage of the normal and
alternative 39-splice sites of TFC3 in wild-type, upf1D and splicing
mutants. Shown is the percentage of transcripts spliced at the
normal 39-splice site (blue) and at the alternative 59-splice site (red). Values shown are the average and standard deviations obtained
from RT-PCR experiments of three independent cultures for each
strain. (TIF) Figure S10
Quantification of the usage of the alternative 59-
splice sites of RPL22B in various normal media (YPD, SDC) or in
stress conditions (Heat shock, amino acid starvation). Values
shown are the average and standard deviations obtained from RT-
PCR experiments of three independent cultures for each strain. (TIF) Figure S11
RT-PCR analysis of the spliced products of TAN1
and TFC3 under stress conditions. Shown are the products for the
unspliced (US), normal spliced product (S), and the alternatively
spliced species (AS) described in Figure 2. (TIF) Figure S4
Quantification of the usage of the two alternative 39-
splice sites of TAN1 in wild-type, upf1D and splicing mutants. Shown is the percentage of transcripts spliced at the alternative 39-
splice site #1 (blue) or #2 (red) compared to all the spliced
transcripts. Values shown are the average and standard deviations
obtained from RT-PCR experiments of three independent
cultures for each strain. Splice site consensus sequence Figure S7
RT-PCR analysis of GCR1 splicing in the prp18 and
prp22-1 mutant strains. The identity of the different spliced
products is labeled according to Figure 2. (TIF) Consensus sequences for 59 and 39 ends of both canonical splice
sites and alternative splice sites were represented as sequence
logos. Sequence logos were constructed using the MATLAB
(MathWorks) seqlogo function. Figure S8
Conservation of the intronic alternative 59-SS in
RPL22B. A. Screen capture of the web browser showing the RNA-
Seq reads mapped for RPL22B that use the alternative 59-SS in
black, and the sequence conservation in closely related yeast
species as blue peaks. The peak showing conservation of the
intronic alternative 59splice site is shown on the right, since
the gene is encoded on the Crick strand. B. Zoomed in view of the
conservation of the sequence of the alternative 59-SS (ACAAC
sequence because of the Crick Strand). (PDF) Quantification of alternative splicing events Alternative splicing events were defined as splicing events that
are within ICGs and are supported by sequencing but that are not
annotated in the Saccharomyces Genome Database (SGD). Counts of
total alternative splicing events and PTC-generating alternative
splicing events were quantified by summing all unique alternative
splicing events in each sample. To determine if an alternative
splicing event is PTC-generating we constructed the splice
product’s sequence using the novel splicing event in the otherwise
canonical transcript sequence. Counts between samples were
normalized by sequencing depth. p-values were calculated by
modeling the wild-type count as a poisson random variable and
calculating the probability of observing each mutant’s count
for both total alternative splicing events and PTC-generating April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 12 Non-productive Alternative Splicing in Yeast alternative splicing events. Venn diagrams of agreement between
samples were generated using BioVenn [51]. standard deviations obtained from RT-PCR experiments of three
independent cultures for each strain. (TIF) standard deviations obtained from RT-PCR experiments of three
independent cultures for each strain. (TIF) Supporting Information (TIF) Figure S12
Quantitation of the use of the alternative 59-splice
site of RPL22B under normal growth conditions (25uC) and after a
20 min heat shock at 42uC. Plotted are the amount of transcript
spliced at the alternative splice site divided by the values obtained
for all spliced species for the indicated strains. Shown are the
average of 4 to 5 independent experiments with the standard
deviations. Figure S5
Validation of the use of the AUG alternative 39 splice
site of GCR1 by RT-PCR. Sequencing of the cloned *D and *E
cDNAs determined the location of the splice junction, while
sequencing of unspliced cDNAs was used to confirm that this
unusual alternative 39-SS was indeed AUG, and not a SNP or other
mutation of the GCR1 gene that would have converted it into an
AAG. RT-PCR confirmation of the use of this AUG 39-SS was
performed using reverse primers spanning the splice junction to
specifically amplify distinct splicing events; either associated with
*D, *E, or unspliced. The use of the AUG 39 SS was also confirmed
using an intronic reverse primer just downstream of the AUG
sequence and detected *D, *E, and unspliced products, as predicted
(Fig. S1). A. RT-PCR strategy. All PCR include the same forward
primer For, and various reverse primers that hybridize to the
indicated regions of GCR1. B. RT-PCR data. Shown are the PCR
products obtained from the different reverse primers shown in A. (TIF) References 17. Mishra SK, Ammon T, Popowicz GM, Krajewski M, Nagel RJ, et al. (2011)
Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative
splicing. Nature 474: 173–178. 1. Isken O, Maquat LE (2007) Quality control of eukaryotic mRNA: safeguarding
cells from abnormal mRNA function. Genes Dev 21: 1833–1856. 2. Kervestin S, Jacobson A (2012) NMD: a multifaceted response to premature
translational termination. Nat Rev Mol Cell Biol 13: 700–712. 18. Meyer M, Plass M, Perez-Valle J, Eyras E, Vilardell J (2011) Deciphering 39ss
selection in the yeast genome reveals an RNA thermosensor that mediates
alternative splicing. Mol Cell 43: 1033–1039. translational termination. Nat Rev Mol Cell Biol 13: 700–712. 3. Green RE, Lewis BP, Hillman RT, Blanchette M, Lareau LF, et al. (2003)
Widespread predicted nonsense-mediated mRNA decay of alternatively-spliced
transcripts of human normal and disease genes. Bioinformatics 19 Suppl 1: i118–
121. 19. Plass M, Codony-Servat C, Ferreira PG, Vilardell J, Eyras E (2012) RNA
secondary structure mediates alternative 39ss selection in Saccharomyces
cerevisiae. RNA 18: 1103–1115. 4. Lareau LF, Inada M, Green RE, Wengrod JC, Brenner SE (2007) Unproductive
splicing of SR genes associated with highly conserved and ultraconserved DNA
elements. Nature 446: 926–929. 20. Marshall AN, Montealegre MC, Jimenez-Lopez C, Lorenz MC, van Hoof A
(2013) Alternative splicing and subfunctionalization generates functional
diversity in fungal proteomes. PLoS Genet 9: e1003376. 5. Ni JZ, Grate L, Donohue JP, Preston C, Nobida N, et al. (2007) Ultraconserved
elements are associated with homeostatic control of splicing regulators by
alternative splicing and nonsense-mediated decay. Genes Dev 21: 708–718. 21. Kawashima T, Pellegrini M, Chanfreau GF (2009) Nonsense-mediated mRNA
decay mutes the splicing defects of spliceosome component mutations. RNA 15:
2236–2247. 6. Mendell JT, Sharifi NA, Meyers JL, Martinez-Murillo F, Dietz HC (2004)
Nonsense surveillance regulates expression of diverse classes of mammalian
transcripts and mutes genomic noise. Nat Genet 36: 1073–1078. 22. Kent WJ (2002) BLAT–the BLAST-like alignment tool. Genome Res 12: 656–
664. 23. Liao XC, Tang J, Rosbash M (1993) An enhancer screen identifies a gene that
encodes the yeast U1 snRNP A protein: implications for snRNP protein function
in pre-mRNA splicing. Genes Dev 7: 419–428. 7. Chan WK, Huang L, Gudikote JP, Chang YF, Imam JS, et al. (2007) An
alternative branch of the nonsense-mediated decay pathway. EMBO J 26: 1820–
1830. 24. Neubauer G, Gottschalk A, Fabrizio P, Seraphin B, Luhrmann R, et al. g
(XLSX) Table S5
List of intron-containing genes for which alternative
splicing events were detected. Shown is the list of intron-
containing genes for which alternative splicing junctions were
detected in at least one of the strains sequenced. Shown is the
number of splice junction sequences counts for the normal (blue)
and alternative (green) splicing events. For the alternative splicing
events, the 59 and/or 39 splice sites which differ from the normal
splice sites are marked by an asterisk. The position of these splice
sites on each chromosome is also indicated. The adjusted number
of reads obtained from each strain (RPKM) and the sequence of
the normal and alternative 59- and 39-splice sites is shown for each
of these genes. Acknowledgments We thank J. Staley for insights, discussions and comments on the
manuscript and K. Roy for critical reading of the manuscript. References (1997)
Identification of the proteins of the yeast U1 small nuclear ribonucleoprotein
complex by mass spectrometry. Proc Natl Acad Sci U S A 94: 385–390. 8. Weischenfeldt J, Damgaard I, Bryder D, Theilgaard-Monch K, Thoren LA, et
al. (2008) NMD is essential for hematopoietic stem and progenitor cells and for
eliminating by-products of programmed DNA rearrangements. Genes Dev 22:
1381–1396. 25. Puig O, Gottschalk A, Fabrizio P, Seraphin B (1999) Interaction of the U1
snRNP with nonconserved intronic sequences affects 59 splice site selection. Genes Dev 13: 569–580. 9. He F, Peltz SW, Donahue JL, Rosbash M, Jacobson A (1993) Stabilization and
ribosome association of unspliced pre-mRNAs in a yeast upf1- mutant. Proc Natl
Acad Sci U S A 90: 7034–7038. 26. Gottschalk A, Tang J, Puig O, Salgado J, Neubauer G, et al. (1998) A
comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals
five novel proteins. RNA 4: 374–393. 10. Mitrovich QM, Anderson P (2000) Unproductively spliced ribosomal protein
mRNAs are natural targets of mRNA surveillance in C. elegans. Genes Dev 14:
2173–2184. 27. Umen JG, Guthrie C (1995) Prp16p, Slu7p, and Prp8p interact with the 39 splice
site in two distinct stages during the second catalytic step of pre-mRNA splicing. RNA 1: 584–597. 11. Jaillon O, Bouhouche K, Gout JF, Aury JM, Noel B, et al. (2008) Translational
control of intron splicing in eukaryotes. Nature 451: 359–362. 28. Aronova A, Bacı´kova´ D, Crotti LB, Horowitz DS, Schwer B (2007) Functional
interactions between Prp8, Prp18, Slu7, and U5 snRNA during the second step
of pre-mRNA splicing. RNA 13: 1437–1444. 12. Sayani S, Janis M, Lee CY, Toesca I, Chanfreau GF (2008) Widespread impact
of nonsense-mediated mRNA decay on the yeast intronome. Mol Cell 31: 360–
370. 13. Sayani S, Chanfreau GF (2012) Sequential RNA degradation pathways provide
a fail-safe mechanism to limit the accumulation of unspliced transcripts in
Saccharomyces cerevisiae. RNA 18: 1563–1572. 29. Villa T, Guthrie C (2005) The Isy1p component of the NineTeen complex
interacts with the ATPase Prp16p to regulate the fidelity of pre-mRNA splicing. Genes Dev 19: 1894–1904. 14. Vijayraghavan U, Company M, Abelson J(1989) Isolation and characterization
of pre-mRNA splicing mutants of Saccharomyces cerevisiae. Genes Dev 3:
1206–1216. 30. Saha D, Banerjee S, Bashir S, Vijayraghavan U (2012) Context dependent
splicing functions of Bud31/Ycr063w define its role in budding and cell cycle
progression. Biochem Biophys Res Commun 424: 579–585. 15. Author Contributions Conceived and designed the experiments: TK SD JG MP GFC. Performed
the experiments: TK JG. Analyzed the data: TK SD JG MP GFC. Wrote
the paper: TK GFC. g
(XLSX) (TIF) Figure
S13
Quantitation of the use of the normal and
alternative
59-splice
site of RPL22B
under normal
growth
conditions in minimal medium (SDC) and after amino acid
starvation (-AA) for the strains expressing the natural (N)
GUUUGU sequence at the alternative 59 splice site of RPL22B,
or the consensus (CS) GUAUGU sequence in the context of wild-
type UPF1 (WT) or when UPF1 has been deleted (D). Plotted are
the amount of transcript spliced at the normal and alternative
splice sites divided by the values obtained for all spliced species. Shown are the average of 3 independent experiments with the
standard deviations. Table S1
Statistics of RNA-Seq analysis sequence alignments. (XLSX) Figure S6
Quantification of the abundance of the major
alternatively spliced forms of GCR1 in wild-type, upf1D and
splicing mutants. Shown is the percentage of the *D (blue), *A (red)
or *C (green) spliced forms. Values shown are the average and Figure S6
Quantification of the abundance of the major
alternatively spliced forms of GCR1 in wild-type, upf1D and
splicing mutants. Shown is the percentage of the *D (blue), *A (red)
or *C (green) spliced forms. Values shown are the average and Table S2
Number of alternative splicing events detected in wild-
type and NMD-deficient strains. (XLSX) Table S2
Number of alternative splicing events detected in wild-
type and NMD-deficient strains. (XLSX) April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 13 Non-productive Alternative Splicing in Yeast Table S3
Mapping of RNA-Seq reads in wild-type and NMD-
deficient strains in various genomic elements and in intron-
containing genes. (XLSX) Table S6
List of alternative proteins potentially generated by
alternative splicing in wild-type or NMD mutants. For each open
reading frame, a portion of the normal protein sequence is shown
on the first line, and the sequence that differs upon the alternative
splicing event is shown below. Amino acids maintained between
the two forms are indicated in red. Amino acids that differ
between the two forms are highlighted in bold and black. In the
case of splicing events inducing a deletion, a delta sign has been
added with a number corresponding to the number of amino acid
deleted. The numbers of the first and last amino acids shown is
indicated before and after each protein sequence, respectively. The numbers in brackets that follow each protein sequence
correspond to the number of reads for the splice junctions in the
wild-type strain, upf1D, upf2D and upf3D mutants. (DOCX) Table
S4
List
of
intron-containing
genes
for
which
no
alternative splicing events were detected. Shown is the list of
intron-containing genes for which no alternative splicing junctions
were detected in any of the strains sequenced. The adjusted
number of reads obtained from each strain (RPKM) and the
sequence of the 59- and 39-splice sites is shown for each of these
genes. Non-productive Alternative Splicing in Yeast 33. Crotti LB, Horowitz DS (2009) Exon sequences at the splice junctions affect
splicing fidelity and alternative splicing. Proc Natl Acad Sci U S A 106: 18954–
18959. 43. Bergkessel M, Whitworth GB, Guthrie C (2011) Diverse environmental stresses
elicit distinct responses at the level of pre-mRNA processing in yeast. RNA 17:
1461–1478. 34. Zhou Y, Chen C, Johansson MJ (2013) The pre-mRNA retention and splicing
complex controls tRNA maturation by promoting TAN1 expression. Nucleic
Acids Res 41: 5669–5678. 44. Pleiss JA, Whitworth GB, Bergkessel M, Guthrie C (2007) Rapid, transcript-
specific changes in splicing in response to environmental stress. Mol Cell 27:
928–937. 35. Yan BC, Westfall BA, Orlean P (2001) Ynl038wp (Gpi15p) is the Saccharomyces
cerevisiae homologue of human Pig-Hp and participates in the first step in
glycosylphosphatidylinositol assembly. Yeast 18: 1383–1389. 45. Li B, Nierras CR, Warner JR (1999) Transcriptional elements involved in the
repression of ribosomal protein synthesis. Mol Cell Biol 19: 5393–5404. 46. Gasch AP, Spellman PT, Kao CM, Carmel-Harel O, Eisen MB, et al. (2000)
Genomic expression programs in the response of yeast cells to environmental
changes. Mol Biol Cell 11: 4241–4257. g y
y p
p
y
y
36. Muhlrad D, Parker R (1999) Aberrant mRNAs with extended 39 UTRs are
substrates for rapid degradation by mRNA surveillance. RNA 5: 1299–1307. 37. Amrani N, Ganesan R, Kervestin S, Mangus DA, Ghosh S, et al. (2004) A faux
39-UTR promotes aberrant termination and triggers nonsense-mediated mRNA
decay. Nature 432: 112–118. 47. Garre E, Romero-Santacreu L, Barneo-Munoz M, Miguel A, Perez-Ortin JE, et
al. (2013) Nonsense-mediated mRNA decay controls the changes in yeast
ribosomal protein pre-mRNAs levels upon osmotic stress. PLoS One 8: e61240. 38. Uemura H, Jigami Y (1995) Mutations in GCR1, a transcriptional activator of
Saccharomyces cerevisiae glycolytic genes, function as suppressors of gcr2
mutations. Genetics 139: 511–521. 48. Yost HJ, Lindquist S (1991) Heat shock proteins affect RNA processing during
the heat shock response of Saccharomyces cerevisiae. Mol Cell Biol 11: 1062–
1068. 39. Holland MJ, Yokoi T, Holland JP, Myambo K, Innis MA (1987) The GCR1
gene encodes a positive transcriptional regulator of the enolase and
glyceraldehyde-3-phosphate dehydrogenase gene families in Saccharomyces
cerevisiae. Mol Cell Biol 7: 813–820. 49. Vogel JL, Parsell DA, Lindquist S (1995) Heat-shock proteins Hsp104 and
Hsp70 reactivate mRNA splicing after heat inactivation. Curr Biol 5: 306–317. 50. References Wahl MC, Will CL, Luhrmann R (2009) The spliceosome: design principles of a
dynamic RNP machine. Cell 136: 701–718. 31. Rodriguez-Navarro S, Igual JC, Perez-Ortin JE (2002) SRC1: an intron-
containing yeast gene involved in sister chromatid segregation. Yeast 19: 43–
54. 16. Grund SE, Fischer T, Cabal GG, Antunez O, Perez-Ortin JE, et al. (2008) The
inner nuclear membrane protein Src1 associates with subtelomeric genes and
alters their regulated gene expression. J Cell Biol 182: 897–910. 32. Chanfreau GF (2010) A dual role for RNA splicing signals. EMBO Rep 11: 720–
721. April 2014 | Volume 10 | Issue 4 | e1004249 April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org April 2014 | Volume 10 | Issue 4 | e1004249 14 Non-productive Alternative Splicing in Yeast Awan AR, Manfredo A, Pleiss JA (2013) Lariat sequencing in a unicellular yeast
identifies regulated alternative splicing of exons that are evolutionarily conserved
with humans. Proc Natl Acad Sci U S A 110: 12762–12767 40. Clifton D, Fraenkel DG (1981) The gcr (glycolysis regulation) mutation of
Saccharomyces cerevisiae. J Biol Chem 256: 13074–13078. with humans. Proc Natl Acad Sci U S A 110: 12762–12767 51. Hulsen T, de Vlieg J, Alkema W (2008) BioVenn - a web application for the
comparison and visualization of biological lists using area-proportional Venn
diagrams. BMC Genomics 9: 488. y
J
41. Mayas RM, Maita H, Staley JP (2006) Exon ligation is proofread by the DExD/
H-box ATPase Prp22p. Nat Struct Mol Biol 13: 482–490. 41. Mayas RM, Maita H, Staley JP (2006) Exon ligation is proofr H-box ATPase Prp22p. Nat Struct Mol Biol 13: 482–490. 42. James SA, Turner W, Schwer B (2002) How Slu7 and Prp18 cooperate in the
second step of yeast pre-mRNA splicing. RNA 8: 1068–1077. April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 15
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Endothelial Insulin Receptors Promote VEGF-A Signaling via ERK1/2 and Sprouting Angiogenesis
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Endocrinology
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Endothelial Insulin Receptors Promote
VEGF-A Signaling via ERK1/2 and Sprouting
Angiogenesis Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6284300 by guest on 01 March 2023 Andrew M. N. Walker,1,* Nele Warmke,1,* Ben Mercer,1,* Nicole T. Watt,1
Romana Mughal,1 Jessica Smith,1 Stacey Galloway,1Natalie J. Haywood,1
Taha
Soomro,1,2
Kathryn
J. Griffin,1
Stephen
B. Wheatcroft,1
Nadira Y. Yuldasheva,1 David J. Beech,1 Peter Carmeliet,3 Mark T. Kearney,1
and Richard M. Cubbon1 Andrew M. N. Walker,1,* Nele Warmke,1,* Ben Mercer,1,* Nicole T. Watt,1
Romana Mughal,1 Jessica Smith,1 Stacey Galloway,1Natalie J. Haywood,1
Taha
Soomro,1,2
Kathryn
J. Griffin,1
Stephen
B. Wheatcroft,1
Nadira Y. Yuldasheva,1 David J. Beech,1 Peter Carmeliet,3 Mark T. Kearney,1
and Richard M. Cubbon1 1Leeds Institute of Cardiovascular and Metabolic Medicine, The University of Leeds, Leeds LS2 9JT, UK;
2Imperial College Ophthalmology Research Group, Western Eye Hospital, London NW1 5QH, UK; and
3Laboratory of Angiogenesis and Vascular Metabolism, Center for Cancer Biology, Vlaams Instituut voor
Biotechnologie (VIB), Department of Oncology, University of Leuven, Leuven 3000, Belgium ORCiD numbers: 0000-0002-6741-9012 (S. B. Wheatcroft); 0000-0001-7844-3600 (R. M. Cubbon). *These authors contributed equally. Abbreviations: Con, control; EC, endothelial cell; EdU, 5-ethynyl-2′-deoxyuridine; eNOS, endothelial nitric oxide synthase;
ERK, extracellular signal-regulated kinase; FCS, fetal calf serum; HUVEC, human umbilical vein endothelial cell; Insr, insulin
receptor; PEC, pulmonary endothelial cell; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth
factor receptor 2; WT, wild-type Received: 7 April 2021; Editorial Decision: 20 May 2021; First Published Online: 25 May 2021; Corrected and Typeset:
24 June 2021. ISSN Online 1945-7170
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium,
provided the original work is properly cited.
© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. https://academic.oup.com/endo Endocrinology, 2021, Vol. 162, No. 8, 1–15
doi:10.1210/endocr/bqab104
Research Article Endocrinology, 2021, Vol. 162, No. 8, 1–15
doi:10.1210/endocr/bqab104
Research Article Research Article Research Article Abstract Endothelial insulin receptors (Insr) promote sprouting angiogenesis, although the under-
pinning cellular and molecular mechanisms are unknown. Comparing mice with whole-
body insulin receptor haploinsufficiency (Insr+/-) against littermate controls, we found
impaired limb perfusion and muscle capillary density after inducing hind-limb ischemia;
this was in spite of increased expression of the proangiogenic growth factor Vegfa. Insr+/-
neonatal retinas exhibited reduced tip cell number and branching complexity during
developmental angiogenesis, which was also found in separate studies of mice with
endothelium-restricted Insr haploinsufficiency. Functional responses to vascular endo-
thelial growth factor A (VEGF-A), including in vitro angiogenesis, were also impaired
in aortic rings and pulmonary endothelial cells from Insr+/- mice. Human umbilical vein
endothelial cells with shRNA-mediated knockdown of Insr also demonstrated impaired
functional angiogenic responses to VEGF-A. VEGF-A signaling to Akt and endothelial ni-
tric oxide synthase was intact, but downstream signaling to extracellular signal-reduced https://academic.oup.com/endo Endocrinology, 2021, Vol. 162, No. 8 2 kinase 1/2 (ERK1/2) was impaired, as was VEGF receptor-2 (VEGFR-2) internalization,
which is required specifically for signaling to ERK1/2. Hence, endothelial insulin recep-
tors facilitate the functional response to VEGF-A during angiogenic sprouting and are
required for appropriate signal transduction from VEGFR-2 to ERK1/2. Key Words: angiogenesis, endothelial, ERK, insulin, vascular, VEGF Key Words: angiogenesis, endothelial, ERK, insulin, vascular, VEGF Insulin receptor (Insr) haploinsufficient (Insr+/-) mice
As we have previously described (15), Insr+/- mice (also
known as IRKO) were obtained from the Medical Research
Council Mammalian Genetics Unit (Harwell, Oxfordshire,
UK), and were maintained as heterozygotes on a C57BL/6J
background. Insr+/- were compared with age-matched wild-
type (WT) littermates. Insulin is a primary regulator of systemic carbohydrate
and lipid metabolism (1), but also has an important role
in vascular function, for example promoting vasodilation
and tissue perfusion (2). Indeed, loss of endothelial insulin
receptors, or perturbation of their signaling function, in-
duces endothelial dysfunction, hypertension, and athero-
sclerosis (3-5). Sprouting angiogenesis, the phenomenon
of new capillary formation, is another fundamental ele-
ment of vascular biology that is intrinsically linked to
metabolism (6). In this highly orchestrated and conserved
process, endothelial “tip cells” emerge from existing ves-
sels, followed by proliferating stalk cells that extend the
sprout and form a lumenized vessel; these neo-vessels then
anastomose into an immature network that remodels to
meet local demands for oxygen and metabolite transport
(7). Tissue collection and processing Retinal angiogenesis was assessed in postnatal day 5 pups by
precisely following the protocol of Pitulescu et al (18). In brief,
all pups from at least 3 litters were included in each experi-
ment, with analysis blinded to the results of genotyping data. Both eyes were processed identically with a mean value from
these to represent that pup. Vascular endothelium was stained
with Isolectin B4 conjugated with Alexa Fluor 488 (I21411;
Thermo Fisher Scientific, Warrington, UK). Costaining with
a rabbit anti-mouse anti-Collagen IV antibody (19) followed
by an Alexa Fluor-647 conjugated goat anti-rabbit antibody
(20) was used to visualize the vascular basement membrane. To define cell proliferation, pups were injected with 125 μg
5-ethynyl-2′-deoxyuridine (EdU) 2 hours before tissue col-
lection; this was stained with Alexa Fluor 647 azide using
Click-iT technology (C10640; Thermo Fisher Scientific). Endothelial cell-specific Insr haploinsufficient (ECInsr+/-)
mice ECInsr+/- mice were generated by crossing mice that have
loxP sites flanking exon 4 of the insulin receptor (Line
006955, The Jackson Laboratory, Bar Harbor, ME) (16)
with mice possessing a Cre transgene driven by the Tie2
promoter/enhancer (Line 004128, The Jackson Laboratory)
(17) and were maintained on a C57BL/6J background. ECInsr+/- were compared with age-matched Cre-negative
Insrlox/+ littermates, which are referred to as WT. Abstract Insulin has been reported to promote angiogenesis
in vitro and in vivo; (8-12) these studies found pro-
angiogenic effects in nanomolar concentrations in vitro,
but did not explore more physiological picomolar concen-
trations. In vivo, insulin receptor expression is known to
be enriched in human tumor endothelial tip cells (13), and
loss of endothelial insulin receptors has been shown to
impair angiogenesis in murine retinopathy (14). However,
it remains unclear how endothelial insulin receptors influ-
ence the cellular and molecular processes of angiogenesis
and so we set out to define this. Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6284300 by guest on 01 March 2023 Materials and Methods
Animal models All experimental mice were kept in a conventional
animal facility with a 12-hour light/dark cycle and
received a standard chow diet. Genotyping was per-
formed using PCR of ear notch (or tail-tip for pups)
genomic DNA by Transnetyx (Cordova, TN). All pro-
cedures were approved by the Animal Welfare and
Ethical Review Committee at the University of Leeds
and were conducted in accordance with The Animals
(Scientific Procedures) Act of 1986 Amendment
Regulations 2012 (SI 2012/3039) under United
Kingdom Home Office project licenses PL40/3523
and P144DD0D6. Confocal microscopy and image analysis
Microscopy was performed using a Zeiss LSM880 up-
right confocal microscope with 10×/0.3NA, 20×/0.8NA, Endocrinology, 2021, Vol. 162, No. 8 3 and 40×/1.4NA objectives and Zen software (Carl Zeiss
Microscopy Ltd, Cambridge, UK). Tile scanning was used
to image entire retinal segments with the 20× objective and
maximum intensity projection of 5 consecutive 1 Airy unit
thickness z-slices was used with the 40× to define tip cells
and filopodia. Image analysis used ImageJ (NIH, Bethesda,
MD). Radial outgrowth was defined as the distance from
the optic disc periphery to the emerging vascular front
measured at 12 points in each retina. Vascular area was de-
fined by binary thresholding of the Isolectin B4 signal and
expressed as a percentage of the region of interest, bounded
either by the peripheral or central half of the vascularized
area. Vascular branching was quantified in multiple 200 ×
200-μm regions of interest placed between arteries and
veins, in the peripheral or central vascular plexus. Tip cell
abundance was normalized to the perimeter of the con-
tiguous vascular front in each image and filopodia were
normalized to tip cell number. Capillary regression was de-
fined as Collagen IV staining without colocalized Isolectin
B4 staining, and expressed as total length per mm2 in com-
plete retinal segments, as per the method of Franco et al
(21). Endothelial proliferation, defined by EdU+ nuclei
costaining with Isolectin B4, was quantified in multiple
200 × 200-μm regions of interest placed between arteries
and veins, in the peripheral vascular plexus. embedded in optimal cutting temperature media (Tissue-
Tek OCT compound, Sakura, Netherlands) before snap
freezing in liquid nitrogen and cryosectioning at 10-µm
thickness. Vascular endothelium was stained with Isolectin
B4 conjugated with Alexa Fluor 488 (I21411; Thermo
Fisher Scientific) and slides were mounted with DAPI-
Fluoromount-G (Southern Biotech, AL) to define nuclei. RT-PCR Snap frozen ischemic adductor muscle was mechanic-
ally lysed in Trizol reagent (Sigma Aldrich, Gillingham,
UK) to isolate RNA. After reverse transcription to gen-
erate cDNA (kit), quantitative PCR was performed (ABI
Prism 7900HT, Applied Biosystems) using Taqman probes
against murine vegfa (Mm01281449-m1), murine insr
(Mm00439688_m1), murine actb (Mm00607939_s1), 18s
(Mm01281449-m1); 18s or actb were used to normalize
gene expression using the equation Surgical procedure Following the protocol we have published (22), 9- to
13-week-old male Insr+/- mice were anesthetized with
isoflurane before dissecting the left femoral artery, ligating
it proximally at the inguinal ligament and distally at the bi-
furcation to saphenous and popliteal vessels, and excising
the intervening segment. Ex vivo aortic ring angiogenesis Aortae were harvested from 8- to 12-week-old Insr+/- mice
under terminal isoflurane anesthesia and then processed
according to the protocol of Baker et al (23). In brief,
after dissection of perivascular fat and overnight storage
in serum free OptiMEM media (Thermo Fisher Scientific),
aortae were cut in to 1-mm-thick rings that were then em-
bedded in rat type I collagen. Rings were incubated for
5 days at 37°C in 5% CO2 in Opti-MEM media containing
2.5% fetal calf serum (FCS), 50 ng/mL VEGF-A165 (R&D
Systems, Abingdon, UK) and penicillin-streptomycin, with
a media change on day 3. Rings were then fixed with 4%
paraformaldehyde, stained with BS-1 lectin-fluorescein
(Sigma Aldrich) to define endothelium, and then imaged
with an inverted confocal microscope (LSM700, Carl
Zeiss Microscopy Ltd.); tiled images were collected using
a 10×/0.2NA objective and stitched using Zen software. Image analysis was performed with Image J (NIH), defining
the number of fluorescein staining sprouts per ring and the
mean length of these sprouts; mean data were then pro-
duced for each experimental animal from at least 4 rings. Laser Doppler perfusion imaging Laser Doppler analysis (Moor LDI2-HR, Moor Systems,
UK) of ischemic and nonischemic limbs was performed
postoperatively in a temperature-controlled environment,
to confirm induction of ischemia, and repeated weekly
until day 21. Images were analyzed (MoorLDI software,
Version 5.3, Moor Systems, UK) to derive an ischemic to
nonischemic limb perfusion ratio, based upon flux below
the level of the inguinal ligament. Materials and Methods
Animal models Confocal microscopy and image analysis
Microscopy was performed using a Zeiss LSM880 up-
right confocal microscope with 20×/0.8NA objective and
Zen software (Carl Zeiss Microscopy Ltd). Image analysis
used ImageJ (NIH). Vascular area was defined by binary
thresholding of the Isolectin B4 signal and expressed as a
percentage of the image area. Assessment of pathological angiogenesis after
hind-limb ischemia 2−deltaCT× 100. 2−deltaCT× 100. Isolation and functional analysis Pulmonary endothelial cells (PECs) were isolated from
both lungs of 8- to 12-week-old Insr+/- mice, precisely
following the protocol of Sobczak et al (24). This uses
immuno-magnetic selection of CD31+ cells, which are
then cultured in EGM2 media (Lonza, Slough, UK)
for 10 to 14 days before a second round of immuno-
magnetic selection from ICAM2+ endothelial cells that
were cultured for a further 5 to 7 days in EGM2 before
functional assays. Human umbilical vein endothelial cells (HUVECs;
PromoCell, Heidelberg, Germany) were cultured at 37°C in
5% CO2 in EGM2 media on 1% gelatin-coated plasticware
and used between passages 3 and 6. Silencing of the in-
sulin receptor was induced using insulin receptor shRNA
introduced by lentiviral particles (SHCLNV-NM_00208,
TRCN0000196786; MISSION, Sigma Aldrich), with GFP-
targeting shRNA lentivirus particles (SHC002H; MISSION,
Sigma Aldrich) serving as control. Both lentiviruses were
applied at 15 multiplicity of infection and HUVECs were
used in downstream experiments 4 days after transduction. Adhesion assay y
HUVECs were seeded on to 1% gelatin-coated 24-well
plates in EBM2 media with 1% FCS, with or without 50 ng/
mL VEGA-A165, at a density of 4 × 104 cells per well and left
for 1 hour before washing 3 times with PBS and fixing with
4% paraformaldehyde. Cells were counterstained with
Hoechst and Phalloidin Alexa Fluor 488 conjugate and
imaged with confocal microscopy (LSM700, Carl Zeiss
Microscopy Ltd.) to count adherent cells per mm2. Scratch wound assay PECs were grown to confluence in EGM2 media on 1%
gelatin coated 96-well plates before forming a scratch
wound using the WoundMaker tool (Essen Bioscience,
Royston, UK) and imaging wound closure hourly in a live
cell imaging system (Incucyte, Essen Bioscience) to define
residual wound area. Scratch wound assay HUVECs were grown to confluence in EGM2 media on
1% gelatin-coated 96-well plates before forming a scratch
wound using the WoundMaker tool (Essen Bioscience) and
imaging wound closure 8 hours later to define percentage
wound closure from baseline. Bead sprouting assay Following the protocol of Nakatsu et al (26), HUVECs
were coated on to the surface of Cytodex-3 microcarrier
beads (Sigma Aldrich) and then embedded in a fibrin matrix
that was overlaid with EGM2 media (Lonza) without the
supplemental bullet kit, but containing 50 ng/mL human
VEGF-A165 (PeproTech, NJ) and 5ng/ml human basic FGF
(PeproTech). After 48 hours of incubation at 37°C in 5%
CO2, 25 beads per condition were imaged with phase con-
trast microscopy (Olympus CX41, Olympus Life Sciences,
Southend-On-Sea, UK) and analyzed with Image J (NIH),
defining sprouts per bead and the mean length of these
sprouts; mean data were then produced for each experi-
mental condition. Matrigel sprouting assay Twenty-four well plates were coated with growth factor
reduced Matrigel (BD Biosciences, Wokingham, UK) be-
fore seeding each well with 2 × 105 PEC suspended in
EBM2 media (Lonza) containing 1% FCS and 50 ng/mL of
VEGF-A165. After 24 hours, phase contrast microscopy was
used to image each well and count tubule-like structures
according to our published protocol (22). Each sample was
run in triplicate, with a single mean datapoint calculated
for each experimental animal. Cell proliferation Sparsely seeded PECs on 1% gelatin coated plastic, cul-
tured in EGM2 media, were exposed to 10 µM EdU 2
hours before fixation with 4% paraformaldehyde and
processing with the Click-iT EdU cell proliferation assay
(Thermo Fisher Scientific) to label nuclei containing ac-
tively forming DNA with Alexa Fluor 488 and a Hoechst
nuclear counterstain. Confocal microscopy (LSM700, Carl
Zeiss Microscopy Ltd.) was used to define the proportion
of EdU+ nuclei. Boyden chamber Following our published protocol (25), 5 × 104 PECs
were seeded in 1% gelatin coated Boyden chamber ap-
paratus to define migration toward 50 ng/mL VEGF-A165. The number of migrating cells per microscopic field
was counted using standard light microscopy and pre-
sented as net migration by subtracting the number of
cells migrating in paired control experiments without
VEGF-A165 gradient. Tissue collection and processing Ischemic and contralateral gastrocnemius muscle was har-
vested and fixed in 4% paraformaldehyde for 48 hours,
whereas adductor muscles were snap frozen with liquid
nitrogen for RNA isolation. Fixed muscle specimens were Endocrinology, 2021, Vol. 162, No. 8 4 Western blotting Western blotting
HUVECs were lysed in cell extraction buffer (FNN0011;
Thermo Fisher Scientific; containing, in mmol/L, 10 mM Endocrinology, 2021, Vol. 162, No. 8 5 Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA,
1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton
X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 2
sodium orthovanadate, 0.5 μg/mL leupeptin, 0.2 PMSF,
0.5 μg/mL aprotinin). Cell extracts were centrifuged for 15
minutes before protein measurement using the biocinochinic
acid assay (Thermo Fisher Scientific). Equal amounts
of protein were resolved on SDS-polyacrylamide gels
(Thermo Fisher Scientific) and transferred to polyvinyldine
difluoride membranes. Immunoblotting was carried out
with primary antibodies against beta-actin (27), insulin
receptor-beta (28), Akt (29), phospho-S473 Akt (30), endo-
thelial nitric oxide synthase (eNOS) (31), phospho-S1177
eNOS (32), extracellular signal-regulated kinase 1/2
(ERK1/2) (33), phospho-T202/Y204 ERK1/2 (34), vas-
cular endothelial growth factor receptor 2 (VEGFR2) (35),
and phospho-Y951 VEGFR2 (36). Blots were incubated
with appropriate peroxidase-conjugated secondary anti-
bodies and developed with enhanced chemiluminescence
(37, 38), and imaged with SynGene chemiluminescence
imaging system (SynGene, Cambridge, UK). Densitometry
of phospho-proteins was normalized to respective total
proteins from the same sample and then these data were
normalized to the value of unstimulated control shRNA-
transduced cells in each experiment (which included paired
control and insulin receptor shRNA-transduced cells, with
samples run on 1 membrane). VEGF-A induced signaling
was calculated by subtracting the data from unstimulated
(control or insulin receptor shRNA transduced) cells from
their respective VEGF-A–exposed cells in each experiment. were washed with radio-immunoprecipitation assay buffer
to remove any unbound antibody before incubating with
cell lysate for 1 hour with rotation at room temperature
to allow the biotinylated antigen-antibody complexes to
form. At the end of the pull-down period, the beads were
washed 5 times with radio-immunoprecipitation assay
buffer (50 mmol/L Tris-HCl [pH 8.0], 150 mmol/L NaCl,
0.5% [w/v] sodium deoxycholate, 0.1% [w/v] SDS, and
1% [v/v] Igepal) to remove any nonspecific binding. The
immunoprecipitated, biotinylated VEGFR2 complexes
were mixed with dissociation buffer and boiled to release
the complexes from the beads. The proteins were resolved
by electrophoresis through 4% to 12% polyacrylamide
gels and then transferred to nitrocellulose membrane. Results Surface VEGFR2 biotinylation, immunoprecipitation, and
VEGFR2 Western blotting were performed according to
our previously published protocol (39). Briefly, HUVECs
were incubated for 1 hour at 4°C with 0.5 mg/mL biotin
sulfo-NHS (Sigma-Aldrich, Gillingham, UK), before being
stimulated with 50 ng/mL VEGF at 37°C for either 5 or
15 minutes. At the end of the exposure period, the cells
were washed 3 times with PBS with calcium and magne-
sium before either: immediate lysis with cell extraction
buffer to enable the measurement of total VEGFR2 in the
sample; or, treated with 0.5 mL of 0.05% trypsin/EDTA to
cleave and remove any remaining biotin-labelled, cell sur-
face VEGFR2, meaning detected biotin-labelled VEFGR2
would define only internalized protein. The trypsinized
cell pellet was lysed using cell extraction buffer as before. Immunoprecipitation of VEGFR2 was carried out using
protein A Dynabeads (Thermo Fisher Scientific) loaded
with anti-VEGFR2 antibody (diluted 1:100) (35) for 30
minutes with rotation at room temperature. The beads Statistics All data are presented as mean (SEM). Comparison be-
tween groups was performed using Student t tests, or
2-way ANOVA for time series data. All tests were 2-sided
and statistical significance was defined as P < 0.05. Western blotting The
membrane was blocked for 1 hour in PBS (1.5 mmol/L
KH2PO4, 2.7 mmol/L Na2HPO4, 150 mmol/L NaCl [pH
7.4]) containing 5% (w/v) dried milk powder and 0.1%
(v/v) Tween-20, followed by incubation with peroxidase-
conjugated streptavidin (1:1000 dilution in PBS containing
0.1% [v/v] Tween-20) for 1 hour. Bound peroxidase conju-
gates were visualized using an enhanced chemiluminescence
detection system (Amersham Biosciences). Quantification
of immunoblots was performed using ImageJ software. Pathological angiogenesis is impaired in
Insr+/- mice and is associated with impaired
responsiveness to VEGF To study pathological angiogenesis, we induced hind-
limb ischemia in Insr+/- mice and quantified limb perfu-
sion recovery every 7 days using laser Doppler imaging. This revealed that, in spite of similar reductions in limb
perfusion immediately postoperatively, Insr+/- exhibited
lower ischemic limb perfusion at all timepoints there-
after (Fig. 1A), being 82% (5.6) in WT and 62% (7.1)
in Insr+/- (P < 0.05) at day 21. Histological analysis of
gastrocnemius muscle also revealed a lower capillary
density in the ischemic limb of Insr+/- at day 21 (vas-
cular area 12% [0.5] vs 8% [0.6] in WT and Insr+/-, re-
spectively, P < 0.05 Fig. 1B), in spite of greater VEGFA
mRNA in the ischemic limb adductor muscle of Insr+/-
(31.4% [6.7] of 18S mRNA in Insr+/- vs 9.8% [2.5] WT
P < 0.05; Fig. 1C). Notably, Vegfa mRNA was similar 6 Endocrinology, 2021, Vol. 162, No. 8 gure 1. Angiogenesis is impaired in Insr+/- mice with hindlimb ischemia. (A) Ischemic to nonischemic limb perfusion defined by laser Doppler
maging, with representative day 21 images, showing impaired recovery from hindlimb ischemia in Insr+/- vs WT (n = 14, 13). (B) Confocal immuno-l
uorescence of ischemic and nonischemic gastrocnemius muscle reveals reduced capillary density in ischemic Insr+/- vs WT muscle. Representative
mages of ischemic muscle show isolectin B4 stained capillaries in red and nuclei in blue. Scale bars denote 50 μm. (n = 8, 7). (C) Vegfa mRNA
ormalized to 18S mRNA is higher in the ischemic limb adductor muscle of Insr+/- vs WT (n = 4, 4). D) Vegfa mRNA normalized to 18S mRNA is higher
the nonischemic limb adductor muscle of Insr+/- vs WT (n = 4, 4). *P < 0.05. Insr, insulin receptor; WT, wild-type. Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6284300 by guest on 01 March 2023 Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6 Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6284300 by guest on 01 March 2023 Figure 1. Angiogenesis is impaired in Insr+/- mice with hindlimb ischemia. (A) Ischemic to nonischemic limb perfusion defined by laser Doppler
imaging, with representative day 21 images, showing impaired recovery from hindlimb ischemia in Insr+/- vs WT (n = 14, 13). (B) Confocal immuno-
fluorescence of ischemic and nonischemic gastrocnemius muscle reveals reduced capillary density in ischemic Insr+/- vs WT muscle. Representative
images of ischemic muscle show isolectin B4 stained capillaries in red and nuclei in blue. Scale bars denote 50 μm. (n = 8, 7). Pathological angiogenesis is impaired in
Insr+/- mice and is associated with impaired
responsiveness to VEGF This revealed fewer capillary sprouts emerging from
Insr+/- aortae in the presence of VEGF (30 [2.2] in WT vs
22 [2.4] in Insr+/-; P < 0.05; Fig. 2A), and the length of
sprouts was also reduced (1037 µm [39] WT vs 847 µm
[38] Insr+/-; P < 0.05; Fig. 2B). Next, we isolated PECs
for functional studies; these exhibited appropriate reduc-
tion in Insr mRNA (85% [5] of 18S in WT vs 49% [4] in
Insr+/-; P < 0.05; Fig. 2C). We then conducted a Matrigel
in vitro angiogenesis assay, which demonstrated reduced
tubule formation in Insr+/- (28 [2] tubules per microscopic
field in WT vs 14.5 [2.4] in Insr+/-; P < 0.05; Fig. 2D). Similarly, a scratch wound assay performed on confluent
PECs revealed significantly slower closure of the wound
formed in Insr+/- PECs (area under curve 4,430,670 arbi-
trary units WT [154,516] vs 5,085,825 [126,748] arbi-
trary units Insr+/-; P < 0.05; Fig. 2E). Because the Matrigel
and scratch wound assays define responses to VEGF-A
with other stimulatory factors, we then performed as-
says to more specifically define functional responses to
VEGF-A. First, we conducted a migration assay using
Boyden chamber apparatus and found fewer Insr+/- PECs
migrated toward VEGF-A165 (7.5 [1.7] WT vs 2.2 [1.1]
Insr+/- net cell migration to VEGF per microscopic field;
P < 0.05; Fig. 2F). Second, we studied VEGF-A165–in-
duced PEC proliferation using EdU incorporation and
elicited no difference between Insr+/- and WT (10.2%
[3.5] WT vs 8.9% [2.6] Insr+/- EdU+ cells; P = 0.77; Fig. 2G). Collectively, these data imply that Insr+/- endothe-
lial cells have selectively impaired migratory responses
to VEGF-A. Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6284300 by guest on 01 March 2023 To discern whether loss of insulin receptors expressed
by ECs contribute to the retinal vascular phenotype of
Insr+/-, we then studied mice with ECInsr+/-. Again, the ra-
dial outgrowth of their retinal vascular plexus was similar
to controls (1377 µm [32] WT vs 1375 µm [40] ECInsr+/-;
P = 0.96; Fig. 4A), although there was reduced vascular
area (front 46.5% [0.5] WT vs 44.0 [0.5] ECInsr+/-,
P < 0.05; center 36.7% [0.4] WT vs 33.9% [1.0] ECInsr+/-,
P < 0.05; Fig. 4B) and branching complexity (front
1192.5 [7.0] WT vs 1043.3 [20.4] ECInsr+/- branch points
per mm2, P < 0.05; center 1083.5 [14.3] WT vs 1000.0
[30.5] ECInsr+/-, P < 0.05; Fig. Pathological angiogenesis is impaired in
Insr+/- mice and is associated with impaired
responsiveness to VEGF 4C) in both the peripheral
and central vascular plexuses of ECInsr+/-. Also mirroring
Insr+/- phenotype, there was a reduction in the number of
sprouting tip cells of ECInsr+/- (21.5 [0.2] WT vs 18.7 [0.3]
ECInsr+/- tip cells/mm; P < 0.05; Fig. 4D), along with fewer
filopodia per length of vascular forefront (21.2 [0.4] WT
vs 18.7 [0.3] ECInsr+/- filopodia per 100 µm; P < 0.05), but
with no difference in filopodia per tip cell between geno-
types (21.8 [0.7] WT vs 22.2 [0.5] ECInsr+/- filopodia per tip
cell; P = 0.65; Fig. 4E). Collectively, these data suggest that
endothelial cell insulin receptor expression is important in
the generation of vascular sprouts, and the branching struc-
ture of the nascent vasculature. Pathological angiogenesis is impaired in
Insr+/- mice and is associated with impaired
responsiveness to VEGF (C) Vegfa mRNA
normalized to 18S mRNA is higher in the ischemic limb adductor muscle of Insr+/- vs WT (n = 4, 4). D) Vegfa mRNA normalized to 18S mRNA is higher
in the nonischemic limb adductor muscle of Insr+/- vs WT (n = 4, 4). *P < 0.05. Insr, insulin receptor; WT, wild-type. 7 Endocrinology, 2021, Vol. 162, No. 8 P < 0.05; Fig. 3B) and reduced vascular branching com-
plexity (front 48.0 [0.4] WT vs 41.9 [0.7] Insr+/- branches
per microscopic field, P < 0.05; center 52.0 [1.3] WT vs
46.4 [1.1] Insr+/- branches per microscopic field, P < 0.05;
Fig. 3C) in both central and peripheral vascular plex-
uses of Insr+/-. High-resolution images of the emerging
vascular plexus revealed a reduction in the number of
sprouting tip cells of Insr+/- (21.2 [0.3] WT vs 18.3 [0.2]
Insr+/- tip cells per mm, P < 0.05; Fig. 3D), fewer filo-
podia per length of vascular forefront (22.0 [0.5] WT vs
18.1 [0.3] Insr+/- filopodia per 100 µm, P < 0.05), and
fewer filipodia per tip cell (22.1 [0.3] WT vs 20.6 [0.4]
Insr+/- filopodia per tip cell, P < 0.05; Fig. 3E). Because
reduced vascularity could also be explained by increased
vessel regression, we quantified the number of “empty”
collagen IV sleeves (ie, collagen IV basement membrane
without overlying Isolectin B4, linking 2 regions of estab-
lished vasculature) in the retinal periphery; we observed
fewer in Insr+/- vs WT, indicating regression was not ex-
aggerated (0.46 [0.02] WT vs 0.40 [0.02] Insr+/- regressed
vessels/100 µm2; P = 0.028; Fig. 3F). Next, we asked if
endothelial cell (EC) proliferation was reduced in Insr+/-,
but found similar numbers of EdU+ ECs in the peripheral
retinal vasculature of both genotypes (781 [44] WT vs
814 [24] Insr+/- EdU+ EC per mm2; P = 0.53; Fig. 3G). Overall, these data are compatible with a reduction in
vascular sprouting in the emerging vasculature of Insr+/-,
resulting in a less branched neovasculature. in the nonischemic limb adductor muscle or WT and
Insr+/- (1.7% [0.3] of 18S mRNA in WT vs. 1.4% [0.3]
in Insr+/-; Fig. 1D). The increased ischemic muscle expres-
sion of Vegfa is indicative of significant residual ischemia
and could also imply an inadequate functional response
to this central regulator of angiogenesis. Pathological angiogenesis is impaired in
Insr+/- mice and is associated with impaired
responsiveness to VEGF To address this
possibility, we explanted aortae from a separate group
of Insr+/- mice to embed rings in a collagen matrix con-
taining VEGF-A165, which induces sprouting angiogen-
esis. This revealed fewer capillary sprouts emerging from
Insr+/- aortae in the presence of VEGF (30 [2.2] in WT vs
22 [2.4] in Insr+/-; P < 0.05; Fig. 2A), and the length of
sprouts was also reduced (1037 µm [39] WT vs 847 µm
[38] Insr+/-; P < 0.05; Fig. 2B). Next, we isolated PECs
for functional studies; these exhibited appropriate reduc-
tion in Insr mRNA (85% [5] of 18S in WT vs 49% [4] in
Insr+/-; P < 0.05; Fig. 2C). We then conducted a Matrigel
in vitro angiogenesis assay, which demonstrated reduced
tubule formation in Insr+/- (28 [2] tubules per microscopic
field in WT vs 14.5 [2.4] in Insr+/-; P < 0.05; Fig. 2D). Similarly, a scratch wound assay performed on confluent
PECs revealed significantly slower closure of the wound
formed in Insr+/- PECs (area under curve 4,430,670 arbi-
trary units WT [154,516] vs 5,085,825 [126,748] arbi-
trary units Insr+/-; P < 0.05; Fig. 2E). Because the Matrigel
and scratch wound assays define responses to VEGF-A
with other stimulatory factors, we then performed as-
says to more specifically define functional responses to
VEGF-A. First, we conducted a migration assay using
Boyden chamber apparatus and found fewer Insr+/- PECs
migrated toward VEGF-A165 (7.5 [1.7] WT vs 2.2 [1.1]
Insr+/- net cell migration to VEGF per microscopic field;
P < 0.05; Fig. 2F). Second, we studied VEGF-A165–in-
duced PEC proliferation using EdU incorporation and
elicited no difference between Insr+/- and WT (10.2%
[3.5] WT vs 8.9% [2.6] Insr+/- EdU+ cells; P = 0.77; Fig. 2G). Collectively, these data imply that Insr+/- endothe-
lial cells have selectively impaired migratory responses
to VEGF-A. in the nonischemic limb adductor muscle or WT and
Insr+/- (1.7% [0.3] of 18S mRNA in WT vs. 1.4% [0.3]
in Insr+/-; Fig. 1D). The increased ischemic muscle expres-
sion of Vegfa is indicative of significant residual ischemia
and could also imply an inadequate functional response
to this central regulator of angiogenesis. To address this
possibility, we explanted aortae from a separate group
of Insr+/- mice to embed rings in a collagen matrix con-
taining VEGF-A165, which induces sprouting angiogen-
esis. Developmental angiogenesis is impaired in Insr+/-
and ECInsr+/- mice Next, we asked whether the abnormalities of patho-
logical angiogenesis in Insr+/- were recapitulated during
developmental angiogenesis, which we assessed using
whole-mounted retinas, at P5 when the vasculature is still
developing. The radial outgrowth of the retinal vascular
plexus was similar in Insr+/- and WT (1367 µm [50] WT
vs 1354 µm [56] Insr+/-, P = 0.87; Fig. 3A), although there
was reduced vascular area in the peripheral vascular
plexus of Insr+/- (48.8% [0.6] WT vs 46.0% [0.7] Insr+/-, Endocrinology, 2021, Vol. 162, No. 8 8 Figure 2. Insr+/- exhibits impaired in vitro functional responses to VEGF. Capillary sprouting from aortic rings embedded in a collagen matrix with
VEGF-A165 is reduced in Insr+/- vs WT (A), as is mean sprout length (B); representative images show isolectin B4 staining of endothelium in green, with
scale bars denoting 1000 μm (n = 5, 5). (C) Insulin receptor (Insr) mRNA normalized to 18S mRNA is reduced in Insr+/- vs WT PEC (n = 5, 5). (D) In vitro
angiogenesis in Matrigel is impaired in Insr+/- vs WT PEC (n = 7, 4). (E) Scratch wound closure is impaired in Insr+/- vs WT PEC (n = 5, 6). (F) Migration
toward VEGF-A165 in Boyden chamber apparatus is impaired in Insr+/- vs WT PEC (n = 5, 4). (G) Proliferation defined by nuclear EdU incorporation is
similar in Insr+/- and WT PEC (n = 11, 7). *P < 0.05. EdU, 5-ethynyl-2′-deoxyuridine; Insr, insulin receptor; PEC, pulmonary endothelial cell; VEGF, vas-
cular endothelial growth factor; WT, wild-type. Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6284300 by guest on 01 March 2023 Figure 2. Insr+/- exhibits impaired in vitro functional responses to VEGF. Capillary sprouting from aortic rings embedded in a collagen matrix with
VEGF-A165 is reduced in Insr+/- vs WT (A), as is mean sprout length (B); representative images show isolectin B4 staining of endothelium in green, with
scale bars denoting 1000 μm (n = 5, 5). (C) Insulin receptor (Insr) mRNA normalized to 18S mRNA is reduced in Insr+/- vs WT PEC (n = 5, 5). (D) In vitro
angiogenesis in Matrigel is impaired in Insr+/- vs WT PEC (n = 7, 4). (E) Scratch wound closure is impaired in Insr+/- vs WT PEC (n = 5, 6). (F) Migration
toward VEGF-A165 in Boyden chamber apparatus is impaired in Insr+/- vs WT PEC (n = 5, 4). Insulin receptor silencing impairs human EC
functional responsiveness to VEGF shRNA vs 95.4 [9.1] Insr shRNA cells per microscopic
field; P < 0.05; Fig. 5E). Overall, these data suggest that
the insulin receptor is also important for VEGF-A–induced
angiogenic sprouting and cell motility in human EC. To explore the relevance of these data in human ECs,
we transduced HUVECs with lentivirus particles to de-
liver insulin receptor targeting shRNA (referred to as Insr
shRNA), or control GFP-targeting shRNA (referred to
as control [Con] shRNA), reducing insulin receptor pro-
tein by 40% (Fig. 5A). Transduced HUVECs were then
coated onto Cytodex-3 carrier beads and embedded in
a fibrin matrix to study VEGF-A165–induced sprouting
angiogenesis in vitro. Mirroring data from Insr+/- aortic
ring sprouting experiments (Fig. 2A-C), we observed that
Insr shRNA HUVECs produced fewer sprouts (6.5 [1.2]
Con shRNA vs 3.4 [1.0] Insr shRNA sprouts per bead;
P < 0.05; Fig. 5B), although the mean length of sprouts was
unaffected (78.3 µm [10.7] Con shRNA vs 65.0 µm [14.0]
Insr shRNA, P > 0.05; Fig. 5C). Insr shRNA HUVECs ex-
hibited impaired scratch wound closure (84.7% [4.7] Con
shRNA vs 74.0% [3.7] Insr shRNA; P < 0.05; Fig. 5D)
and impaired adhesion to gelatin coated plates, which
was more marked in the presence of VEGF-A165 (vehicle
138.7 [5.5] Con shRNA vs 85.6 [6.9] Insr shRNA cells per
microscopic field; P < 0.05; VEGF-A165 184.3 [6.6] Con Developmental angiogenesis is impaired in Insr+/-
and ECInsr+/- mice (G) Proliferation defined by nuclear EdU incorporation is
similar in Insr+/- and WT PEC (n = 11, 7). *P < 0.05. EdU, 5-ethynyl-2′-deoxyuridine; Insr, insulin receptor; PEC, pulmonary endothelial cell; VEGF, vas-
cular endothelial growth factor; WT, wild-type. Endothelial insulin receptors are required for
VEGFR2 internalization and subsequent ERK
signaling Because we had demonstrated that insulin receptor expres-
sion influenced functional responses to VEGF-A in human
and murine ECs, we then asked if this was associated with
altered VEGF-A signaling. VEGF-A promotes angiogen-
esis by binding to VEGFR2, a cell membrane-bound re-
ceptor tyrosine kinase that initiates a complex intracellular
signaling cascade. We therefore stimulated Insr shRNA and
control shRNA HUVECs with 50 ng/mL VEGF-A165 and
studied major VEGF-A signaling nodes 5 and 15 minutes
later, along with unstimulated cells (Fig. 6A). Insr shRNA
HUVECs exhibited unaffected activation of VEGFR2
(measured by phosphorylation at Y951), or the down-
stream nodes Akt (measured by phosphorylation at S473)
and eNOS (measured by phosphorylation at S1177) (data 9 Endocrinology, 2021, Vol. 162, No. 8 ure 3. Developmental angiogenesis is impaired in the neonatal P5 retina of Insr+/- mice. (A) Radial outgrowth of the developing retinal vascula-
is comparable in Insr+/- and WT, with representative images showing white isolectin B4 staining of endothelium and scale bars denoting 500 μm
7, 13). (B) Vascular endothelial area is reduced in the peripheral half of the retinal vasculature in Insr+/- vs WT (n = 7, 13). (C) Vascular branching
educed in the peripheral and central zones of the retinal vasculature in Insr+/- vs WT (n = 7, 13). (D) Emerging tip cells per millimeter of vascular
t perimeter are reduced in Insr+/- vs WT, with representative images showing white isolectin B4 staining of endothelium and scale bars denoting
m (n = 7, 13). (E) The number of filopodia per tip cell is similar in Insr+/- vs WT (n = 7, 13). (F) The number of regressed vessels, defined as Collagen
eeves (red) without overlying isolectin B4 (green) in representative images, is lower in Insr+/- than WT (n = 11, 10). (G) The number of proliferating
othelial cells, defined as EdU+ nuclei (red) overlying isolectin B4 (white) in representative images, is similar in Insr+/- and WT (n = 13, 5). *P < 0.05. 5
th
l 2′ d
idi
I
i
li
t
WT
ild t
Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6284300 by guest on 01 March 2023 Downloaded from https://academic.oup.com/endo/article/162/8 Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6284300 by guest on 01 March 2023 Figure 3. Developmental angiogenesis is impaired in the neonatal P5 retina of Insr+/- mice. Endothelial insulin receptors are required for
VEGFR2 internalization and subsequent ERK
signaling (A) Radial outgrowth of the developing retinal vascula-
ture is comparable in Insr+/- and WT, with representative images showing white isolectin B4 staining of endothelium and scale bars denoting 500 μm
(n = 7, 13). (B) Vascular endothelial area is reduced in the peripheral half of the retinal vasculature in Insr+/- vs WT (n = 7, 13). (C) Vascular branching
is reduced in the peripheral and central zones of the retinal vasculature in Insr+/- vs WT (n = 7, 13). (D) Emerging tip cells per millimeter of vascular
front perimeter are reduced in Insr+/- vs WT, with representative images showing white isolectin B4 staining of endothelium and scale bars denoting
50 μm (n = 7, 13). (E) The number of filopodia per tip cell is similar in Insr+/- vs WT (n = 7, 13). (F) The number of regressed vessels, defined as Collagen
IV sleeves (red) without overlying isolectin B4 (green) in representative images, is lower in Insr+/- than WT (n = 11, 10). (G) The number of proliferating
endothelial cells, defined as EdU+ nuclei (red) overlying isolectin B4 (white) in representative images, is similar in Insr+/- and WT (n = 13, 5). *P < 0.05. EdU, 5-ethynyl-2′-deoxyuridine; Insr, insulin receptor; WT, wild-type. Endocrinology, 2021, Vol. 162, No. 8 10 Figure 4. Developmental angiogenesis is impaired in the neonatal P5 retina of ECInsr+/- mice. (A) Radial outgrowth of the developing retinal vascu-
lature is comparable in ECInsr+/- and WT, with representative images showing white isolectin B4 staining of endothelium and scale bars denoting
500 μm (n = 7, 6). (B) Vascular endothelial area is reduced in the peripheral and central zones of the retinal vasculature in ECInsr+/- vs WT (n = 7, 6). (C) Vascular branching is reduced in the peripheral and central zones of the retinal vasculature in ECInsr+/- vs WT (n = 7, 6). (D) Emerging tip cells per
millimeter of vascular front perimeter are reduced in ECInsr+/- vs WT, with representative images showing white isolectin B4 staining of endothelium
and scale bars denoting 50 μm (n = 7, 6). (E) The number of filopodia per tip cell is similar in ECInsr+/- vs WT (n = 7, 6). ECInsr, endothelial cell insulin
receptor; WT, wild-type. Figure 4. Developmental angiogenesis is impaired in the neonatal P5 retina of ECInsr+/- mice. Endothelial insulin receptors are required for
VEGFR2 internalization and subsequent ERK
signaling (A) Radial outgrowth of the developing retinal vascu-
lature is comparable in ECInsr+/- and WT, with representative images showing white isolectin B4 staining of endothelium and scale bars denoting
500 μm (n = 7, 6). (B) Vascular endothelial area is reduced in the peripheral and central zones of the retinal vasculature in ECInsr+/- vs WT (n = 7, 6). (C) Vascular branching is reduced in the peripheral and central zones of the retinal vasculature in ECInsr+/- vs WT (n = 7, 6). (D) Emerging tip cells per
millimeter of vascular front perimeter are reduced in ECInsr+/- vs WT, with representative images showing white isolectin B4 staining of endothelium
and scale bars denoting 50 μm (n = 7, 6). (E) The number of filopodia per tip cell is similar in ECInsr+/- vs WT (n = 7, 6). ECInsr, endothelial cell insulin
receptor; WT, wild-type. [15.6] Con shRNA vs 87.4% [3.8] Insr shRNA surface-
retained VEFGR2; P < 0.05; 15 minutes: 20.6% [12.9]
Con shRNA vs 89.8% [0.9] Insr shRNA surface-retained
VEFGR2; P < 0.05; Fig. 6D,E), which is known to select-
ively impede ERK1/2 activation. Overall, these data reveal
a selective deficit in VEGF-A signal transduction in Insr
shRNA HUVECs, which is likely to result from impaired
internalization of activated VEGFR2. not shown). However, downstream activation of ERK1/2
(measured by phosphorylation at T202/Y204) was im-
paired in Insr shRNA HUVECs at 5 and 15 minutes after
stimulation (5-minute change: 3.6 [0.95] Con shRNA vs 1.9
[0.53] Insr shRNA pERK/ERK ratio; P < 0.05; 15-minute
change: 4.9 [0.98] Con shRNA vs 2.5 [1.02] Insr shRNA
pERK/ERK ratio; P < 0.05; Fig. 6B). Control experiments
defining the signaling response to insulin revealed clear re-
duction in Akt activation after 15 minutes, confirming that
Insr shRNA induced the expected changes in activation of
this major insulin signaling node (Fig. 6C). Importantly,
the activation of Akt and ERK1/2 downstream of VEGFR2
follows highly distinct pathways, with internalization of
VEGFR2 being essential for only the latter (40); more-
over, integrins are known to influence this process (41). Hence, we then asked whether VEGF-A induced internal-
ization of VEGFR2 was impaired in Insr shRNA HUVEC
by performing a surface biotinylation assay to quantify
surface-resident and internalized VEGFR2 protein. This
revealed that, although baseline surface located VEGFR2
was similar in both groups, there was reduced VEGFR2
internalization in Insr shRNA HUVECs (5 minutes: 41.6% Major findings and implications Our data reveal for the first time that endothelial insulin
receptors are required for appropriate migration and
angiogenic sprouting in response to VEGF-A, both in vitro
and in vivo. At a molecular level, we found that insulin re-
ceptor expression promotes the internalization of VEGF-A–
activated VEGFR2, allowing signaling to ERK1/2. Our data
suggest that the proangiogenic effects of insulin receptors
relate to crosstalk with VEGF-A signaling, although the na-
ture of this interaction, and whether insulin participates in
the process, requires further study (Fig. 7). This previously Endocrinology, 2021, Vol. 162, No. 8 11 Figure 5. Insr knockdown in HUVECs impairs functional responses to VEGF. (A) Insulin receptor protein knockdown of 40% was achieved in Insr
shRNA HUVECs vs control shRNA HUVECs, with representative gel (n = 7, 7). (B) Angiogenic sprout numbers were reduced from Cytodex beads
coated with Insr shRNA HUVECs vs control shRNA HUVECs, with representative microscopy images (n = 3, 3). (C) Angiogenic sprout length was
similar from Cytodex beads coated with Insr shRNA HUVECs vs control shRNA HUVECs (n = 3, 3). (D) Scratch wound closure was impaired in Insr
shRNA HUVECs vs control shRNA HUVECs (n = 4, 4). (E) Adhesion to gelatin was impaired in Insr shRNA HUVECs vs control shRNA HUVECs, espe-
cially in context of media supplemented with VEGF-A165; representative microscopy images show DAPI-defined nuclei in blue and phalloidin-defined
filamentous actin in red, with scale bars denoting 250 μm (n = 4, 4). *P < 0.05. HUVEC, human umbilical vein endothelial cell; Insr, insulin receptor;
VEGF, vascular endothelial growth factor. Figure 5. Insr knockdown in HUVECs impairs functional responses to VEGF. (A) Insulin receptor protein knockdown of 40% was achieved in Insr
shRNA HUVECs vs control shRNA HUVECs, with representative gel (n = 7, 7). (B) Angiogenic sprout numbers were reduced from Cytodex beads
coated with Insr shRNA HUVECs vs control shRNA HUVECs, with representative microscopy images (n = 3, 3). (C) Angiogenic sprout length was
similar from Cytodex beads coated with Insr shRNA HUVECs vs control shRNA HUVECs (n = 3, 3). (D) Scratch wound closure was impaired in Insr
shRNA HUVECs vs control shRNA HUVECs (n = 4, 4). Major findings and implications (E) Adhesion to gelatin was impaired in Insr shRNA HUVECs vs control shRNA HUVECs, espe-
cially in context of media supplemented with VEGF-A165; representative microscopy images show DAPI-defined nuclei in blue and phalloidin-defined
filamentous actin in red, with scale bars denoting 250 μm (n = 4, 4). *P < 0.05. HUVEC, human umbilical vein endothelial cell; Insr, insulin receptor;
VEGF, vascular endothelial growth factor. unappreciated crosstalk establishes a further link between
systems regulating metabolism and angiogenesis. Ronald Kahn’s group indicate a ligand-independent
role of insulin receptors in the membrane trafficking of
brown pre-adipocytes (46), potentially aligning with our
findings. However, although the concentrations of in-
sulin experienced by sprouting endothelial cells in vivo
are unknown, our in vitro data are likely to reflect low
picomolar concentrations of insulin because of its pres-
ence in fetal calf serum. This may suggest a role for in-
sulin in promoting VEGF-induced ERK signaling, as has
been shown for epidermal growth factor signaling (47). Another explanation may be that insulin regulates a
common endocytic mechanism for its own receptor and
VEGFR2, as discussed later. Insulin and angiogenesis A number of studies have shown insulin exerts
proangiogenic effects, although they did not dissect
the role of endothelial insulin receptors (8-12). These
mainly in vitro studies revealed proangiogenic effects
of nanomolar range insulin, but did not explore more
physiological picomolar concentrations. The extent to
which picomolar insulin augments ERK signaling in
endothelial cells is a source of disagreement in the litera-
ture (11, 42-44), probably reflecting known heterogen-
eity between endothelial populations, including in their
insulin receptor expression. Notably, insulin receptor
expression is reported to be enriched in endothelial tip
cells of human tumors (13), yet tip cells generally lack a
lumen (45), so may be exposed to lower concentrations
of insulin than other endothelial cells. Hence, one pos-
sible explanation for our data is a ligand-independent
role of tip cell insulin receptors. Indeed, recent data from The only existing data describing the role of endothe-
lial insulin receptors in angiogenesis were published by
Kahn’s group in 2003. Using mice with complete deletion
of endothelial insulin receptors, they found a 57% reduc-
tion in retinal neovascularization during oxygen-induced
retinopathy (14). However, they did not study the indi-
vidual cellular processes contributing to angiogenesis, or
examine VEGF signaling. Our in vivo data suggest that
insulin receptors regulate endothelial tip cell emergence 12 Endocrinology, 2021, Vol. 162, No. 8 Figure 6. Insr knockdown in HUVECs impairs signaling responses to VEGF. (A) Representative blots illustrating major VEGF signaling nodes in
Insr shRNA HUVECs and control shRNA HUVECs grown on gelatin at baseline, 5 minutes, and 15 minutes after stimulation with VEGF-A165 50 ng/
mL, (B) with quantification of VEGF-induced phosphorylation of ERK1/2 at 5 and 15 minutes (n = 4, 4). (C) Representative blot illustrating impaired
insulin-stimulated Akt phosphorylation in Insr shRNA HUVECs vs control shRNA HUVECs after 15 minutes’ exposure to 10 and 100 nm insulin. (D)
Representative blots and (E) quantification from surface biotinylation experiment illustrating impaired internalization of VEGFR2 in Insr shRNA
HUVECs vs control shRNA HUVECs (n = 4, 4). (D) Detected biotin-labelled VEGFR2 in the presence or absence of TE and so is not directly represented
in €, which presents derived internalization data. HUVEC, human umbilical vein endothelial cell; Insr, insulin receptor; TE, trypsin exposure; VEGF,
vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2. Figure 6. Insr knockdown in HUVECs impairs signaling responses to VEGF. Insulin and angiogenesis (A) Representative blots illustrating major VEGF signaling nodes in
Insr shRNA HUVECs and control shRNA HUVECs grown on gelatin at baseline, 5 minutes, and 15 minutes after stimulation with VEGF-A165 50 ng/
mL, (B) with quantification of VEGF-induced phosphorylation of ERK1/2 at 5 and 15 minutes (n = 4, 4). (C) Representative blot illustrating impaired
insulin-stimulated Akt phosphorylation in Insr shRNA HUVECs vs control shRNA HUVECs after 15 minutes’ exposure to 10 and 100 nm insulin. (D)
Representative blots and (E) quantification from surface biotinylation experiment illustrating impaired internalization of VEGFR2 in Insr shRNA
HUVECs vs control shRNA HUVECs (n = 4, 4). (D) Detected biotin-labelled VEGFR2 in the presence or absence of TE and so is not directly represented
in €, which presents derived internalization data. HUVEC, human umbilical vein endothelial cell; Insr, insulin receptor; TE, trypsin exposure; VEGF,
vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2. studied, ERK1/2 activation has recently emerged as regu-
lating endothelial tip cell function and sprouting angiogen-
esis; (54, 55) importantly, tip cells are exposed to the highest
VEGF-A concentrations during angiogenesis, and tip cell
ERK phosphorylation is prevented in vivo by VEGF inhib-
ition (7, 55). There are many known interacting partners
of VEGFR2, which can modify its signaling and internal-
ization (41, 56). The insulin receptor signaling adaptor, in-
sulin receptor substrate-1, has been implicated in receptor
endocytosis (57), and is reported to interact with VEGFR2
(58). Interestingly, recent data show that insulin signaling
to ERK1/2 (and Src homology phosphatase 2) feeds back
via serine phosphorylation of insulin receptor substrate-1
to induce insulin receptor internalization, which aug-
ments ERK1/2 signaling (59). Therefore, it is possible that
VEGFR2 internalization is similarly affected, and this pu-
tative insulin-dependent mechanism also warrants further
exploration. Integrins are also well established to modulate
the propagation of ERK1/2 signals downstream of many
growth factor receptors, including during angiogenesis, so and migration, although with no major impact on endo-
thelial cell proliferation. Notably, we found no reduction
in nonischemic muscle tissue vascularity of Insr+/- mice,
implying that impaired vascularization ultimately catches
up; this is seen in many published examples of impaired
angiogenesis (48-50), presumably reflecting persistent acti-
vation of proangiogenic programmes. Limitations Although we show that internalization of activated
VEGFR2 is impaired, the underlying mechanism of this
requires further investigation. It will also be interesting
to explore signal transduction downstream of other re-
ceptor tyrosine kinases to assess the generalizability of
this phenomenon. As alluded to earlier, it is also im-
portant to acknowledge that impaired VEGFR2 intern-
alization may not be the only mechanism by which Insr
silencing impairs VEGF-A responses; indeed, intracellular
signaling networks are highly complex, as is their per-
turbation. Moreover, our work only sought to describe
the fundamental links between insulin receptors and
VEGF signaling during angiogenesis, and so we cannot
comment on disease relevance. However, obesity-induced
insulin resistance is associated with reduced vascular in-
sulin receptor expression and impaired angiogenesis
(63), so it would be interesting to explore endothelial
VEGFR2 internalization and ERK signal transduction Conclusions We show that endothelial insulin receptors are required
for appropriate migration and angiogenic sprouting in re-
sponse to VEGF-A, along with internalization of activated
VEGFR2 and downstream signaling to ERK1/2. This novel
link between major regulators of systemic metabolism and
angiogenesis warrants further mechanistic exploration to
understand its wider relevance. VEGFR2 signaling VEGF-A binding to VEGFR2 induces a complex intracel-
lular signaling cascade (51), a crucial element of which is
internalization (endocytosis) of ligand-bound VEGFR2. This moves the phosphorylated receptor to a domain
where it is less susceptible to the phosphatase PTP1B,
hence sustaining signal transmission, which is particularly
important for ERK1/2 signaling (40). VEGFR2 internaliza-
tion, and subsequent ERK1/2 signaling, is known to be cru-
cial to vascular biology (eg, during arterial morphogenesis
and lymphatic specification) (52, 53). Although less well Endocrinology, 2021, Vol. 162, No. 8 13 Figure 7. Schematic illustration of the proposed role of Insr during sprouting angiogenesis. Insr expression is known to be enriched in tip ECs, which
migrate along VEGF gradients, leading emerging sprouts during angiogenesis. Knockdown of Insr in ECs impairs VEGF signaling to ERK1/2 as a result
of impaired VEGFR2 internalization, which manifests as diminished sprout formation and EC migration. EC, endothelial cell; ERK, extracellular signal-
regulated kinase; Insr, insulin receptor; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2. Figure 7. Schematic illustration of the proposed role of Insr during sprouting angiogenesis. Insr expression is known to be enriched in tip ECs, which
migrate along VEGF gradients, leading emerging sprouts during angiogenesis. Knockdown of Insr in ECs impairs VEGF signaling to ERK1/2 as a result
of impaired VEGFR2 internalization, which manifests as diminished sprout formation and EC migration. EC, endothelial cell; ERK, extracellular signal-
regulated kinase; Insr, insulin receptor; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2. in this setting. Finally, our ECInsr+/- control data come
from littermates expressing Tie2-Cre, and recent data
show that Cre is not biologically inert (64); however, the
similar retinal vascular phenotype of Insr+/- and ECInsr+/-
mice provides some reassurance that off-target Cre ef-
fects do not underpin our findings. warrant future assessment (60). Finally, the insulin receptor
can regulate cytoskeletal actin remodeling (61), another
process that influences endocytosis (62), warranting further
exploration in future. References 18. Pitulescu ME, Schmidt I, Benedito R, Adams RH. Inducible
gene targeting in the neonatal vasculature and analysis of ret-
inal angiogenesis in mice. Nat Protoc. 2010;5(9):1518-1534. 1. Belfiore A, Frasca F, Pandini G, Sciacca L, Vigneri R. Insulin
receptor isoforms and insulin receptor/insulin-like growth
factor receptor hybrids in physiology and disease. Endocr Rev. 2009;30(6):586-623. 19. RRID:AB_2082660. 20. RRID:AB_2535814. 2. Manrique C, Lastra G, Sowers JR. New insights into insulin
action and resistance in the vasculature. Ann N Y Acad Sci. 2014;1311:138-150. 21. Franco CA, Jones ML, Bernabeu MO, et al. Dynamic endothe-
lial cell rearrangements drive developmental vessel regression. PLoS Biol. 2015;13(4):1–19. 3. Duncan ER, Walker SJ, Ezzat VA, et al. Accelerated endothe-
lial dysfunction in mild prediabetic insulin resistance: the early
role of reactive oxygen species. Am J Physiol Endocrinol Metab. 2007;293(5):E1311-E1319. 22. Cubbon RM, Yuldasheva NY, Viswambharan H, et al. Restoring Akt1 activity in outgrowth endothelial cells from
South Asian men rescues vascular reparative potential. Stem
Cells. 2014;32(10):2714-2723. 4. Gage MC, Yuldasheva NY, Viswambharan H, et al. Endothelium-
specific insulin resistance leads to accelerated atherosclerosis in
areas with disturbed flow patterns: a role for reactive oxygen
species. Atherosclerosis. 2013;230(1):131-139. 23. Baker M, Robinson SD, Lechertier T, et al. Use of the mouse aortic
ring assay to study angiogenesis. Nat Protoc. 2011;7(1):89-104. 24. Sobczak M, Dargatz J, Chrzanowska-Wodnicka M. Isolation
and culture of pulmonary endothelial cells from neonatal mice. J Vis Exp. 2010;(46):e2316. 5. Rask-Madsen C, Li Q, Freund B, et al. Loss of insulin signaling
in vascular endothelial cells accelerates atherosclerosis in
apolipoprotein E null mice. Cell Metab. 2010;11(5):379-389. 25. Sengupta A, Patel PA, Yuldasheva NY, et al. Endothelial in-
sulin receptor restoration rescues vascular function in male
insulin
receptor
haploinsufficient
mice. Endocrinology. 2018;159(8):2917-2925. 6. Eelen G, de Zeeuw P, Treps L, Harjes U, Wong BW, Carmeliet P. Endothelial cell metabolism. Physiol Rev. 2018;98(1):3-58. 7. Potente M, Gerhardt H, Carmeliet P. Basic and therapeutic
aspects of angiogenesis. Cell. 2011;146(6):873-887. 26. Nakatsu MN, Davis J, Hughes CCW. Optimized fibrin gel bead
assay for the study of angiogenesis. J Vis Exp. 2007;(3):e186. 8. Rensing KL, Houttuijn Bloemendaal FM, Weijers EM, et al. Could recombinant insulin compounds contribute to adeno-
carcinoma progression by stimulating local angiogenesis? Diabetologia. 2010;53(5):966-970. 27. RRID:AB_2714189. 28. RRID:AB_2280448. 29. RRID:AB_329827. 30. RRID:AB_329825. 9. Pellegatta F, Brambilla C, Reduzzi A, Bragheri M, Zerbini G,
Catapano AL. Endothelin-1 does not impair insulin-induced
angiogenesis in vitro. Acknowledgments Financial Support: This work was funded by a British Heart
Foundation Intermediate Clinical Fellowship awarded to R.M.C. (FS/12/80/29821). A.M.N.W., B.M., and K.J.G. were supported by
British Heart Foundation Clinical Research Training Fellowships. N.W. was supported by a University of Leeds Alumni PhD fellow-
ship. M.T..K is a British Heart Foundation professor. The Bioimaging
Facility has received equipment grants from the Wellcome Trust to
purchase confocal microscopes used in this project. 14 Endocrinology, 2021, Vol. 162, No. 8 10. Wei X, Song H, Semenkovich CF. Insulin-regulated protein
palmitoylation impacts endothelial cell function. Arterioscler
Thromb Vasc Biol. 2014;34(2):346-354. Author Contributions: A.M.N.W. conducted experiments, ana-
lyzed data, and drafted manuscript. N.W. conducted experiments,
analyzed data, and drafted manuscript. B.M. conducted experi-
ments, analyzed data, and drafted manuscript. N.T.W. conducted ex-
periments, analyzed data, and drafted manuscript. R.M. conducted
experiments, analyzed data, and drafted manuscript. J.S. conducted
experiments, analyzed data, and critically revised the manuscript. S.G. conducted experiments, analyzed data, and critically revised
the manuscript. N.J.H. conducted experiments, analyzed data, and
critically the revised manuscript. K.J.G. conducted experiments, ana-
lyzed data, and critically revised the manuscript. S.B.W. acquired
ethical approvals and critically revised the manuscript. N.Y.Y. con-
ducted experiments, analyzed data, and critically revised the manu-
script. D.J.B. critically revised the manuscript. P.C. critically revised
the manuscript. M.T.K. critically revised the manuscript. R.M.C. de-
signed the study, acquired funding, conducted experiments, analyzed
data, and drafted the manuscript. 11. Lassance L, Miedl H, Absenger M, et al. Hyperinsulinemia
stimulates angiogenesis of human fetoplacental endothelial
cells: a possible role of insulin in placental hypervascularization
in diabetes mellitus. J Clin Endocrinol Metab. 2013;98(9):
E1438-E1447. 12. Liu Y, Petreaca M, Martins-Green M. Cell and molecular
mechanisms of insulin-induced angiogenesis. J Cell Mol Med. 2009;13(11-12):4492-4504. 13. Goveia J, Rohlenova K, Taverna F, et al. An integrated gene ex-
pression landscape profiling approach to identify lung tumor
endothelial cell heterogeneity and angiogenic candidates. Cancer Cell. 2020;37(3):421. Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6284300 by guest on 01 March 2023 14. Kondo T, Vicent D, Suzuma K, et al. Knockout of insulin and
IGF-1 receptors on vascular endothelial cells protects against ret-
inal neovascularization. J Clin Invest. 2003;111(12):1835-1842. 15. Kahn MB, Yuldasheva NY, Cubbon RM, et al. Insulin resistance
impairs circulating angiogenic progenitor cell function and de-
lays endothelial regeneration. Diabetes. 2011;60(4):1295-1303. Additional Information Correspondence: Richard M. Cubbon, PhD, LIGHT Laborator-
ies 7.04, The University of Leeds, Clarendon Way, Leeds, LS2 9JT,
United Kingdom. E-mail: r.cubbon@leeds.ac.uk. 16. Brüning JC, Michael MD, Winnay JN, et al. A muscle-specific
insulin receptor knockout exhibits features of the metabolic
syndrome of NIDDM without altering glucose tolerance. Mol
Cell. 1998;2(5):559-569. Disclosures: The authors have nothing to disclose. Data Availability: All datasets generated during and/or analyzed
during the current study are not publicly available but are available
from the corresponding author on reasonable request. 17. Koni PA, Joshi SK, Temann UA, Olson D, Burkly L, Flavell RA. Conditional vascular cell adhesion molecule 1 deletion in mice:
impaired lymphocyte migration to bone marrow. J Exp Med. 2001;193(6):741-754. References Int J Mol Med. 2011;28(3):443-448. 31. RRID:AB_397690. 32. RRID:AB_399751. 33. RRID:AB_330744. 15 Endocrinology, 2021, Vol. 162, No. 8 34. RRID:AB_331646. 50. Saab S, Buteau B, Leclère L, et al. Involvement of plasmalogens
in post-natal retinal vascular development. Plos One. 2014;9(6):e101076. 35. RRID:AB_2212507. 36. RRID:AB_331367. 51. Koch S, Claesson-Welsh L. Signal transduction by vascular
endothelial growth factor receptors. Cold Spring Harb Perspect
Med. 2012;2(7):a006502. 37. RRID:AB_772210. 38. RRID:AB_772206. 39. McNally BD, Moran A, Watt NT, et al. Inorganic nitrate pro-
motes glucose uptake and oxidative catabolism in white adi-
pose tissue through the XOR-catalyzed nitric oxide pathway. Diabetes. 2020;69(5):893-901. 52. Deng Y, Atri D, Eichmann A, Simons M. Endothelial ERK
signaling controls lymphatic fate specification. J Clin Invest. 2013;123(3):1202-1215. 53. Simons M, Eichmann A. Molecular controls of arterial morpho-
genesis. Circ Res. 2015;116(10):1712-1724. 40. Simons M, Gordon E, Claesson-Welsh L. Mechanisms and regu-
lation of endothelial VEGF receptor signalling. Nat Rev Mol
Cell Biol. 2016;17(10):611-625. 54. Mavria G, Vercoulen Y, Yeo M, et al. ERK-MAPK signaling
opposes Rho-kinase to promote endothelial cell survival and
sprouting during angiogenesis. Cancer Cell. 2006;9(1):33-44. Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6284300 by guest on 01 March 2023 41. Jones MC, Caswell PT, Norman JC. Endocytic recycling path-
ways: emerging regulators of cell migration. Curr Opin Cell
Biol. 2006;18(5):549-557. 55. Shin M, Beane TJ, Quillien A, Male I, Zhu LJ, Lawson ND. Vegfa signals through ERK to promote angiogenesis, but not
artery differentiation. Development. 2016;143(20):3796-3805. 42. Johansson GS, Chisalita SI, Arnqvist HJ. Human microvascular
endothelial cells are sensitive to IGF-I but resistant to insulin at
the receptor level. Mol Cell Endocrinol. 2008;296(1-2):58-63. 56. Simons M. An inside view: VEGF receptor trafficking and
signaling. Physiology (Bethesda). 2012;27(4):213-222. 43. De Nigris V, Pujadas G, La Sala L, Testa R, Genovese S,
Ceriello A. Short-term high glucose exposure impairs in-
sulin signaling in endothelial cells. Cardiovasc Diabetol. 2015;14:114. 57. Yoneyama Y, Lanzerstorfer P, Niwa H, et al. IRS-1 acts as an
endocytic regulator of IGF-I receptor to facilitate sustained IGF
signaling. Elife. 2018;7:e32893. 44. Li G, Barrett EJ, Wang H, Chai W, Liu Z. Insulin at physiological
concentrations selectively activates insulin but not insulin-like
growth factor I (IGF-I) or insulin/IGF-I hybrid receptors in
endothelial cells. Endocrinology. 2005;146(11):4690-4696. 58. Senthil D, Ghosh Choudhury G, Bhandari BK, Kasinath BS. The
type 2 vascular endothelial growth factor receptor recruits in-
sulin receptor substrate-1 in its signalling pathway. Biochem J. 2002;368(Pt 1):49-56. 45. Iruela-Arispe ML, Davis GE. References Cellular and molecular mechanisms
of vascular lumen formation. Dev Cell. 2009;16(2):222-231. 59. Choi E, Kikuchi S, Gao H, et al. Mitotic regulators and the
SHP2-MAPK pathway promote IR endocytosis and feedback
regulation of insulin signaling. Nat Commun. 2019;10(1):1473. 46. Nagao H, Cai W, Wewer Albrechtsen NJ, et al. Distinct
signaling by insulin and IGF-1 receptors and their extra-
and intracellular domains. Proc Natl Acad Sci USA. 2021;118(17):e2019474118. 60. Schwartz MA, Ginsberg MH. Networks and crosstalk: integrin
signalling spreads. Nat Cell Biol. 2002;4(4):E65-E68. 61. Hu H, Juvekar A, Lyssiotis CA, et al. Phosphoinositide 3-kinase
regulates glycolysis through mobilization of aldolase from the
actin cytoskeleton. Cell. 2016;164(3):433-446. 47. Borisov N, Aksamitiene E, Kiyatkin A, et al. Systems-level
interactions between insulin-EGF networks amplify mitogenic
signaling. Mol Syst Biol. 2009;5:256. 62. Smythe E, Ayscough KR. Actin regulation in endocytosis. J Cell
Sci. 2006;119(Pt 22):4589-4598. 48. Ngo MH, Borowska-Fielding J, Heathcote G, et al. Fzd4
haploinsufficiency delays retinal revascularization in the
mouse model of oxygen induced retinopathy. Plos One. 2016;11(8):e0158320. 63. Cubbon RM, Ali N, Sengupta A, Kearney MT. Insulin- and
growth factor-resistance impairs vascular regeneration in dia-
betes mellitus. Curr Vasc Pharmacol. 2012;10(3):271-284. 49. Napp LC, Jabs O, Höckelmann A, et al. Normal endothelial but
impaired arterial development in MAP-kinase activated protein
kinase 2 (MK2) deficient mice. Vasc Cell. 2016;8:4. 64. Brash JT, Bolton RL, Rashbrook VS, Denti L, Kubota Y, Ruhrberg C. Tamoxifen-activated CreERT impairs retinal angiogenesis inde-
pendently of gene deletion. Circ Res. 2020;127(6):849-850.
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https://openalex.org/W4385207978
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https://jurnal.unimed.ac.id/2012/index.php/handayani/article/download/6528/9534
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Indonesian
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UPAYA MENINGKATKAN BELAJAR PKN MELALUI PENERAPAN MODEL NHT DI KELAS V SDN 050676 KEBUN BALOK
|
Jurnal handayani
| 2,017
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cc-by-sa
| 3,954
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UPAYA MENINGKATKAN BELAJAR PKN MELALUI PENERAPAN
MODEL NHT DI KELAS V SDN 050676 KEBUN BALOK Zaitun
Guru Kelas di SD Negeri 050676 Kebun Balok
Surel : tesiaite@yahoo.com Abstract: Efforts To Improve Student Learning Outcomes Pkn Through
Cooperative Learning Model Application Type NHT In Class V SDN 050676
Garden State Beam.This study aims to improve students' understanding of the
material. The research subject of sixth grade students by the number of students as
many as 13 people. Student activity data according to the second observation
observer in the cycle I and II are Activities writing / reading (37.1%), working LKS
(25.9%), asked their peers (13.5%), to ask the teacher (11.8 %), and are not relevant
to KBM (11.8%). Activities of students in the second cycle of observation by both
observers, among others: writing / reading (24.7%), Working LKS (49.9%), asked
their peers (16.5%), ask the teacher (10.6%), and are not relevant to KBM (2.4% ). Keywords: Cooperative Learning type Numbered Heads Together, Learning
Outcomes, Learning Activities Keywords: Cooperative Learning type Numbered Heads Together, Learning
Outcomes, Learning Activities Abstrak: Upaya Meningkatkan Belajar Pkn Melalui Penerapan Model
Pembelajaran Kooperatif Tipe NHT Di Kelas V SDN 050676 Kebun Balok. Penelitian ini bertujuan untuk meningkatkan pemahaman siswa terhadap materi. Subjek penelitian siswa kelas VI dengan jumlah siswa sebanyak 13 orang. Data
aktivitas siswa menurut kedua pengamatan pengamat pada siklus I dan II adalah
Aktivitas menulis/membaca (37,1%), mengerjakan LKS (25,9% ), bertanya sesama
teman (13,5%), bertanya kepada guru (11,8%), dan yang tidak relevan dengan KBM
(11,8%). Aktivitas siswa pada Siklus II menurut pengamatan kedua pengamat
antara lain: menulis/membaca (24,7%), Mengerjakan LKS (49,9%), bertanya
sesama teman (16,5%), bertanya kepada guru (10,6%), dan yang tidak relevan
dengan KBM (2,4%). Kata Kunci : Pembelajaran Kooperatif tipe Numbered Heads Together, Hasil
Belajar Siswa, Aktivitas Belajar Kata Kunci : Pembelajaran Kooperatif tipe Numbered Heads Together, Hasil
Belajar Siswa, Aktivitas Belajar PENDAHULUAN oleh ombak dan tergulung oleh gelombang,
serta
mudah
kehilangan
arah
dalam
melangkah, kecuali bagi yang memiliki daya
tahan dan daya-suai yang tinggi serta
pedoman dan pegangan hidup yang kuat. Oleh karena itu pembentukan karakter siswa
yang aktif, kreatif dan terampil sangat
diperlukan,
agar
kelak
siswa
mampu
bersaing di era globalisasi yang juga
mencerminkan
kualiatas
bangsa
dan
negaranya. Dalam kehidupan suatu negara,
pendidikan memegang peranan yang amat
penting
untuk
menjamin
kelangsungan
hidup bangsa dan negara, karena pendidikan
merupakan wahana untuk meningkatkan dan
mengembangkan
kualitas
sumber
daya
manusia. Pendidikan adalah usaha sadar dan
terencana
untuk
mewujudkan
suasana
belajar dan proses pembelajaran agar
peserta didik secara aktif mengembangkan
potensi dirinya untuk memiliki kekuatan
spiritiual keagamaan, pengendalian diri,
kepribadian, kecerdasan, akhlak mulia, serta
keterampilan
yang
diperlukan
dirinya,
masyarakat, bangsa dan negara. Berdasarkan uraian di atas, penulis
sebagai tenaga pendidik terdorong agar
secara langsung ikut dalam mempersiapkan
siswa/siswi yang aktif, kreatif dan terampil
demi kelangsungan bangsa dan negara
sesuai dengan tujuan pendidikan nasional
Indonesia. Hal yang paling nyata penulis
lakukan yakni dengan cara meningkatkan
kualitas pembelajaran di kelas agar kiranya
pembelajaran
di
sekolah
menjadi Di era globalisasi dan pasar bebas
sekarang ini manusia dihadapkan pada
perubahan-perubahan
besar
yang
tidak
menentu dan sulit diprediksi. Manusia ibarat
buih di lautan lepas yang mudah terseret p-ISSN: 2355 - 1739
e-ISSN: 2407 - 6295 p-ISSN: 2355 - 1739
e-ISSN: 2407 - 6295 146 Jurnal Handayani (JH).Vol 6 (2) Desember 2016, Halm 146- 152 pembelajaran yang menyenangkan bagi
siswa yang mampu meningkatkan motivasi
belajar siswa, aktivitas belajar siswa dan
hasil belajar siswa. (NHT)
Numbered
Heads
Together
digunakan untuk mengajarkan isi akademik
atau
untuk
mengetahui
sejauh
mana
pemahaman siswa terhadap suatu materi
pelajaran yang diajarkan. Guru menciptakan
interaksi yang mendorong rasa ingin tahu,
ingin mencoba, bersikap mandiri dan ingin
maju. Guru memberikan suatu informasi
yang mendasar saja sebagai dasar pemikiran
bagi
anak
didik
dalam
mencari
dan
menemukan sendiri informasi lainnya. j
Berdasarkan
pengalaman
penulis
selama mengajar, masalah belajar pasti
selalu di temukan pada setiap kelas yang
penulis ajar. Pada umumnya masalah belajar
siswa tergolong pada tiga bagian yakni
rendahnya motivasi dan minat belajar siswa,
rendahnya aktivitas belajar siswa dan
rendahnya hasil belajar siswa. Pembelajaran
yang telah dilakukan selama dua bulan
setelah memasuki ajaran baru tampak
masalah yang dominan dalam pembelajaran
adalah aktivitas belajar dan hasil belajar
yang rendah. PENDAHULUAN Dalam proses pembelajaran
dari 13 siswa yang ada di kelas V hanya 3
orang yang aktif dalam proses pembelajaran,
10 lainnya tampak pasif dan ada seorang
siswa yang tidak perduli sama sekali tentang
materi pelajaran dan tugas-tugas serta tidak
ada interaksi di dalam kelas. Siswa tersebut
jarang sekali berbicara dan tugas di sekolah
tidak dikerjakan dengan baik seperti teman-
teman yang lain. Disamping itu akibat
rendahnya aktivitas belajar siswa berdampak
besar terhadap hasil belajar siswa. Dari 11
orang hanya 45,5% yang nilai sehari-hari
dan ujian bulanan yang mencapai KKM dan
sisanya harus mengulang pelajaran untuk
memperbaiki nilai. Hal tersebut membuat
proses pelajaran terhambat. Penulis telah
mengupayakan beberapa inovasi untuk lebih
meningkatakan kualitas pembelajaran seperti
penerapan
model
pembelajaran
dan
pemanfaatan media ajar, namun upaya
tersebut belum dapat semaksimal mungkin
meningkatakn kualiatas pembelajaran yang
akan berimbas pada peningkatan hasil
belajar
siswa. Keberhasilan
penelitian
sebelumya
yang
menerapkan
model
pembelajaran Inkuiri mendapat pengaruh
positif terhadap peningkatan hasil belajar
siswa, selanjutnya akan dilakukan penelitin
lanjutan pada kelas yang sama dengan
model yang berbeda. Oleh karena itu perlu
kiranya di lakukan sebuah penelitian untuk
melihat efektivitas dari inovasi yang penulis
terapkan
agar
dapat
dilihat
letak
kesalahannya sehingga dapat ditemukan
tindakan perbaikan yang tepat agar inovasi
yang dilakukan benar-benar optimal. Adapun
alternatif
pemecahan
masalah belajar siswa dalam penelitian ini Dari uraian latar belakang, maka
penulis dapat merumuskan permasalahan
yang akan dijawab yaitu 1) Apakah
penerapan model pembelajaran Numbered
Heads Together (NHT) dapat meningkatkan
hasil belajar siswa pada bidang studi PKn di
kelas
VI
SD
Negeri
050676
Kebun
Balok?;2)
Apakah
penerapan
model
pembelajaran Numbered Heads Together
(NHT) dapat meningkatkan aktivitas belajar
siswa pada bidang studi PKn di kelas V SD
Negeri 050676 Kebun Balok?. Sehingga
berdasarkan rumusan masalah tersebut,
penelitian
ini
bertujuan
untuk;
1)
Mengetahuui
apakah
penerapan
model
pembelajaran Numbered Heads Together
(NHT) dapat meningkatkan hasil belajar
siswa pada bidang studi PKn di kelas V SD
Negeri 050676 Kebun Balok; 2) Mengetahui
apakah
penerapan
model
pembelajaran
Numbered Heads Together (NHT) dapat
meningkatkan aktivitas belajar siswa pada
bidang studi PKn di kelas V SD Negeri
050676 Kebun Balok. Model Pembelajaran Kooperatif Tipe
Numbered Heads Together (NHT) atau
penomoran
berpikir
bersama
adalah
merupakan jenis pembelajaran kooperatif
yang dirancang untuk mempengaruhi pola
interaksi
siswa
dan
sebagai
alternatif
terhadap struktur kelas. PEMBAHASAN Pembelajaran menggunakan model
pembelajaran
kooperatif
pendekatan
struktural
Numbered
Head
Together
memberikan
kesempatan
kepada
siswa
untuk saling membagikan ide-ide dan
mempertimbangkan jawaban yang paling
tepat
sehingga
secara
tidak
langsung
menuntut siswa untuk mau dan mampu
mengkonstruksi pengetahuan melalui proses
kelompok maupun individu melalui setiap
tahapan model pembelajaran di setiap
Siklus. Penelitian ini dilaksanakan dalam
dua
siklus. Masing-masing
siklus
dilaksanakan dua kali pertemuan, pertemuan
digunakan untuk pembahasan materi dengan
alokasi
waktu
2x35
menit. Hal
ini
disesuaikan dengan jadwal pelajaran PKn di
kelas V SD Negeri 050676 Kebun Balok. Alat
pengumpul
data
dalam
penelitian ini adalah: tes hasil belajar untuk
mengetahui kemampuan kognitif dan lembar
observasi untuk mengamati aktivitas belajar
siswa. Penelitian ini berbentuk Penelitian
Tindakan Kelas (PTK). PTK pertama kali
diperkenalkanoleh psikologi sosial Amerika
yang bernama Kurt Lewin pada tahun 1946
(Aqib, 2006 :13). Menurut Lewin dalam
Aqib (2006 : 21) menyatakan bahwa dalam
satu Siklus terdiri atas empat langkah, yaitu
perencanaan (planning), tindakan (acting),
observasi
(observing)
dan
refleksi
(reflecting). Langkah-langkah pengolahan data
sebagai berikut: Sebelum
dilaksanakan
Siklus
I
dilakukan
uji
awal
untuk
menjajaki
kemampuan
awal
siswa. Hasil
pretes
menunjukkan nilai rata-rata 39,2 dengan
nilai terendah 30 dan tertinggi 60. Dengan
ketuntasan minimal (KKM) sebesar 70 maka
ketuntasan klasikal hanya sebesar 0%. 1. Merekapitulasi nilai pretes sebelum
tindakan dan nilai tes akhir Siklus I dan
Siklus II. 2. Menghitung nilai rerata atau persentase
hasil belajar siswa sebelum dilakukan
tindakan dengan hasil belajar setelah
dilakukan tindakan pada Siklus I dan
Siklus II untuk mengetahui adanya
peningkatan hasil belajar. 2. Menghitung nilai rerata atau persentase
hasil belajar siswa sebelum dilakukan
tindakan dengan hasil belajar setelah
dilakukan tindakan pada Siklus I dan
Siklus II untuk mengetahui adanya
peningkatan hasil belajar. Nilai hasil Formatif dalam Siklus I
disajikan dalam tabel. Tabel Deskripsi Data Formatif I
Nilai
Frekuensi
Rata-rata
40
3
61,5
60
6
80
4
Jumlah
13 3. Untuk penilaian aktivitas digunakan
rumus sebagai berikut: 3. Untuk penilaian aktivitas digunakan
rumus sebagai berikut: % 𝑃𝑟𝑜𝑝𝑜𝑟𝑠𝑖 𝐴𝑘𝑡𝑖𝑣𝑖𝑡𝑎𝑠
𝑗𝑢𝑚𝑙𝑎ℎ𝑠𝑘𝑜𝑟𝑦𝑎𝑛𝑔𝑑𝑖𝑝 % 𝑃𝑟𝑜𝑝𝑜𝑟𝑠𝑖 𝐴𝑘𝑡𝑖𝑣𝑖𝑡𝑎𝑠 % 𝑃𝑟𝑜𝑝𝑜𝑟𝑠𝑖 𝐴𝑘𝑡𝑖𝑣𝑖𝑡𝑎𝑠
𝑗𝑢𝑚𝑙𝑎ℎ 𝑠𝑘𝑜𝑟 𝑦𝑎𝑛𝑔 𝑑𝑖𝑝𝑒𝑟𝑜𝑙𝑒ℎ % 𝑃𝑟𝑜𝑝𝑜𝑟𝑠𝑖 𝐴𝑘𝑡𝑖𝑣𝑖𝑡𝑎𝑠
𝑗𝑢𝑚𝑙𝑎ℎ 𝑠𝑘𝑜𝑟 𝑦𝑎𝑛𝑔 𝑑𝑖𝑝𝑒𝑟𝑜𝑙𝑒ℎ 𝑗𝑢𝑚𝑙𝑎ℎ 𝑠𝑘𝑜𝑟 𝑦𝑎𝑛𝑔 𝑑𝑖𝑝𝑒𝑟𝑜𝑙𝑒ℎ𝑥 = 𝑗𝑢𝑚𝑙𝑎ℎ 𝑠𝑘𝑜𝑟 𝑦𝑎𝑛𝑔 𝑑𝑖𝑝𝑒𝑟𝑜𝑙𝑒ℎ = 𝑗
𝑦
𝑔
𝑝
𝑗𝑢𝑚𝑙𝑎ℎ 𝑠𝑘𝑜𝑟 𝑖𝑑𝑒𝑎𝑙
𝑥 100%
(Majid, 2009:268) 4. Ketentuan persentase ketuntasan belajar
kelas: 4. Ketentuan persentase ketuntasan belajar
kelas: Merujuk pada tabel di atas, nilai
terendah formatif I adalah 40 berjumlah 3
orang siswa dan nilai tertinggi 80 berjumlah
4 orang siswa. Zaitun, Upaya Meningkatkan Belajar ... Zaitun, Upaya Meningkatkan Belajar ... demikian, dalam kelompok siswa diberi
nomor masing-masing sesuai urutannya. Berkaitan dengan indikator kinerja
Suwandi dan Madyo Eko Susilo (2007:36)
menyatakan
bahwa
”Indikator
kinerja
merupakan rumusan kinerja yang akan
dijadikan dalam menentukan keberhasilan
atau keefektifan penelitian. Dalam penelitian
ini indikator pencapaian apabila nilai siswa
secara individu mencapai KKM Bahasa
Indonesia yaitu 70 yang ditetapkan sekolah
dan secara klasikal ≥ 85% siswa mencapai
KKM tersebut. METODE Pengambilan data untuk penelitian ini
dilakukan di SD Negeri 050676 Kebun
Balok di Jalam Pendidikan Desa Kebun
Balok
Kecamatan
Wampu
Kabupaten
Langkat. Waktu penelitian dilakukan selama
5 bulan terhitung mulai bulan Februari
sampai bulan Juni tahun 2016. Subjek penelitian adalah siswa-siswi
Kelas V SD Negeri 050676 Kebun Balok
yang berjumlah 13 orang Tahun Pelajaran
2015/2016. PEMBAHASAN 9 orang siswa mendapat
nilai di bawah kriteria ketuntasan minimal
dan ketuntasan klasikal sebesar 40,7%. Nilai
ini berada di bawah kriteria keberhasilan
sehingga dapat dikatakan KBM siklus I
kurang berhasil memberi ketuntasan belajar. PENDAHULUAN Numbered Heads
Together (NHT) pertama kali dikembangkan
oleh
Spenser
Kagen
(1993)
untuk
melibatkan lebih banyak siswa dalam
menelaah materi yang tercakup dalam suatu
pelajaran dan mengecek pemahaman mereka
terhadap isi pelajaran tersebut (Trianto,
2007: 62). Pengertian lain dari model ini
(Istarani, 2011: 12) merupakan rangkaian
penyampaian materi dengan menggunakan
kelompok sebagai wadah dalam menyatakan
persepsi/pikiran siswa terhadap pertanyaan
yang dilontarkan atau diajukan guru, yang
kemudian akan dipertanggungjawabkan oleh
siswa sesuai dengan nomor permintaan guru
dari masing-masing kelompok. Dengan Adapun
alternatif
pemecahan
masalah belajar siswa dalam penelitian ini
yakni
dengan
menerapan
model
pembelajaran Numbered Heads Together
(NHT). Model pembelajaran kooperatif tipe p-ISSN: 2355 - 1739
e-ISSN: 2407 - 6295 p-ISSN: 2355 - 1739
e-ISSN: 2407 - 6295 147 kelas
belajar
Ketuntasan %
100
K
Sb
ΣSb = Jumlah siswa yang mendapat
nilai ≥ KKM
ΣK = Jumlah siswa %
100
K
Sb
ΣSb = Jumlah siswa yang mendapat
nilai ≥ KKM
ΣK
J
l h i K
ΣSb = Jumlah siswa yang mendapat
nilai ≥ KKM ΣK = Jumlah siswa 148 Jurnal Handayani (JH).Vol 6 (2) Desember 2016, Halm 146-152 Data hasil formatif I ini dapat disajikan
kembali dalam grafik histogram berikut ini. Data hasil formatif I ini dapat disajikan
kembali dalam grafik histogram berikut ini. Merujuk
pada
hasil
belajar
dan
aktivitas belajar siswa serta dokumentasi
penelitian, belum tercapainya hasil belajar
siswa secara klasikal 85% seperti indikator
ketercapaian pada penelitian ini disebabkan
oleh : Grafik Data Hasil Formatif I Grafik Data Hasil Formatif I 1. Masih
terjadi
kesalahan-kesalahan
dalam
penarikan
kesimpulan
oleh
siswa
yang
menandakan
terjadi
kesalahpahaman
dalam
diskusi
kelompok. p
2. Jawaban siswa yang sama dengan
jawaban temannya masih banyak hal
ini
menunjukkan
bahwa
siswa
cenderung untuk menyalin jawaban
temannya. Grafik Data Hasil Formatif I Penilaian aktivitas diperoleh dari
lembar observasi aktivitas yang dilakukan
pada saat siswa bekerja kelompok. Pengamatan dilakukan oleh dua pengamat
selama 20 menit kerja kelompok dalam
setiap kegiatan belajar-mengajar (KBM). 3. Sebagian siswa masih belum terbiasa
dengan alur pembelajaran atau masih
bingung diperlihatkan dengan besarnya
aktivitas menulis dan membaca yang
baru dilakukan di sekolah (42%) yang
seharusnya sudah dilakukan di rumah. p
g
j
g j
Tabel Aktivitas Belajar Siswa Siklus I Tabel Aktivitas Belajar Siswa Siklus I Tabel Aktivitas Belajar Siswa Siklus I
No
Aktivitas
Jumlah
Pro-
porsi
1
Menulis/membaca
63
37,1%
2
Mengerjakan LKS
44
25,9%
3
Bertanya pada teman
23
13,5%
4
Bertanya pada guru
20
11,8%
5
Yang tidak relevan
dengan KBM
20
11,8%
JUMLAH
170
100% 4. Sebagian siswa belum memiliki rasa
tanggung jawab terhadap tugasnya
dalam kelompok diperlihatkan dengan
aktivitas mengerjakan LKS yang hanya
mencapai 25,9%. 5. Ada
siswa
yang
lebih
suka
mengerjakan soal sendiri. 6. Pada
tahap
pengajuan
pertanyaan
sebagian besar siswa tidak memusatkan
perhatian dalam mengerjakan LKS,
terbukti
dari
tingginya
persentase
aktivitas yang tidak relevan dengan
KBM yaitu 11,8%. Dengan
pengamatan
setiap
dua
menit, maka nilai maksimum yang mungkin
teramati untuk satu kategori aktivitas selama
20 menit tersebut adalah 10 kali. Nilai
aktivitas untuk setiap KBM adalah rata-rata
dari nilai aktivitas kedua pengamat. Karena
dalam satu siklus terdapat dua KBM, maka
nilai aktivitas tiap kategori untuk satu siklus
adalah rata-rata dari aktivitas kedua KBM. (5) Pengelolaan waktu diharapkan lebih
efisien. Data hasil belajar Siklus II disajikan dalam
tabel berikut ini. P d Sikl
II
t l h
k Tabel Deskripsi Data Hasil Formatif II
Nilai
Frekuensi
Rata-rata
80
10
84,6
100
3
Jumlah
13 Tabel Deskripsi Data Hasil Formatif II Tabel Deskripsi Data Hasil Formatif II
Nilai
Frekuensi
Rata-rata
80
10
84,6
100
3
Jumlah
13
Data hasil formatif II ini dapat
disajikan kembali dalam bentuk grafik
histogram dan dapat dilihat pada grafik
berikut ini Data hasil formatif II ini dapat
disajikan kembali dalam bentuk grafik
histogram dan dapat dilihat pada grafik
berikut ini. Pada Siklus II guru telah menerapkan
model pembelajaran kooperatit tipe tutor
sebaya dengan baik dan dilihat dari aktivitas
siswa serta hasil belajar siswa pelaksanaan
proses belajar mengajar sudah berjalan
dengan baik. Maka tidak diperlukan revisi
terlalu
banyak,
tetapi
yang
perlu
diperhatikan untuk tindakah selanjutnya
adalah memaksimalkan dan mepertahankan
apa yang telah ada dengan tujuan agar pada
pelaksanaan
proses
belajar
mengajar
selanjutnya penerapan pembelajaran tuntas
dapat meningkatkan proses belajar mengajar
sehingga tujuan pembelajaran dapat tercapai. Akan tetapi karena keterbatasan biaya dan
waktu
dalam
desain
penelitian
maka
penelitian direncanakan dalam dua siklus
saja. Grafik Hasil Formatif Siklus II Nilai rata-rata tes siswa sebelum
menerapkan model pembelajaran kooperatif
tipe Numbered Head Together adalah 39,2
dan setelah diterapkan model pembelajaran
kooperatif tipe Numbered Head Together
meningkat menjadi 61,5 pada Siklus I dan
84,8 pada Siklus II. Ketuntasan belajar
secara klasikal juga mengalami peningkatan,
sebelumnya hanya ada lima siswa tuntas 70
(ketuntasan secara klasikal 30,7%) pada
Siklus I dan pada siklus II menjadi 100%. Dapat disimpulkan KBM Siklus I gagal
memberikan ketuntasan klasikal pada siswa
sedangkan
KBM
Siklus
II
berhasil
memberikan ketuntasan klasikal. j
Pembelajaran
pertemuan
pertama
pada
Siklus
I
diawali
dengan
pengelompokkan
siswa
menjadi
3
kelompok,
masing-masing
kelompok
berjumlah 4-5 orang siswa. Kemudian dari
masing-masing kelompok tersebut diberi
nomor identitas dalam kelompok dari 1-4. Saat
pembelajaran
berlangsung
masih
terlihat siswa belum biasa menangkap alur
dan konsep yang diberikan guru saat
pembelajaran. Hal ini terlihat dari selama
proses pembelajaran sebagian siswa hanya
sibuk dengan kegiatan mereka masing-
masing meskipun guru sudah menegur
mereka. Saat presentasi kelompok, masih
banyak siswa yang salah dalam menarik
kesimpulan dan ada siswa yang tidak
mengikuti alur jalannya diskusi sehingga
siswa tersebut tidak dapat memahami isi
pelajaran yang sedang berlangsung. Sedangkan, data hasil aktivitas siswa
dapat dilihat pada tabel berikut ini. kelas
belajar
Ketuntasan 7. Waktu yang digunakan dalam kegiatan
pembelajaran
tidak
sesuai
dengan
waktu yang direncanakan sebelumnya. Perbaikan yang akan dilakukan pada
sebelum melakukan Siklus II adalah sebagai
berikut: Perbaikan yang akan dilakukan pada
sebelum melakukan Siklus II adalah sebagai
berikut: (1) Penjelasan dan penekanan kepada
siswa tentang pembelajaran kooperatif
di
antaranya
kerja
sama
dalam
menyelesaikan tugas, saling membantu,
dan berdiskusi harus diperhatikan. Data
aktivitas
belajar
siswa
ditampilkan dalam bentuk diagram dapat
dilihat pada grafik berikut ini. Grafik Aktivitas Belajar Siswa Siklus I (2) Pada pelaksanaan siklus II penomoran
siswa pada masing-masing kelompok
sama dengan pelaksanaan siklus I
untuk
menghindari
siswa
saling
berebut dalam penentuan nomor. (3) Guru harus lebih sering mengunjungi
kelompok pada tahap berpikir bersama
berlangsung untuk menghindari siswa
ngobrol sendiri di luar materi pelajaran. (4) Guru harus memberikan penghargaan
kepada
siswa/kelompok
yang
mendapatkan skor tertinggi. Grafik Aktivitas Belajar Siswa Siklus I p-ISSN: 2355 - 1739
e-ISSN: 2407 - 6295 149 Zaitun, Upaya Meningkatkan Belajar ... (5) Pengelolaan waktu diharapkan lebih
efisien. Data hasil belajar Siklus II disajikan dalam
tabel berikut ini. Tabel Deskripsi Data Hasil Formatif II
Nilai
Frekuensi
Rata-rata
80
10
84,6
100
3
Jumlah
13
Data hasil formatif II ini dapat
disajikan kembali dalam bentuk grafik
histogram dan dapat dilihat pada grafik
berikut ini. Grafik Hasil Formatif Siklus II
Nilai rata-rata tes siswa sebelum
menerapkan model pembelajaran kooperatif
tipe Numbered Head Together adalah 39,2
dan setelah diterapkan model pembelajaran
kooperatif tipe Numbered Head Together
meningkat menjadi 61,5 pada Siklus I dan
84,8 pada Siklus II. Ketuntasan belajar
secara klasikal juga mengalami peningkatan,
sebelumnya hanya ada lima siswa tuntas 70
(ketuntasan secara klasikal 30,7%) pada
Siklus I dan pada siklus II menjadi 100%. Dapat disimpulkan KBM Siklus I gagal
memberikan ketuntasan klasikal pada siswa
sedangkan
KBM
Siklus
II
berhasil
memberikan ketuntasan klasikal. Sedangkan, data hasil aktivitas siswa
dapat dilihat pada tabel berikut ini. Tabel Data Aktivitas Belajar Siswa Siklus
II
No
Aktivitas
Jumlah
Pro-
porsi
1
Menulis/membaca
42
24,7%
2
Mengerjakan LKS
78
45,9%
3
Bertanya pada
teman
28
16,5%
4
Bertanya pada
guru
18
10,6%
Yang tidak
relevan dengan (5) Pengelolaan waktu diharapkan lebih
efisien. Data hasil belajar Siklus II disajikan dalam
tabel berikut ini. Tabel di
atas
dapat dikonversi
menjadi diagram histogram seperti berikut
ini. (5) Pengelolaan waktu diharapkan lebih
efisien. Data hasil belajar Siklus II disajikan dalam
tabel berikut ini. (5) Pengelolaan waktu diharapkan lebih
efisien. Tabel Data Aktivitas Belajar Siswa Siklus Tabel Data Aktivitas Belajar Siswa Siklus
II
No
Aktivitas
Jumlah
Pro-
porsi
1
Menulis/membaca
42
24,7%
2
Mengerjakan LKS
78
45,9%
3
Bertanya pada
teman
28
16,5%
4
Bertanya pada
guru
18
10,6%
5
Yang tidak
relevan dengan
KBM
4
2,4%
JUMLAH
170
100% Pertemuan kedua pada Siklus I,
kegiatan pembelajaran dilaksanakan sama
seperti pada pertemuan pertama. Mulai dari
pengelompokkan, pemberian nomor pada
masing-masing siswa. Pertemuan kedua ini
siswa sudah mulai beradaptasi dan aktif saat p-ISSN: 2355 - 1739
e-ISSN: 2407 - 6295 p-ISSN: 2355 - 1739
e-ISSN: 2407 - 6295 150 Jurnal Handayani (JH).Vol 6 (2) Desember 2016, Halm 146-152 menyelesaikan tugas, saling membantu,
dan berdiskusi harus diperhatikan. menyelesaikan tugas, saling membantu,
dan berdiskusi harus diperhatikan. pembelajaran berlangsung. Guru juga ikut
memberikan
masukkan
dalam
kerja
kelompok jika terdapat perselisihan atau
perbedaan pendapat saat diskusi kelompok
berlangsung. Kerja sama antarkelompok
sudah mulai terlihat. Presentasi kelompok
juga berlangsung dengan baik. Namun
demikian perolehan nilai rata-rata kelas 61,5
dengan ketuntasan kelas hanya mencapai
30,7%. Sehingga dapat dikatakan ketuntasan
klasikal belum tercapai. Hal ini karena
pembelajaran Siklus I masih terkendala pada (2) Pada pelaksanaan siklus II, penomoran
siswa pada masing-masing kelompok
sama dengan pelaksanaan siklus I
untuk
menghindari
siswa
saling
berebut dalam penentuan nomor. (3) Guru harus lebih sering mengunjungi
kelompok pada tahap berpikir bersama
berlangsung untuk menghindari siswa
bercerita di luar materi pelajaran. (3) Guru harus lebih sering mengunjungi
kelompok pada tahap berpikir bersama
berlangsung untuk menghindari siswa
bercerita di luar materi pelajaran. (3) Guru harus lebih sering mengunjungi
kelompok pada tahap berpikir bersama
berlangsung untuk menghindari siswa
bercerita di luar materi pelajaran. (4) Guru harus memberikan penghargaan
kepada
siswa/kelompok
yang
mendapatkan skor tertinggi. (1) Masih
terjadi
kesalahan-kesalahan
dalam
penarikan
kesimpulan
oleh
siswa
yang
menandakan
terjadi
kesalahpahaman
dalam
diskusi
kelompok. (5) Pengelolaan waktu diharapkan lebih
efisien. Hasil yang diperoleh setelah melakukan
Siklus II yaitu adanya peningkatan aktivitas
belajar
siswa. Aktivitas
menulis
dan
membaca menurun sedikit dari 31,7%
menjadi 24,7%. Aktivitas mengerjakan LKS
dari siklus I dan II memiliki proporsi yang
berbeda yaitu 25,9% meningkat pada siklus
II 45,9%. Sementara aktivitas bertanya pada
teman mengalami peningkatan dari 13,5%
menjadi
16,5%. Bertanya
pada
guru
mengalami penurunan dari 11,8% menjadi
10,6%. Dan
aktivitas
tidak
relevan
mengalami penurunan dari 11,8% menjadi
2,4%. (2) Jawaban siswa yang sama dengan
jawaban temannya masih banyak. Hal
ini
menunjukkan
bahwa
siswa
cenderung untuk menyalin jawaban
temannya. (5) Pengelolaan waktu diharapkan lebih
efisien. (3) Sebagian siswa masih belum terbiasa
dengan alur pembelajaran atau masih
bingung,
diperlihatkan
dengan
besarnya
aktivitas
menulis
dan
membaca yang baru dilakukan di
sekolah
(37,1%)
yang
seharusnya
sudah dilakukan di rumah. (4) Sebagian siswa belum memiliki rasa
tanggung jawab terhadap tugasnya
dalam kelompok diperlihatkan dengan
aktivitas mengerjakan LKS yang hanya
mencapai 25,9%. Nilai rata-rata tes siswa sebelum
menerapkan model pembelajaran kooperatif
tipe Numbered Head Together adalah 39,2
dan setelah diterapkan model pembelajaran
kooperatif tipe Numbered Head Together
meningkat menjadi 61,5 pada Siklus I dan
84,6 pada Siklus II. Ketuntasan belajar
secara klasikal juga mengalami peningkatan,
ketuntasan secara klasikal Pretest 0%
menjadi 61,5% pada Siklus I dan pada siklus
II menjadi 84,6%. Dapat disimpulkan KBM
Siklus I gagal memberikan ketuntasan
klasikal sedangkan KBM Siklus II berhasil
memberikan ketuntasan klasikal. (5) Ada
siswa
yang
lebih
suka
mengerjakan soal sendiri. (5) Ada
siswa
yang
lebih
suka
mengerjakan soal sendiri. (6) Pada
tahap
pengajuan
pertanyaan
sebagian besar siswa tidak memusatkan
perhatian dalam mengerjakan LKS,
terbukti
dari
tingginya
persentase
aktivitas yang tidak relevan dengan
KBM yaitu 12,67%. (7) Waktu yang digunakan dalam kegiatan
pembelajaran
tidak
sesuai
dengan
waktu yang direncanakan sebelumnya. Dengan demikian dapat dikatakan
bahwa proses pembelajaran menggunakan
model
pembelajaran
kooperatif
tipe
Numbered Heads Together (NHT) dapat
meningkatkan
aktivitas
belajar
dan
ketuntasan
pembelajaran
siswa
pada
pembelajaran PKn di Kelas V SD Negeri
050676 Kebun Balok. Namun, dapat juga
peneliti
sampaikan
bahwa
terjadinya
peningkatan aktivitas belajar dan hasil
belajar siswa ini dimungkinkan karenakan
siklus yang berulang. Dengan demikian, hal
ini juga dapat dijadikan sebagai bahan kajian
lanjutan. Sehingga pada penelitian ini masih
dilanjutkan pada Siklus II untuk mencapai
ketuntasan
kelas
minimal
85%. Pembelajaran
pertemuan
pertama
pada
Siklus II dikondisikan sama seperti pada
Siklus I, namun ada beberapa perbaikan
pada kelemahan-kelemahan yang terjadi saat
pembelajaran pada Siklus I, diantaranya : (1) Penjelasan dan penekanan kepada
siswa tentang pembelajaran kooperatif
di
antaranya
kerja
sama
dalam p-ISSN: 2355 - 1739
e-ISSN: 2407 - 6295 151 Zaitun, Upaya Meningkatkan Belajar ... Suwandi, Sarwiji dan Madyo Ekosusilo,
2007, Pendidikan dan Latihan
Profesi
Guru
(PLPG),
Panitia
Sertifikasi
Guru
Rayon
13,
Surakarta. Zaitun, (2016),
Implementasi
Model
Pembelajaran
Kooperatif
Tipe
Numbered Heads Together (NHT)
Untuk
Meningkatkan
Aktivitas
Belajar Pkn Di Kelas V Sd Negeri
050676 Kebun Balok Semester
Genap T.P. 2015/2016, UD Toma,
Medan. Data aktivitas siswa menurut kedua
pengamatan pengamat pada siklus I dan II
adalah Aktivitas menulis/membaca (37,1%),
mengerjakan LKS (25,9% ), bertanya
sesama teman (13,5%), bertanya kepada
guru (11,8%), dan yang tidak relevan
dengan KBM (11,8%). Aktivitas siswa pada
Siklus II menurut pengamatan kedua
pengamat antara lain: menulis/membaca
(24,7%),
Mengerjakan
LKS
(49,9%),
bertanya sesama teman (16,5%), bertanya
kepada guru (10,6%), dan yang tidak relevan
dengan KBM (2,4%). Dengan menerapkan
model
pembelajaran
Kooperatif
tipe
numbered heads together, aktivitas belajar
siswa dari Siklus I ke Siklus berikutnya
mengalami peningkatan. KESIMPULAN Berdasarkan
analisis
data
dan
pembahasan maka dapat disimpulkan hasil
penerapan model pembelajaran kooperatif
tipe Numbered Heads Together dalam
pembelajaran PKn di Kelas V SD Negeri
050676 Kebun Balok tahun pembelajaran
2015/2016 berikut ini: Trianto,
(2010),
Mendesain
Model
Pembelajaran
Inovatif-Progresif,
Penerbit Kencana Prenada Media
Group, Jakarta. Hasil penelitian menunjukkan bahwa
pembelajaran dengan menggunakan model
pembelajaran Numbered Heads Together
dapat meningkatkan hasil belajar PKn siswa
kelas V SDN. Melihat hasil analisis data
penelitian menunjukkan bahawa hasil tes
hasil belajar siswa pada Siklus I meningkat
siknifikan dbandingkan dengan siklus II. Dari hasil tes hasil belajar pada Siklus I
yang tuntas sebanyak 4 orang (30,7%),
sedangkan secara kelas belum tuntas. Dari
hasil tes hasil belajar pada Siklus I yang
tuntas sebanyak 13 orang (100%) secara
kelas dikatakan tuntas karena jumlah siswa
yang memperoleh nilai diatatas nilai KKM
(70) lebih dari 85%. DAFTAR RUJUKAN Aqib, Zainal, 2006, Penelitian Tindakan
Kelas, Penerbit Yrama Widya,
Bandung. Istarani,(2012).58
Model
Pembelajaran
Inovatif, Media Persada, Medan. Istarani,(2012).58
Model
Pembelajaran
Inovatif, Media Persada, Medan. Majid,
Abdul,
2009,
Perencanaan
Pembelajaran
Mengembangkan
Standar
Kompetensi Guru, Penerbit PT. Remaja
Rosdakarya
Offset,
Bandung. 152
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English
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Healthcare workers’ compliance with COVID-19 preventive and control measures at De Martino Hospital, Mogadishu, Somalia: a cross-sectional study
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Healthcare workers’ compliance with COVID-19
preventive and control measures at De Martino
Hospital, Mogadishu, Somalia: a cross-sectional
study Abdullahi Ibrahim Janay Dokuz Eylül University
Bulent Kilic
Dokuz Eylül University
Belgin Unal
Dokuz Eylül University DOI: https://doi.org/10.21203/rs.3.rs-4232936/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read
Full License License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read
Full License Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Page 1/19 Page 1/19 Research Article Keywords: Compliance, Healthcare workers, COVID-19 prevention and control, Hospital
Posted Date: April 19th, 2024
DOI: https://doi.org/10.21203/rs.3.rs-4232936/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read
Full License Results In total, 58.3% of the participants had good compliance with IPC. There were significant associations
between IPC compliance and the type of healthcare worker (doctors and doctor assistants: 72.3%, nurses
and paramedical staff: 67.3%, non-clinical staff: 5.7%, p < 0.01). After adjusting for potential confounding
factors, compared to non-clinical staff, doctors and doctor assistants (OR: 12.11, 95% CI: 2.23–65.84) and
nurses and paramedical staff (OR: 21.38, 95% CI: 4.23–108.01) had higher compliance with infection
prevention and control measures. There were no significant associations between compliance and sex,
marital status, vaccination status, or smoking (p > 0.05 for all). Conclusions Inadequate compliance with COVID-19 IPC measures was observed among hospital workers. Prioritizing
awareness campaigns and behavior change interventions, especially among non-clinical staff, is crucial for
effective COVID-19 infection prevention and control within hospitals. Methods A cross-sectional study was conducted at De Martino Public Hospital, Mogadishu, Somalia from August to
October 2022, with the participation of 204 healthcare workers (response rate = 97%). Compliance was
assessed using responses to 25 questions on a five-point Likert-type scale, and a median score of 20 was
used to dichotomize compliance scores. A chi-square test and logistic regression analysis were performed
to check the associations between healthcare workers’ sociodemographic information, related factors to
IPC, work conditions and practices on COVID-19, and IPC compliance during healthcare interventions using
SPSS 23 version. Background Healthcare workers are a high-risk group for COVID-19 and protecting them is crucial for healthcare delivery. Limited studies have explored compliance with infection prevention and control (IPC) practices among
Somali healthcare workers. This study aimed to determine compliance with IPC practices among healthcare
workers in De Martino Public Hospital, Somalia. Introduction All age
groups are susceptible to the virus, but elderly people with underlying health conditions are at greater risk of
severe illness [4]. Healthcare workers (HCWs) are also vulnerable, emphasizing the importance of infection
prevention and control (IPC) practices and personal protective equipment (PPE) use [5]. IPC strategies include non-pharmaceutical interventions such as school and workplace closures, event
bans, stay-at-home orders, and movement restrictions, which have reduced transmission [6]. Personal
protective measures are also an important component of COVID-19 infection prevention and control [7]. Vaccines have been developed, but their effectiveness varies by variant, with better protection against
severe outcomes. Vaccine effectiveness against symptomatic disease was greater for the delta variant than
for the omicron variant. With previous variants, vaccine effectiveness against severe disease, including
hospitalization and death, has been higher and retained for longer than effectiveness against mild disease
[8]. The World Health Organization (WHO) declared that COVID-19 is an emergency public health problem of
international concern that posed a high risk to countries with vulnerable health systems on January 30,
2020 [9]. Although the Director General of the WHO declared the end of COVID-19 as a public health
emergency and is no longer a global threat on May 5th 2023, he said that COVID-19 is still killing and
warned of the risks from the new emerging variants [10]. In fragile settings like Somalia where the number
of HCWs is significantly lower than that required for Sustainable Development Goals, the protection of
health workers is vital [11]. To protect HCWs, Enough supplies of PPE should be ensured and HCWs should
be trained in using it. The environmental hygiene of hospitals and the personal hygiene of HCWs should be
maintained [2]. HCWs should maintain contact reduction to reduce the number of infections [7]. It was reported from China that the potential risk of COVID-19 has largely improved the IPC behaviors of
HCWs working in hospitals [12]. Determination of the compliance of hospital workers with the COVID-19 IPC
practices and the factors affecting this compliance is important for the protection of hospital workers
during the pandemic. Studies assessing COVID-19 IPC practices in Somali hospitals and HCWs are limited. This cross-sectional study assessed COVID-19 IPC compliance among HCWs at De Martino Public Hospital
during healthcare interventions and determined the factors affecting compliance to improve hospital
workers’ compliance with COVID-19 IPC practices. Introduction The COVID-19 pandemic was the most recent and worst pandemic in the 21st century, and resulted in
765 million confirmed cases and 6.9 million deaths worldwide as of May 3, 2023 [1]. Page 2/19 Page 2/19 This highly infectious virus can be transmitted by asymptomatic carr
making isolation until testing negative crucial [2]. Quarantine measur
endemic areas or with confirmed contact and for mild cases not requ
groups are susceptible to the virus, but elderly people with underlying
severe illness [4]. Healthcare workers (HCWs) are also vulnerable, em
prevention and control (IPC) practices and personal protective equip
IPC strategies include non-pharmaceutical interventions such as sch
bans, stay-at-home orders, and movement restrictions, which have re
protective measures are also an important component of COVID-19 i
Vaccines have been developed, but their effectiveness varies by varia
severe outcomes. Vaccine effectiveness against symptomatic diseas
for the omicron variant. With previous variants, vaccine effectiveness
hospitalization and death, has been higher and retained for longer th
[8]. The World Health Organization (WHO) declared that COVID-19 is an e
international concern that posed a high risk to countries with vulnera
2020 [9]. Although the Director General of the WHO declared the end
emergency and is no longer a global threat on May 5th 2023, he said
warned of the risks from the new emerging variants [10]. In fragile se
of HCWs is significantly lower than that required for Sustainable Dev
health workers is vital [11]. To protect HCWs, Enough supplies of PPE
be trained in using it. The environmental hygiene of hospitals and the
maintained [2]. HCWs should maintain contact reduction to reduce th
It was reported from China that the potential risk of COVID-19 has lar
HCWs working in hospitals [12]. Determination of the compliance of
practices and the factors affecting this compliance is important for t
during the pandemic. Studies assessing COVID-19 IPC practices in S
This cross-sectional study assessed COVID-19 IPC compliance amon
during healthcare interventions and determined the factors affecting
workers’ compliance with COVID-19 IPC practices. Methods This highly infectious virus can be transmitted by asymptomatic carriers and those in the incubation period,
making isolation until testing negative crucial [2]. Quarantine measures are essential for persons from
endemic areas or with confirmed contact and for mild cases not requiring medical attention [3]. Study population and sample size The study population is 210 HCWs working at De Martino Hospital between August and November 2022. HCWs include all hospital staff who provide health services directly or indirectly, such as managers,
secretaries, doctors, nurses, laboratory technicians, radiologists, pharmacists, cleaners, security personel, or
other personnel. All HCWs working in the hospital were included in the study. In total, 204 HCWs were
reached during data collection in the study and their informed consent was obtained using a written form. Methods This hospital-based cross-sectional study aimed to assess COVID-19 IPC compliance among HCWs at De
Martino Public Hospital during healthcare interventions and to determine the factors affecting their
compliance. De Martino Public Hospital is located in Mogadishu, Somalia’s capital. It was built during Italy’s
colonial era and is currently under the control of the Ministry of Health and Human Services of the Federal
Government of Somalia. It provides free medical care, especially for inpatients. During the COVID-19 Page 3/19 Page 3/19 pandemic, the hospital exclusively served COVID-19 patients. The hospital has 115 beds and offers a range
of services, including polyclinics, inpatient care, emergency services, and intensive care. In 2019, there were a total of 61 health facilities run by the government in the Benadir region, which
encompasses the capital city [13]. De Martino Hospital in this region serves as a secondary referral hospital
(RH) indicating its role in providing specialized care. It’s important to note that while RHs like De Martino
Hospital offer valuable specialist services, they may face challenges in meeting the extensive demand due
to limited capacity and resources [14]. During the COVID-19 pandemic, De Martino Hospital emerged as a
key center for managing COVID-19 cases, playing a vital role in the national response. This underscores its
importance in addressing public health challenges, even during unprecedented events like a global
pandemic. In 2019, there were a total of 61 health facilities run by the government in the Benadir region, which
encompasses the capital city [13]. De Martino Hospital in this region serves as a secondary referral hospital
(RH) indicating its role in providing specialized care. It’s important to note that while RHs like De Martino
Hospital offer valuable specialist services, they may face challenges in meeting the extensive demand due
to limited capacity and resources [14]. During the COVID-19 pandemic, De Martino Hospital emerged as a
key center for managing COVID-19 cases, playing a vital role in the national response. This underscores its
importance in addressing public health challenges, even during unprecedented events like a global
pandemic. The study variables To assess HCWs’ compliance with COVID-19 prevention and control practices (IPC), the use of personal
practices during their healthcare interactions in the hospital was measured using a self-report
questionnaire. Accordingly, the questionnaire was created under three main headings; personal protective
equipment (PPE), hand washing and hygiene (HH) and other COVID-19 IPC practices. The data on personal
characteristics, related factors to IPC, work conditions and practices on COVID-19 were also included in the
questionnaire. The questionnaire was adapted from the WHO’s risk assessment tool for healthcare workers
in the context of COVID-19 [15], with some modifications according to the suitability of the hospital facility
and using literature guidelines. The questionnaire is attached as "Supplementary file 1". The anonymity and confidentiality of the participants were ensured; a specific number was given to every
questionnaire, and there was no information that could identify individual participants during or after data
collection. A pilot study was conducted initially; then, the questionnaire was distributed to the hospital staff
and the data were collected between August and November 2022. There were 25 questions measuring the
compliance of HCWs with COVID-19; 12 questions for PPE, 7 questions for HH and 6 questions for other
COVID-19 IPC IPC on the questionnaire. Responses to each question were measured using a 5-point Likert
scale: "always (5 points), often (4 points), sometimes (3 points), rarely (2 points), or never (1 point)". The
scores of each person’s responses were categorized into two categories and the answers "always" and
"most of the time" were taken as compliance. Then, the compliance responses of each participant were
summed and the median was taken into account as the cut-off point; scores above the median were
considered “high compliance”. Data analysis Page 4/19 For descriptive analyses, the variables measured by scale values were converted into categorical ones, and
all independent variables are presented as percentages. To determine the association between independent
variables and high compliance with the COVID-19 IPC measures, the chi-square test was used by converting
the scores of compliance with the COVID-19 IPC measures into two categories, and the answers "always"
and "most of the time" were taken as “compliance”. Then, the compliance responses of each participant
were collected and the median was considered the cut-off point. Scores above the median were considered
“high compliance” and presented by percentage for descriptive analysis. To identify the key independent factors associated with a high level of compliance with COVID-19 IPC
measures, variables that showed significant associations with high compliance in univariate analyses were
included in logistic regression models. The backward LR method was used to identify predictive variables
associated with compliance with IPC measures and the association was presented with odds ratio and 95%
confidence interval. The Statistical Package for Social Sciences (SPSS 23) was used for the data analysis. P values lower than
0.05 were considered significant. Results In total, data from 204 HCWs were analyzed, achieving a response rate of 97%. Among the participants, 51%
were male (Table 1, column 4). The majority of HCWs (46.1%) fell in the 20 to 29 age group, while only
15.7% were aged 40 and above (Table 1, column 5). Approximately, 77.8% held bachelor’s degrees or higher
(Table 1, column 6). Nurses and paramedical staff constituted the largest group (51%), followed by (31.9%)
doctors and doctor assistants (Table 2, column 4). Outpatient workers accounted for 20.1% of the
participants, while inpatient workers made up 26.5%. HCWs in other clinical departments comprised 29.9%
of the total (Table 2, column 5). Compliance with COVID-19 IPC practices among healthcare
workers The total IPC compliance rate was 58.3% among the participants. HCWs demonstrated 55.9% compliance
with PPE during healthcare interactions, 55.4% compliance with HH, and 52% compliance other COVID-19
IPC measures during healthcare interactions (Fig. 1). Figure 1. COVID-19 IPC compliance in the HCWs (total IPC, PPE use, HH, other IPC), % Results Page 5/19 Table 1
COVID-19 IPC compliance in HCWs during healthcare provision by sociodemographic characteristics and
related factors to IPC
Independent variables
n
%
COVID-19 IPC compliance
Total IPC%
PPE use%
HH%
Other IPC%
High
p*
High
p*
High
p*
High
p*
Sex
Male
Female
105
98
51.7
48.3
60
56.1
0.58
61
51
0.15
55.2
55.1
0.98
52.4
51.0
0.85
Age group
20–29
30–39
40 and above
94
78
32
46.1
38.2
15.7
62.6
61.5
37.5
0.03
59.6
59
37.5
0.07
62.8
55.1
34.4
0.02
57.4
44.9
53.1
0.26
Education level
-Secondary degree
and below
-Associate degree
-Undergraduate and
graduate degrees
23
22
158
11.3
10.8
77.8
8.7
54.5
66.5
<
0.01
13
50
63.3
<
0.01
8.7
45.5
63.9
<
0.01
26.1
54.5
55.7
0.03
Experience years
1–5
6–10
11 and above
118
59
20
59.9
29.9
10.2
58.5
64.4
50
0.50
57.6
59.3
45
0.52
58.5
57.6
45
0.53
55.9
45.8
50
0.43
Marital status
Married
Unmarried
Divorced/Widow
102
16
84
50.5
7.9
41.6
57.8
56.3
59.5
0.96
55.1
50
59.5
0.66
53.9
64.3
57.1
0.91
50
31.3
58.3
0.12
Having a child
Yes
No
107
94
53.2
46.8
57.9
58.5
0.94
55.1
57.4
0.74
54.2
57.4
0.64
47.7
57.4
0.17
Training on COVID-19
171
83.8
61.4
0.04
59.1
0.04
56.9
0.63
53.2
0.41 Table 1 Independent variables
n
%
COVID-19 IPC compliance
Total IPC%
PPE use%
HH%
Other IPC%
High
p*
High
p*
High
p*
High
p*
Yes
No
33
16.2
42.4
39.5
51.5
45.5
COVID-19 information
From official sources
From social media
85
113
42.9
57.1
48.2
67.3
0.01
41.2
69
<
0.01
40
69
<
0.01
43.5
60.2
0.02
COVID-19 infection
Yes
No
Unknown
95
46
63
46.6
22.5
30.9
66.3
41.3
58.7
0.02
62.1
26.2
60.3
0.01
58.9
39.1
61.9
0.04
50.5
47.8
57.1
0.59
COVID-19 Vaccination
Yes
No
180
24
88.2
11.8
59.4
50
0.38
57.2
45.8
0.29
56.7
45.8
0.32
50.6
62.5
0.27
Vaccination doses
0
1
2 and above
24
56
124
11.8
27.5
60.8
50
48.2
64.5
0.08
45.8
55.3
58.1
0.54
45.8
55.4
57.3
0.59
62.5
42.9
54
0.21
Smoking cigarette
Yes
No
33
168
16.4
83.6
48.5
60.1
0.22
48.5
57.1
0.36
39.4
58.3
0.05
36.4
54.8
0.05
An old /chronic patient
in the family
Yes
No
103
96
50.8
48.2
52.5
63.5
0.11
52.4
58.3
0.40
50.5
59.4
0.21
50.5
54.2
0.60
*Chi
t
t Page 7/19 Table 2
COVID-19 IPC compliance in HCWs during healthcare provision by working conditions and practices on
COVID-19
Independent variables
n
%
COVID-19 IPC compliance
Total IPC%
PPE use%
HH%
Other IPC%
High
p*
High
p*
High
p*
High
p*
Profession
Doctors and doctor
assistants
Nurses and
Paramedical staff
Non-clinical staff
65
104
35
31.9
51
17.2
72.3
67.3
5.7
<
0.01
66.2
66.3
5.7
<
0.01
70.8
60.2
11.4
<
0.01
46.2
61.5
34.3
0.01
Department
Outpatient
Inpatient
Other clinical
departments
Non-clinical
departments
41
54
61
48
20.1
26.5
29.9
23.5
68.3
70.4
70.5
20.8
<
0.01
65.9
64.8
70.5
18.8
<
0.01
65.9
62.9
70.5
18.8
<
0.01
46.9
53.7
59
45.8
0.47
Direct COVID-19 care
Yes
No
Unknown
109
56
39
53.4
27.5
19.1
67.9
37.5
61.5
<
0.01
66.1
30.4
64.1
<
0.01
62.4
35.7
64.1
<
0.01
49.5
51.8
59
0.60
Face-to-face contact
with COVID-19 patient
Yes
No
Unknown
109
63
30
53.9
31.2
14.9
75.2
31.7
53.3
<
0.01
72.5
28.6
53.3
<
0.01
68.8
33.3
53.3
<
0.01
54.1
50.8
50
0.88
Direct contact with
COVİD-19 environment
Yes
No
95
66
41
47
32.7
25.3
69.5
41
58.5
<
0.01
70.5
33.3
56.1
<
0.01
57.9
45.5
63.4
<
0.14
52.6
47
58.5
0.50
h Page 8/19 Independent variables
n
%
COVID-19 IPC compliance
Total IPC%
PPE use%
HH%
Other IPC%
High
p*
High
p*
High
p*
High
p*
Unknown
Presence at AGP
performance
Yes
No
Unknown
112
76
13
55.7
37.8
6.5
82.1
27.6
30.8
<
0.01
77.7
26.3
38.5
<
0.01
78.6
31.6
7.7
<
0.01
56.3
44.7
53.8
0.30
Type of AGP procedure
-Not applicable
-Open airway aspiration
+Sputum collection
+Tracheotomy
-Tracheal intubation
+Cardiopulmonary
resuscitation
-Nebulizer treatment
-More than one
89
12
25
42
32
44.5
6
12.5
21
16
28.1
83.3
84
73.8
93.8
<
0.01
28.1
83.3
80
69
84.4
<
0.01
28.1
83.3
80
69
90.6
<
0.01
46.1
50
60
50
65.6
0.36
*Chi square test
Note: N: Number of participants, IPC: COVID-19 Infection Prevention and Control, PPE: Personal Protective Note IPC: infection prevention and control, PPE: personal protective equipment, HH: hand hygiene. Page 9/19 Page 9/19 Age and educational status were significantly associated with overall COVID-19 IPC and HH compliance (P
< 0.05 for all). Educational status was associated with compliance with PPE (p < 0.01) and other COVID-19
IPC measures (p = 0.03). Younger age groups and higher education level groups expressed better
compliance (Table 1). There was no association between any of the IPC, PPE, HH compliance and marital
status, having a child at home, having an old/chronic disease patient at home, or having COVID-19
vaccination. There was no significant association between the IPC dimensions and work experience years
(Table 1). Participants who received training on COVID-19 reported higher compliance with total IPC and
PPE than those who did not receive training (p < 0.05). There was a significant association between the type of HCW and overall compliance with COVID-19 IPC,
PPE and HH; non-clinical staff members showed significantly lower levels of compliance for all three
dimensions (p < 0.01 for all). The same pattern was observed in the departments where HCWs worked; non-
clinical departments showed lower compliance (p < 0.01 for all). HCWs not providing direct care to COVID-
19 patients, not having face-to-face contact, and not being present during the AGP performance reported
lower compliance (p < 0.01 for all). HCWs not having direct contact with the COVID-19 environment reported
lower compliance in total IPC and PPE use (p < 0.01 for all). There was no significant association with any of
the working conditions or practices on COVID-19 and compliance with other IPC measures except the type
of HCW (p = 0.01) (Table 2). In the logistic regression model, profession, having training on COVID-19, providing direct care to COVID-19
patients and presence when performing AGP were independent factors associated with total IPC
compliance among HCWs. Doctors and doctor assistants and nurses and paramedical staff expressed a
higher level of compliance with overall IPC compared to non-clinical staff (Doctors OR: 12.11, 95% CI: 2.23–
65.84, nurses and paramedical staff OR: 21.38, 95% CI: 4.23–108.01). HCWs who received training on
COVID-19 exhibited higher COVID-19 compliance than those who did not (OR: 3.48, 95% CI: 1.06–11.35). HCWs who were present when AGPs were performed exhibited higher compliance compared to those
uncertain about that (OR: 12.45, 95% CI: 12.16–71.76) (Table 3). Page 9/19 In the logistic regression model, profession, having training on COVID-19, providing direct care to COVID-19
patients and presence when performing AGP were independent factors associated with total IPC
compliance among HCWs. Doctors and doctor assistants and nurses and paramedical staff expressed a
higher level of compliance with overall IPC compared to non-clinical staff (Doctors OR: 12.11, 95% CI: 2.23–
65.84, nurses and paramedical staff OR: 21.38, 95% CI: 4.23–108.01). HCWs who received training on
COVID-19 exhibited higher COVID-19 compliance than those who did not (OR: 3.48, 95% CI: 1.06–11.35). HCWs who were present when AGPs were performed exhibited higher compliance compared to those
uncertain about that (OR: 12.45, 95% CI: 12.16–71.76) (Table 3). Page 10/19 Page 10/19 Page 10/19 Table 3
Independent determinants of COVID-19 IPC compliance in HCWs during healthcare provision: Results of
multivariate logistic regression analysis* (Backward elimination method)
COVID-19 IPC compliance
Variables in the model
Total IPC
OR (95% CI)
PPE use
OR (95% CI)
HH
OR (95% CI)
Other IPC
OR (95% CI)
Profession
Doctors & doctor assistants
12.11 (2.23–
65.84)
11.59 (1.94–
69.01)
-
1.35 (0.56–
3.24)
Nurses & paramedical staff
21.38 (4.23–
108.01)
17.91 (3.22–
99.64)
-
2.59 (1.13–
5.93)
Non-clinical staff
Ref. Ref. -
Ref. Department
Inpatient
-
-
4.97 (1.62–
15.21)
-
Other clinical departments
-
-
6.2 (2.1–
18.35)
-
Outpatient
-
-
4.21 (1.34–
13.23)
-
Non-clinical departments
-
-
Ref. -
COVID-19 material
From official sources
-
0.38 (0.17–
0.83)
0.45 (0.22–
0.92)
-
From social media
-
Ref. Ref. -
Direct COVID-19 care
Provide
0.26 (0.07–
0.98)
1.02 (0.38–
2.76)
-
-
Unknown
0.9 (0.2–3.99)
4.10 (1.12–
15.03)
-
-
Does not provide
Ref. Ref. -
-
Face-to-face contact with
COVID-19 patient
Have
1.5 (0.27–8.07)
-
1.06 (0.32–
3.51)
-
Does not have
0 37 (0 07–
0 4 (0 12– Table 3 Independent determinants of COVID-19 IPC compliance in HCWs during healthcare provision: Results of
multivariate logistic regression analysis* (Backward elimination method) of COVID-19 IPC compliance in HCWs during healthcare provision: Results of
logistic regression analysis* (Backward elimination method) Independent determinants of COVID-19 IPC compliance in HCWs during heal
multivariate logistic regression analysis* (Backward eliminati COVID-19 IPC compliance COVID-19 IPC compliance
Direct contact with COVID-19
environment
Have
-
2.59 (0.88–
7.6)
-
-
Does not have
-
1.08 (0.36–
3.23)
-
-
Unknown
-
Ref. Page 9/19 Nurses and paramedical staff expressed
higher compliance compared to non-clinical staff (OR: 2.59, 95% CI: 1.13–5.93) (Table 3). Page 9/19 -
-
Presence at AGP performance
Present
12.45 (2.16–
71.76)
4.36 (0.8–
23.79)
-
-
Not present
1.59 (0.3–8.21)
0.7 (0.13–
3.52)
-
-
Unknown
Ref. Ref. -
-
Training on COVID-19
Received
3.48 (1.06–
11.35)
2.66 (0.93–
7.66)
-
-
Did not receive
Ref. Ref. -
-
Education level
Undergraduate and graduate
degrees
-
-
8.48 (1.64–
43.94)
-
Associate degree
-
-
3.67 (0.56–
24.04)
-
Secondary degree and below
-
-
Ref. -
*Variables included in the logistic regression model
For the total COVID-19 IPC: age group, education, receiving training, source of COVID-19 information,
covid infection, type of HCW, type of department, providing direct COVID-19 care, Having face-to-face
contact with COVID-19 patients, having direct contact with the COVID-19 environment, presence at AGP
performance and type of AGP. For PPE: education, training, source of COVID-19 information, covid infection, Type of HCW, Type of
department, providing direct COVID-19 care, having face-to-face contact with COVID-19 patients, having
direct contact with the COVID-19 environment, presence at AGP performance and type of AGP. For HH: age group, education, source of COVID-19 information, COVID-19 infection, type of HCW, type of
department, providing direct COVID-19 care, having face-to-face contact with COVID-19 patients. For Other COVID-19 IPC: education, source of COVID-19 information, Type of HCW. Note: IPC: Infection Prevention and Control, OR: Odds Ratio, CI: Confidence Interval, PPE: Personal
Protective Equipment HH: Hand Hygiene AGP: Aerosol Generating Procedure Page 12/19 Page 12/19 For PPE; profession, the source of COVID-19 material and providing direct care to COVID-19 patient were
independent factors. Compliance with PPE showed significant differences among HCWs in various roles. Doctors and doctor assistants (OR: 11.59, 95% CI: 1.94–69.01) and nurses and paramedical staff (OR:
17.91, 95% CI: 3.22–99.64) exhibited higher compliance compared to non-clinical staff. HCWs who
accessed COVID-19 information from official sources expressed lower compliance compared to those who
accessed information from social media (OR: 0.38, 95% CI: 0.17–0.83) (Table 3). For HH, departments where HCWs worked, source of COVID-19 material and education level were
independent factors. Clinical departments showed higher compliance compared to non-clinical
departments (inpatient OR: 4.97, %95 CI: 1.62–15.21, outpatient OR: 4.21, %95 GA: 1.34–13.23, other
clinical departments OR: 6.2, %95 GA: 2.1–18.35). HCW participants who read COVID-19 related material
from Official sources reported lower compliance than those who read from social media (OR: 0.45, 95% CI:
0.22–0.92) (Table 3). For Other IPC compliance, profession was the only predictor. Discussion This study was designed to assess COVID-19 IPC compliance among HCWs during healthcare interventions
and to determine the factors affecting compliance. The study showed that 58.3% of HCWs had high
compliance with COVID-19 IPC measures during healthcare interventions. The compliance was over 50% in
all domains (PPE use, HH, and other IPC). Non-clinical staff had lower compliance compared with clinical
staff. There are several studies that reported high compliance with COVID-19 IPC among HCWs. In Ghana, a study
with 424 HCWs in COVID-19 treatment centers reported high compliance with hand hygiene (88.4%) and
PPE usage (90.6%) [16]. Two Ethiopian studies, involving 403 and 422 participants, reported good COVID-19
infection prevention practices in 64.3% and 63.5% of healthcare workers, respectively. The first study
revealed 96.1% compliance with hand hygiene but only 45.2% compliance with PPE usage, possibly due to
PPE availability, comfort, negligence, or education [17, 18]. Compared to previous studies, our findings indicated lower IPC compliance rates. This variance may be
attributed to differences in study methods and the timing of data collection. While our study used
compliance scores above the median for each domain, the referenced studies used either above-average
scores or cutoff points of 60% or 75% of total compliance scores. It is also possible that healthcare
workers’ adherence to IPC measures decreased over time since the pandemic’s onset. A study on
healthcare worker HH practices revealed a 13.7% increase upon room exit during the initial COVID-19 wave. Compliance decreased by 9.9% post-lockdown but rebounded by 2.8% in the second wave [19]. Some studies have reported low HCW compliance with COVID-19 IPC measures. For instance, in a study
involving 422 HCWs at COVID-19 referral hospitals in Ethiopia, overall compliance with COVID-19 prevention
practices was only 22% [20]. In this study, only 63.4% of the participating HCWs received training on COVID- Page 13/19 Page 13/19 19, 58.2% read COVID-19 materials and 83.2% of the HCWs felt a shortage of appropriate PPE in the
hospital. 19, 58.2% read COVID-19 materials and 83.2% of the HCWs felt a shortage of appropriate PPE in the
hospital. A review identified various barriers to HCWs’ compliance with IPC guidelines for respiratory infectious
diseases, including the availability of training programs, PPE supply, and individual factors such as
knowledge, attitudes, beliefs, and PPE discomfort [21]. Discussion Additionally, a study conducted in Uganda; involving
657 HCWs at community hospitals, revealed that only 37.0% of participants had good COVID-19 IPC
practices, despite high rates of mask usage and hand washing [22]. In our study, doctors, doctor assistants, nurses, and paramedical staff demonstrated higher COVID-19 IPC
compliance than non-clinical staff, except in HH practices. This aligns with prior research. For instance, a
study performed in private-not-for-profit community hospitals in Uganda revealed a significant association
between clinical HCWs and good COVID-19 IPC practices [22]. Similarly, a study in COVID-19 treatment
centers in Ghana revealed that non-clinical staff exhibited significantly lower compliance with hand hygiene
and PPE usage compared to clinical staff [16]. The difference in compliance may be due to the higher risk faced by clinical healthcare workers in close
contact with COVID-19 patients. A study from Somalia, reported that ancillary staff, including security
workers and cleaners, have a higher infection risk due to lower knowledge and adherence to infection
control measures when handling suspected COVID-19 patients. Healthcare assistants are often informally
employed and receive less attention than formal employees such as doctors, nurses, and technologists [11]. Our study showed that HCWs who were present during AGPs exhibited higher compliance with overall IPC
measures compared to those who were uncertain about that. Our study aligns with Ashinyo ME et al.’s study
that reported high compliance with COVID-19 IPC protocols during AGPs [16]. Healthcare workers
performing AGPs face a higher risk of COVID-19 infection, possibly explaining their heightened compliance
[23]. Our study also showed that HCWs who had received training on COVID-19 exhibited higher COVID-19
compliance with overall IPC than those who did not. This is in line with 2 studies on 422 HCWs in Ethiopia;
Etafa W. et al [20] and Arsemahagn MA [24], and a review study by Cooper S. et al. [25]. Our study also showed that HCWs who had received training on COVID-19 exhibited higher COVID-19
compliance with overall IPC than those who did not. This is in line with 2 studies on 422 HCWs in Ethiopia;
Etafa W. et al [20] and Arsemahagn MA [24], and a review study by Cooper S. et al. [25]. Interestingly, our study revealed that HCWs who obtained COVID-19 information from official sources
showed lower compliance with PPE and HH compared to those who accessed information from social
media. Discussion In contrast, a study on the Somali population reported the opposite, where HCWs who relied on
social media for COVID-19 information exhibited lower compliance, likely due to misinformation [26]. However, De Martino HCWs may follow specific social media pages they trust for COVID-19 information. To improve compliance, HCWs must receive continuous awareness and training in COVID-19 IPC guidelines. Policymakers should develop comprehensive programs to increase awareness among HCWs at all levels
and provide the necessary equipment and supplies for effective IPC practices in healthcare settings. This study has limitations. Firstly, there could be recall bias; because participants were asked about their
compliance with COVID-19 IPC measures during the late stages of the pandemic when no COVID-19 Page 14/19 Page 14/19 patients were likely admitted, and some PPE like respirators, gowns, and face shields might not have been
used. To minimize this bias, we asked about daily IPC practices and included specific questions for
suspected or confirmed COVID-19 patients. Since the study was a single-center study, De Martino Public
Hospital may not fully represent other pandemic hospitals in Somalia, although it plays a significant role in
treating COVID-19 patients. Nonetheless, the inclusion of all staff members and the high response rate
enhances the generalizability of the results within the hospital. Furthermore, the limited number of
participants resulted in a wide confidence interval for the odds ratio derived from the model. Abbreviations IPC: infection prevention and control
HCWs: healthcare workers
PPE: personal protective equipment
WHO: World Health Organization
RH: referral hospital
HH: Hand washing and Hygiene Conclusions A notable portion of hospital workers reported inadequate compliance with COVID-19 IPC measures, with
particularly low adherence among non-clinical staff. This lower compliance might be linked to their
perception of low risk and a lack of awareness regarding COVID-19. It is essential to recognize that all
HCWs hospitals are susceptible to COVID-19 infection. Therefore, prioritizing awareness campaigns and
behavior change interventions, especially among non-clinical staff, is crucial for effective COVID-19
prevention and control within hospitals. Consent for publication Not applicable Data availability All data generated or analyzed during this study are included in this published article and its supplementary
information files. Funding There is no funding support for this research. Competing interests The authors declare that they have no competing interests. Author contributions All authors (AIJ, BK, BU) contributed equally to the conception and design of the research, data collection,
data analysis, data interpretation and creating the manuscript. All authors revised the manuscript and
approved the final version of the article. Ethics approval and consent to participate The study received ethical approval from Dokuz Eylul University’s Non-Interventional Research Ethics
Committee (approval date: 17.08.2022, decision number: 2022/26-08) and adhered to the principles of the
Declaration of Helsinki and local institutional guidelines. Official permission was granted by De Martino
Public Hospital in Mogadishu, Somalia. Research participants were informed about the study’s purpose and
methodology, and their consent was obtained during the data collection process. Declarations Supplementary information Supplementary file 1: Questionnaire for compliance of healthcare workers with COVID-19 prevention and
control measures.docx: is the questionnaire used for data collection in English. Acknowledgments First, we would like to express our sincere gratitude to the managers of De Martino Hospital for allowing us
to conduct our study at the hospital. Second, we thank Dr. Lul Ahmed Abdi, Maternity Department, De Martino hospital who was the link person
between us and the hospital administration. She also supported us in delivering the questionnaire papers to Page 15/19 the hospital workers and collecting it after filling during the data collection process, since she works at the
hospital and knows the hospital well. the hospital workers and collecting it after filling during the data collection process, since she works at the
hospital and knows the hospital well. Third, we extend our gratitude to the different groups of healthcare workers at De Martino hospital for
participating in the study. References 1. World Health Organization. WHO Coronavirus (COVID-19) Dashboard. Updated May 3, 2023 [Cited 2023,
May 5]. Available from: https://covid19.who.int/ 1. World Health Organization. WHO Coronavirus (COVID 19) Dashboard. Updated May 3, 2023 [Cited 2023,
May 5]. Available from: https://covid19.who.int/
2. Yang Y, Peng F, Wang R, Guan K, Jiang T, Xu G, et al. The deadly coronaviruses: The 2003 SARS
pandemic and the 2020 novel coronavirus epidemic in China. Journal of Autoimmunity, 2020;111:2-3. https://doi.org/10.1016/j.jaut.2020.102434 2. Yang Y, Peng F, Wang R, Guan K, Jiang T, Xu G, et al. The deadly coronaviruses: The 2003 SARS
pandemic and the 2020 novel coronavirus epidemic in China. Journal of Autoimmunity, 2020;111:2-3. https://doi.org/10.1016/j.jaut.2020.102434 2. Yang Y, Peng F, Wang R, Guan K, Jiang T, Xu G, et al. The deadly coronaviruses: The 2003 SARS
pandemic and the 2020 novel coronavirus epidemic in China. Journal of Autoimmunity, 2020;111:2-3. https://doi.org/10.1016/j.jaut.2020.102434 2. Yang Y, Peng F, Wang R, Guan K, Jiang T, Xu G, et al. The deadly coronaviruses: The 2003 SARS
pandemic and the 2020 novel coronavirus epidemic in China. Journal of Autoimmunity, 2020;111:2-3. https://doi.org/10.1016/j.jaut.2020.102434 Page 16/19 Page 16/19 3. Lombardi A, Bozzi G, Mangioni D, Muscatello A, Peri AM, Taramasso L, et al. Duration of quarantine in
hospitalized patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection: a
question needing an answer. Journal of Hospital Infection, 2020; 105: 404. https://doi.org/10.1016/j.jhin.2020.03.003 4. Han Q, Lin Q, Jin S, You, L. Coronavirus 2019-nCoV: A brief perspective from the front line. journal of
infection, 2020; 80 (4): 374. https://doi.org/10.1016/j.jinf.2020.02.010 4. Han Q, Lin Q, Jin S, You, L. Coronavirus 2019-nCoV: A brief perspective from the front line. journal of
infection, 2020; 80 (4): 374. https://doi.org/10.1016/j.jinf.2020.02.010 5. Wang J, Zhou M, Liu F. Exploring the reasons for healthcare workers infected with novel coronavirus
disease 2019 (COVID-19) in China. Journal of Hospital Infection, 2020; 105(1):100-101. https://doi.org/10.1016/j.jhin.2020.03.002 5. Wang J, Zhou M, Liu F. Exploring the reasons for healthcare workers infected with novel coronavirus
disease 2019 (COVID-19) in China. Journal of Hospital Infection, 2020; 105(1):100-101. https://doi.org/10.1016/j.jhin.2020.03.002 6. Li Y, Campbell H, Kulkarni D, Harpur A, Nundy M, Wang X, Nair H. The temporal association of
introducing and lifting non-pharmaceutical interventions with the time-varying reproduction number (R)
of SARS-CoV-2: a modeling study across 131 countries. The Lancet Infectious Diseases,
2021;21(2):193–200. https://doi.org/10.1016/S1473-3099(20)30785-4 6. Li Y, Campbell H, Kulkarni D, Harpur A, Nundy M, Wang X, Nair H. References The temporal association of
introducing and lifting non-pharmaceutical interventions with the time-varying reproduction number (R)
of SARS-CoV-2: a modeling study across 131 countries. The Lancet Infectious Diseases,
2021;21(2):193–200. https://doi.org/10.1016/S1473-3099(20)30785-4 (R) 7. Goyal R, Gautam RK, Chopra H, Dubey AK, Singla RK, Rayan RA, Kamal M.. Comparative highlights on
MERS-COV, SARS-COV-1, SARS-COV-2 and NEO-COV. EXCLI Journal 2022;21:1260. https://dx.doi.org/10.17179/excli2022-5355 7. Goyal R, Gautam RK, Chopra H, Dubey AK, Singla RK, Rayan RA, Kamal M.. Comparative highlights on
MERS-COV, SARS-COV-1, SARS-COV-2 and NEO-COV. EXCLI Journal 2022;21:1260. https://dx.doi.org/10.17179/excli2022-5355 8. Andrews N, Stowe J, Kirsebom F, Toffa S, Rickeard T, Gallagher E, et al. Covid-19 Vaccine Effectiveness
against the Omicron (B.1.1.529) Variant. The New England Journal of Medicine, 2022;386(16):1532-
1541. DOI: 10.1056/NEJMoa2119451 8. Andrews N, Stowe J, Kirsebom F, Toffa S, Rickeard T, Gallagher E, et al. Covid-19 Vaccine Effectiveness
against the Omicron (B.1.1.529) Variant. The New England Journal of Medicine, 2022;386(16):1532-
1541. DOI: 10.1056/NEJMoa2119451 9. Sohrabi C, Alsafi Z, O’neill N, Khan M, Kerwan A, Al-jabir A, et al. Review World Health Organization
declares global emergency: A review of the 2019 novel coronavirus (COVID-19). International Journal of
Surgery, 2020;76:71. https://doi.org/10.1016/j.ijsu.2020.02.034 10. United Nations. WHO chief declares end to COVID-19 as a global health emergency. Published May 5,
2023 [Cited 2023, May 7]. Available from:
https://news.un.org/en/story/2023/05/1136367#:~:text=%E2%80%9CLast%20week%2C%20COVID%2D
19,the%20agency’s%20headquarters%20in%20Geneva. 11. Abdi A, Ahmed AY, Abdulmunima M, Karanjaa MJ, Solomona A, Muhammad F, et al. Preliminary findings
of COVID-19 infection in health workers in Somalia: A reason for concern. International Journal of
Infectious Diseases, 2021;104:735–736. https://doi.org/10.1016/j.ijid.2021.01.066 11. Abdi A, Ahmed AY, Abdulmunima M, Karanjaa MJ, Solomona A, Muhammad F, et al. Preliminary findings
of COVID-19 infection in health workers in Somalia: A reason for concern. International Journal of
Infectious Diseases, 2021;104:735–736. https://doi.org/10.1016/j.ijid.2021.01.066 12. Deressa W, Worku A, Abebe W, Gizaw M, Amogne W. Risk perceptions and preventive practices of
COVID-19 among healthcare professionals in public hospitals in Addis Ababa, Ethiopia. PLOS ONE,
2021;16(6):11-12. https://doi.org/10.1371/journal.pone.0242471 12. Deressa W, Worku A, Abebe W, Gizaw M, Amogne W. Risk perceptions and preventive practices of
COVID-19 among healthcare professionals in public hospitals in Addis Ababa, Ethiopia. PLOS ONE,
2021;16(6):11-12. https://doi.org/10.1371/journal.pone.0242471 13. Warsame A. Somalia’s Healthcare System: A Baseline Study & Human Capital Development
Strategy Heritage Institute for Policy Studies and City University of Mogadishu. Mogadishu; 2020. Pp. 39-41. 13. Warsame A. Somalia’s Healthcare System: A Baseline Study & Human Capital Development
Strategy Heritage Institute for Policy Studies and City University of Mogadishu. References Mogadishu; 2020. Pp. 39-41. 14. WHO Regional Office for the Eastern Mediterranean. Somalia health profile 2015, Egypt; 2017. Pp.23. Available from: https://iris.who.int/handle/10665/254906 14. WHO Regional Office for the Eastern Mediterranean. Somalia health profile 2015, Egypt; 2017. Pp.23. Available from: https://iris.who.int/handle/10665/254906 15. World Health Organization. Risk assessment and management of exposure of health care workers in
the context of COVID-19: Interim Guidance March 19, 2020. Pp.1-6. Available from: 15. World Health Organization. Risk assessment and management of exposure of health care workers in
the context of COVID-19: Interim Guidance March 19, 2020. Pp.1-6. Available from: Page 17/19 https://www.who.int/publications/i/item/WHO-2019-nCov-HCW_risk_assessment-2020.2 https://www.who.int/publications/i/item/WHO-2019-nCov-HCW_risk_assessment-2020.2 https://www.who.int/publications/i/item/WHO-2019-nCov-HCW_risk_assessment-2020.2
16. Ashinyo ME, Dubik SD, Duti V, Amegah KE, Ashinyo A, Asare BA, et al. Infection prevention and control
compliance among exposed healthcare workers in COVID-19 treatment centres in Ghana: A descriptive
cross-sectional study. PLOS ONE, 2021;16(3):16. https://doi.org/10.1371/journal.pone.0248282
17. Gebremeskel TG, Kiros K, Gesesew HA, Ward PR. Assessment of Knowledge and Practices Toward
COVID-19 Prevention Among Healthcare Workers in Tigray, North Ethiopia. Frontiers of Public Health,
2021;9:1-8. doi: 10.3389/fpubh.2021.614321 16. Ashinyo ME, Dubik SD, Duti V, Amegah KE, Ashinyo A, Asare BA, et al. Infection prevention and control
compliance among exposed healthcare workers in COVID-19 treatment centres in Ghana: A descriptive
cross-sectional study. PLOS ONE, 2021;16(3):16. https://doi.org/10.1371/journal.pone.0248282 17. Gebremeskel TG, Kiros K, Gesesew HA, Ward PR. Assessment of Knowledge and Practices Toward
COVID-19 Prevention Among Healthcare Workers in Tigray, North Ethiopia. Frontiers of Public Health,
2021;9:1-8. doi: 10.3389/fpubh.2021.614321 18. Jemal B, Aweke Z, Mola S, Hailu S, Abiy S, Dendir G. Knowledge, attitude, and practice of healthcare
workers toward COVID-19 and its prevention in Ethiopia: A multicenter study. SAGE Open Medicine,
2021;9:4. https://doi.org/10.1177/20503121211034389 19. Huang F, Armando M, Dufau S, Florea O, Brouqui P, Boudjema S. COVID-19 outbreak and healthcare
worker behavioural change toward hand hygiene practices. Journal of Hospital Infection, 2021;111:27-
31. https://doi.org/10.1016/j.jhin.2021.03.004 20. Etafa W, Gadisal G, Jabessa S, Takele T. Healthcare workers’ compliance and its potential determinants
to prevent COVID-19 in public hospitals in Western Ethiopia. BMC Infectious Diseases, 2021;21(1):1-7. https://doi.org/10.1186/s12879-021-06149-w 21. Houghton C, Meskell P, Delaney H, Smalle M, Glenton C, Booth A, et al. Barriers and facilitators to
healthcare workers’ adherence with infection prevention and control (IPC) guidelines for respiratory
infectious diseases: a rapid qualitative evidence synthesis. Cochrane Database of Systematic Reviews,
2020;4(4):8-15. DOI: 10.1002/14651858.CD013582. 21. Houghton C, Meskell P, Delaney H, Smalle M, Glenton C, Booth A, et al. References Barriers and facilitators to
healthcare workers’ adherence with infection prevention and control (IPC) guidelines for respiratory
infectious diseases: a rapid qualitative evidence synthesis. Cochrane Database of Systematic Reviews,
2020;4(4):8-15. DOI: 10.1002/14651858.CD013582. 22. Kamacooko O, Kitonsa J, Bahemuka UM, Kibengo FM, Wajja A, Basajja V, et al. Knowledge, Attitudes,
and Practices Regarding COVID-19 among Healthcare Workers in Uganda: A Cross-Sectional Survey. International Journal of Environtal Research and Public Health, 2021;18(13):1-10. https://doi.org/10.3390/ijerph18137004. 22. Kamacooko O, Kitonsa J, Bahemuka UM, Kibengo FM, Wajja A, Basajja V, et al. Knowledge, Attitudes,
and Practices Regarding COVID-19 among Healthcare Workers in Uganda: A Cross-Sectional Survey. International Journal of Environtal Research and Public Health, 2021;18(13):1-10. https://doi.org/10.3390/ijerph18137004. 23. Harrison D, Muradalia K, El Sahlyb H, Bozkurtc B, Jneid H. Impact of the SARS-CoV-2 pandemic on
health-care workers. Hospital Practice, 2020;48(4):162. DOI: 10.1080/21548331.2020.1771010 23. Harrison D, Muradalia K, El Sahlyb H, Bozkurtc B, Jneid H. Impact of the SARS-CoV-2 pandemic on
health-care workers. Hospital Practice, 2020;48(4):162. DOI: 10.1080/21548331.2020.1771010 24. Asemahagn MA. Factors determining the knowledge and prevention practice of healthcare workers
towards COVID-19 in Amhara region, Ethiopia: a cross-sectional survey. Tropical Medicine and Health,
2020;48:1-9. https://doi.org/10.1186/s41182-020-00254-3 24. Asemahagn MA. Factors determining the knowledge and prevention practice of healthcare workers
towards COVID-19 in Amhara region, Ethiopia: a cross-sectional survey. Tropical Medicine and Health,
2020;48:1-9. https://doi.org/10.1186/s41182-020-00254-3 25. Cooper S, Wiyeh A, Schmidt B, Wiysonge CS. Cochrane corner: factors that influence compliance by
healthcare workers with infection prevention and control guidelines for COVID-19 and other respiratory
infections. Pan African Medical Journal,2020;35(2):2. DOI: 10.11604/pamj.supp.2020.35.2.23012 25. Cooper S, Wiyeh A, Schmidt B, Wiysonge CS. Cochrane corner: factors that influence compliance by
healthcare workers with infection prevention and control guidelines for COVID-19 and other respiratory
infections. Pan African Medical Journal,2020;35(2):2. DOI: 10.11604/pamj.supp.2020.35.2.23012 26. Ahmed M, Fodjo J, Gele A, Farah A, Osman S, Guled I, et al. COVID-19 in Somalia: Adherence to
Preventive Measures and Evolution of the Disease Burden. Pathogens, 2020;9(9):10. doi:10.3390/pathogens9090735 26. Ahmed M, Fodjo J, Gele A, Farah A, Osman S, Guled I, et al. COVID-19 in Somalia: Adherence to
Preventive Measures and Evolution of the Disease Burden. Pathogens, 2020;9(9):10. doi:10.3390/pathogens9090735 COVID-19 IPC compliance in the HCWs (total IPC, PPE use, HH, other IPC), % Note: IPC: infection prevention and control, PPE: personal protective equipm Figures Page 18/19 Fi
1 Figure 1 COVID-19 IPC compliance in the HCWs (total IPC, PPE use, HH, other IPC), % COVID-19 IPC compliance in the HCWs (total IPC, PPE use, HH, other IPC), % Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to download. BMCPublicHealthSupplementaryfile1Questionnaire.docx.docx BMCPublicHealthSupplementaryfile1Questionnaire.docx.docx Page 19/19
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Синдром диссеминированного внутрисосудистого
свертывания при хирургическом лечении больных
злокачественными опухолями печени Сомонова О.В.1 • Елизарова А.Л.1 • Матвеева И.И.1 Сомонова О.В.1 • Елизарова А.Л.1 • Матвеева И.И.1 Исследования системы гемостаза проводились
до операции и на 1–20-е сутки после опера-
ции на автоматическом анализаторе гемостаза
“STA-R Evolution” и на агрегометре “Chrono-log”. Результаты. После хирургического вмеша-
тельства на печени подострый синдром дис-
семинированного внутрисосудистого сверты-
вания (ДВС) выявлен у 34 больных. Наиболее
часто (65%) он развивался после правосто-
ронней гемигепатэктомии и характеризовал-
ся снижением концентрации фибриногена до
121 мг/дл (p < 0,001), факторов протромбино-
вого комплекса до 45% (р < 0,05), антитромби-
на III до 48% (р < 0,05) на фоне значительного
повышения D-димера до 14,5 мкг/мл (р < 0,05). У 12 больных с подострой формой синдрома
ДВС развились тромбозы вен нижних конеч-
ностей, у 9 – тяжелая печеночная недоста-
точность. У пациентов с тяжелой печеночной недостаточностью наблюдалось статистически
значимое (p < 0,05) снижение протромбиновой
активности до 45%, антитромбина III до 44%,
плазминогена ниже 50% при высоком уровне
D-димера (> 20 мкл/мл) и фактора Виллебранда. Заключение. Хирургические
вмешатель-
ства у больных злокачественными опухолями
печени вызывают развитие синдрома ДВС. Своевременная диагностика и коррекция гемо-
стазиологических факторов риска развития тя-
желой печеночной недостаточности позволяют
улучшить результаты хирургического лечения
больных вторичными злокачественными опу-
холями печени. Актуальность. В структуре хирургических вме-
шательств у больных злокачественными опухо-
лями печени преобладают обширные резекции
или расширенные гемигепатэктомии, сопрово-
ждающиеся серьезными послеоперационными
осложнениями. Цель – выяснить роль нару-
шений системы гемостаза в патогенезе после-
операционной печеночной недостаточности,
а также тромбогеморрагических осложнений
у больных злокачественными опухолями пе-
чени. Материал и методы. Под наблюдением
находились 120 больных злокачественными
опухолями печени (20 пациентов с первичными
опухолями печени и 100 – с метастатическим
колоректальным раком печени). Обширные
резекции печени (право- и левосторонняя
гемигепатэктомия простая и расширенная)
были выполнены у 100 (84%) больных, резек-
ция нескольких сегментов печени – у 20 (16%). Ключевые слова: резекция печени, система
гемостаза, печеночная недостаточность doi: 10.18786/2072-0505-2016-44-5-640-646 З Такие вмешательства характеризуются высокой
травматичностью, длительностью, обширной ра-
невой поверхностью, развитием острой порталь-
ной гипертензии в раннем послеоперационном
периоде [6–8]. З
аболеваемость
колоректальным
раком
в России составляет порядка 50 тыс. слу-
чаев в год, при этом от 20 до 50% паци-
ентов при первичном обращении имеют
метастазы в печень [1, 2]. Резекция печени оста-
ется единственным методом, позволяющим зна-
чительно увеличить продолжительность жизни
(до уровня 5-летней выживаемости 74%) [3, 4]. Частота
послеоперационных
осложнений
у больных, перенесших расширенные гемиге-
патэктомии, варьирует от 14,4 до 45% [9–11]. Альманах клинической медицины. 2016 Июнь-июль; 44 (5): 640–646 Альманах клинической медицины. 2016 Июнь-июль; 44 (5): 640–646 ренная) были выполнены у 100 (84%) больных, ре-
зекция нескольких сегментов печени – у 20 (16%). При гистологическом исследовании первич-
ных опухолей установлен низкодифференци-
рованный
или
высокодифференцированный
гепатоцеллюлярный рак (10 больных) и холангио
целлюлярный рак (10 больных). При гистологи-
ческом исследовании метастатических очагов
обнаружена аденокарцинома кишечного типа
с различной степенью дифференцировки. ренная) были выполнены у 100 (84%) больных, ре-
зекция нескольких сегментов печени – у 20 (16%). Сомонова Оксана
Васильевна – д-р
мед. наук, вед. науч. сотр., клинико-
диагностическая
лаборатория1
*
* 115478, г. Москва,
Каширское шоссе, 24,
Российская Федерация. Тел.: +7 (499) 324 28 90. E-mail: somonova@mail.ru недостаточность характеризуется протромбино-
вой активностью по Квику < 50% (соответствует
международному нормализованному отноше-
нию > 1,7) и повышением уровня билирубина
> 50 ммоль/л (2,9 мг/дл) на 5-е сутки после опера-
ции [14]. Если состояние пациента отвечает кри-
териям «50/50», риск смерти составляет 59%, если
нет – то только 1,2% [15]. При гистологическом исследовании первич-
ных опухолей установлен низкодифференци-
рованный
или
высокодифференцированный
гепатоцеллюлярный рак (10 больных) и холангио
целлюлярный рак (10 больных). При гистологи-
ческом исследовании метастатических очагов
обнаружена аденокарцинома кишечного типа
с различной степенью дифференцировки. К другим тяжелым осложнениям резекции
печени относят острые кровотечения, тромбоз
воротной вены, тромбоз глубоких вен нижних
конечностей, тромбоэмболию легочной артерии,
в патогенезе которых немаловажное значение
принадлежит нарушениям в системе гемостаза
[15, 16]. Причины активации свертывания крови
у больных злокачественными новообразовани-
ями многообразны. Среди них следует отметить
выделение опухолевыми клетками высокоактив-
ного тканевого фактора и ракового прокоагулян-
та, а также прокоагулянтную активность моно-
цитов, тромбоцитов и эндотелиальных клеток
в ответ на опухоль. Активация прокоагулянт-
ного и тромбоцитарного звеньев гемостаза ведет
к появлению тромбина и локальному отложению
фибрина вокруг опухолевых клеток. Это форми-
рует матрицу для опухолевого роста и ангиоге-
неза, способствует развитию венозного тромбоза
и синдрома диссеминированного свертывания
крови (ДВС) [17, 18]. Елизарова Анна
Львовна – канд. биол. наук, ст. науч. сотр., клинико-
диагностическая
лаборатория1 Елизарова Анна
Львовна – канд. биол. наук, ст. науч. сотр., клинико-
диагностическая
лаборатория1
Матвеева Ирина
Ивановна – д-р мед. наук, заведующая
клинико-
диагностической
лабораторией1 Сопутствующая патология диагностирована
в 58 (48%) наблюдениях, в том числе сахарный ди-
абет (у 10 больных), гепатит В (7), гепатит С (4),
ишемическая болезнь сердца, артериальная ги-
пертония (20), варикозная болезнь, хроническая
венозная недостаточность (12). Неоадъювантная
химиотерапия была проведена у 20 больных. Показатели системы гемостаза оценивали по
10 параметрам, наиболее точно отражающим со-
стояние ее основных звеньев: прокоагулянтного,
тромбоцитарного, антикоагулянтного, фибри-
нолитического с маркерами внутрисосудистого
свертывания крови. Альманах клинической медицины. 2016 Июнь-июль; 44 (5): 640–646 Исследования системы гемо-
стаза проводились на автоматических коагулоло-
гических анализаторах “STA-R Evolution” и агре-
гометре фирмы “Chrono-log” до операции и на
1–20-е сутки после операции. На автоматическом
анализаторе “STA-R Evolution” определяли акти-
вированное частичное тромбопластиновое время
(АЧТВ), протромбиновую активность по Квику,
концентрацию фибриногена по Клауссу, актив-
ность антитромбина III, протеина С, плазмино-
гена, α2-антиплазмина, концентрацию D-димера,
фактор Виллебранда. Агрегацию тромбоцитов
исследовали на агрегометре фирмы “Chrono-log”. Печень играет ключевую роль в регуляции ге-
мостаза. Учитывая, что в печени синтезируется
большинство факторов свертывания крови и ин-
гибиторов, а также белков, участвующих в фи-
бринолизе, своевременная диагностика и кор-
рекция нарушений в системе гемостаза позволят
снизить частоту осложнений и тем самым улуч-
шить результаты хирургического лечения боль-
ных злокачественными опухолями печени. При статистической обработке результатов
рассчитывали средние арифметические значения
и стандартное отклонение (M ± SD). Сравнение
количественных данных в двух несвязанных
группах проводилось с использованием t-кри-
терия Стьюдента для независимых выборок. Достоверными считались результаты с ошибкой
менее 5% (p < 0,05). Цель исследования – выяснить роль наруше-
ний системы гемостаза в патогенезе послеопера-
ционной печеночной недостаточности, а также
тромбогеморрагических осложнений у больных
злокачественными опухолями печени. Исследования выполнялись в соответствии
с принципами Хельсинкской декларации, разра-
ботанной Всемирной медицинской ассоциацией. Материал и методы Под нашим наблюдением находились 120 боль-
ных злокачественными опухолями печени (муж-
чин – 56, женщин – 64). Из них 20 пациентов были
с первичными опухолями печени и 100 пациен-
тов с метастатическим колоректальным раком
печени. Контрольная группа состояла из 40 прак-
тически здоровых людей. 1 ФГБУ «Российский он-
кологический научный
центр им. Н.Н. Блохина»
Минздрава России;
115478, г. Москва,
Каширское шоссе, 24,
Российская Федерация Сомонова О.В., Елизарова А.Л., Матвеева И.И.
Синдром диссеминированного внутрисосудистого свертывания при хирургическом лечении больных злокачественными опухолями печени Сомонова О.В., Елизарова А.Л., Матвеева И.И. Синдром диссеминированного внутрисосудистого
свертывания при хирургическом лечении больных
злокачественными опухолями печени В структуре послеоперационных осложнений
и причин летальности (3–14%) ведущее место
занимает печеночная недостаточность разной
степени выраженности [12, 13]. Однозначных
и общепринятых критериев пострезекционной
печеночной недостаточности в настоящее время
нет. Обычно под этим термином понимают нару-
шение одной или нескольких синтетических или
экскреторных функций печени и/или печеноч-
ную энцефалопатию разной степени. Достаточно
точным представляется критерий «50/50», со-
гласно которому пострезекционная печеночная ур
В мире ежегодно выявляют около 1 млн
новых наблюдений первичного рака печени. Гепатоцеллюлярный рак по частоте составляет
около 85–90% всех первичных злокачественных
опухолей печени. Резекции печени может быть
подвергнуто от 13 до 30% пациентов, 5-летняя
выживаемость после этой операции достигает
14–61% [5]. ур
В мире ежегодно выявляют около 1 млн
новых наблюдений первичного рака печени. Гепатоцеллюлярный рак по частоте составляет
около 85–90% всех первичных злокачественных
опухолей печени. Резекции печени может быть
подвергнуто от 13 до 30% пациентов, 5-летняя
выживаемость после этой операции достигает
14–61% [5]. В последние 20 лет хирургическое лечение
больных злокачественными опухолями печени
представлено в основном обширными резекци-
ями или расширенными гемигепатэктомиями. 640 Оригинальные статьи Альманах клинической медицины. 2016 Июнь-июль; 44 (5): 640–646 Результаты Как видно из данных табл. 1, при изучении ис-
ходного состояния у больных первичными и вто-
ричными злокачественными опухолями печени
установлено достоверное укорочение АЧТВ и уве-
личение концентрации фибриногена в 1,3 раза,
что указывает на активацию прокоагулянтного Обширные резекции печени (право- и лево-
сторонняя гемигепатэктомия простая и расши- 641 еминированного внутрисосудистого свертывания при хирургическом лечении больных злокачественными опухолями печени Альманах клинической медицины. 2016 Июнь-июль; 44 (5): 640–646 Таблица 1. Показатели системы гемостаза у больных первичным и метастатическим раком
печени до начала лечения
Показатель
Здоровые доноры
(n = 40)
Больные раком
печени (n = 120)
Активированное частичное тромбопластиновое
время, с
38 ± 0,8
34 ± 0,9*
Протромбиновая активность по Квику, %
92 ± 2
91 ± 1,3
Концентрация фибриногена, мг/дл
300 ± 8,5
384 ± 14*
Агрегация тромбоцитов: степень агрегации МА, %
65 ± 1,9
80 ± 1*
Антитромбин III, %
87 ± 1,7
81 ± 0,9*
Протеин С, %
130 ± 2,5
75 ± 2,3*
Плазминоген, %
110 ± 1,9
90 ± 1,8*
α2-антиплазмин, %
93 ± 3,8
99 ± 2
D-димер, мкг/мл
0,4 ± 0,2
1,6 ± 0,4*
Фактор Виллебранда, %
110 ± 10
234 ± 10*
Данные представлены в виде среднего арифметического значения и стандартного отклонения
(M ± SD)
* Различия статистически значимы (p<0 05) по сравнению со здоровыми донорами Таблица 1. Показатели системы гемостаза у больных первичным и метастатическим раком
печени до начала лечения преимущественно в виде подострых и хрониче-
ских форм синдрома ДВС. Подострый синдром
ДВС выявлен у 34 больных; наиболее часто
(65%) он развивался после правосторонней ге-
мигепатэктомии и характеризовался существен-
ным и статистически значимым повышением
уровня D-димера (до 12 мкг/мл на 3–4-е сутки
после операции, до 14 мкг/мл на 9–10-е сутки)
и фактора Виллебранда (до 415% на 3–4-е сут-
ки), что свидетельствовало о выраженной акти-
вации внутрисосудистого свертывания крови
(табл. 2). Наблюдалось умеренное укорочение
АЧТВ с 1-х суток послеоперационного пери-
ода, максимально – на 3–6-е сутки (p < 0,05). Одновременно выявлено существенное сниже-
ние активности факторов протромбинового ком-
плекса: протромбиновая активность по Квику
снижалась до 53% на 2-е сутки после операции
и оставалась резко сниженной до 9–10-х суток
(в 2 раза на 7–8-е сутки). Концентрация фибрино-
гена также существенно снижалась после резек-
ции печени (до 121 мг/дл на 7–8-е сутки после опе-
рации) (p < 0,001). Результаты В послеоперационный период
обращало на себя внимание значительное умень-
шение уровня естественных антикоагулянтов
(антитромбина III до 48%, протеина С до 50%),
а также компонентов фибринолитической систе-
мы (плазминогена до 52%), защищающих орга-
низм от тромбообразования. * Различия статистически значимы (p < 0,05) по сравнению со здоровыми донорами звена свертывающей системы крови. Отмечены
изменения со стороны тромбоцитарного зве-
на в виде усиления агрегационной способно-
сти тромбоцитов. Активация прокоагулянтного
и тромбоцитарного звеньев системы гемостаза
ведет к появлению тромбина, повышенному от-
ложению фибрина с последующим его лизисом,
о чем свидетельствует увеличение маркеров вну-
трисосудистого свертывания крови. У больных
метастатическим колоректальным раком печени
концентрация D-димера, одного из надежных
и чувствительных маркеров тромбообразования,
была увеличена в 4 раза. Что касается фактора
Виллебранда – показателя повреждения стенки
сосуда и активации системы гемостаза, он был
повышен в 2,1 раза. В ответ на усиленное внутри-
сосудистое свертывание крови происходит рас-
ходование естественных ингибиторов тромбина
и других активных сериновых протеаз: у боль-
ных раком печени выявлено снижение уровня ан-
титромбина III и протеина С (в 1,7 раза). Таким
образом, можно говорить о развитии у больных
с первичными и метастатическими опухолями
печени гиперкоагуляции с признаками хрониче-
ского внутрисосудистого свертывания крови. У 12 больных с подострой формой синдрома
ДВС развились тромбоэмболические осложне-
ния. По локализации они распределились следу-
ющим образом: тромбоз поверхностных и глубо-
ких вен нижних конечностей зарегистрирован
в 10 наблюдениях, тромбозы воротной вены пе-
чени и левой печеночной вены – по 1 случаю. Несмотря на снижение факторов свертывания
крови, применение низкомолекулярных гепа-
ринов в сочетании с концентратом антитромби-
на III и свежезамороженной плазмой у больных
с подострой формой синдрома ДВС восстанав-
ливало факторы свертывания крови – по данным
ультразвукового допплеровского сканирования,
венозные тромбозы не определялись либо наблю-
далась реканализация просвета сосуда. У остальных пациентов (преимущественно
больные с резекцией нескольких сегментов пече-
ни) отмечались менее выраженные изменения си-
стемы гемостаза, характерные для хронической
формы синдрома ДВС. У больных с подострым диссеминирован-
ным внутрисосудистым свертыванием крови,
оперированных на печени, в раннем послеопе-
рационном периоде отмечались клинические Установлено,
что
хирургические
вмеша-
тельства на печени вызывали глубокие измене-
ния системы гемостаза, которые развивались 642 Оригинальные статьи Альманах клинической медицины. 2016 Июнь-июль; 44 (5): 640–646 а у больных злокачественными опухолями печени с подострым синдромом диссеминированного внутрисосудистого Таблица 2. Результаты Показатели системы гемостаза у больных злокачественными опухолями печени с подострым синдромом диссеминированного внутрисосудистого
свертывания (n = 34) после операции
Показатель
До операции
Послеоперационный период, сутки
1-е
2-е
3–4-е
5–6-е
7–8-е
9–10-е
Активированное частичное
тромбопластиновое время, с
33 ± 0,3
29 ± 0,5*
28 ± 0,6*
26 ± 0,7*
26 ± 1*
27 ± 0,9*
30 ± 1,4*
Протромбиновая активность
по Квику, %
92 ± 2,4
69 ± 1,4*
53 ± 2,4*
52 ± 2,1*
50 ± 3,6*
45 ± 2,6*
48 ± 2,9*
Концентрация фибриногена,
мг/дл
418 ± 9
207 ± 12*
197 ± 15,1*
145 ± 10,2*
151 ± 11*
121 ± 17*
125 ± 16*
Агрегация тромбоцитов:
степень агрегации МА, %
80 ± 2,3
68 ± 1,9*
60 ± 2*
67 ± 2,5*
53 ± 3*
54 ± 2,4*
56 ± 3,4*
Антитромбин III, %
82 ± 1,9
61 ± 2,1*
58 ± 1,9*
50 ± 2*
48 ± 1,8*
51 ± 2,3*
54 ± 2,4*
Протеин С, %
76 ± 2,2
58 ± 2,5*
48 ± 2,8*
50 ± 2,8*
52 ± 2,9*
57 ± 2,4*
54 ± 3*
Плазминоген, %
91 ± 2,1
69 ± 2,7*
52 ± 2,7*
55 ± 3*
52 ± 2,8*
55 ± 2,4*
68 ± 2,9*
D-димер, мкг/мл
1,6 ± 0,2
4 ± 0,4*
11 ± 0,5*
12,1 ± 0,9*
13,2 ± 0,7*
12,3 ± 0,6*
14,5 ± 0,8*
Фактор Виллебранда, %
234 ± 6,2
243 ± 8,1
380 ± 13*
415 ± 11*
393 ± 10*
340 ± 12*
300 ± 13,3*
Данные представлены в виде среднего арифметического значения и стандартного отклонения (M ± SD)
* Различия статистически значимы (p < 0,05) по сравнению с дооперационным периодом Таблица 2. Показатели системы гемостаза у больных злокачественными опухолями печени с подострым синдромом диссеминированного внутрисосудистого
свертывания (n = 34) после операции достоверно отличаясь от показателей пациентов
без тяжелой печеночной недостаточности (табл. 4). Одновременно в этой группе больных отмечалось
резкое снижение активности факторов протром-
бинового комплекса, уровня антитромбина III
и плазминогена по сравнению с аналогичными
показателями больных без тяжелой печеночной
недостаточности (p < 0,05). Следовательно, у па-
циентов с тяжелой печеночной недостаточностью
наблюдалось снижение протромбиновой актив-
ности до 45%, антитромбина III до 44%, плазми-
ногена ниже 50% при высоком уровне D-димера
(> 20 мкл/мл) и фактора Виллебранда. Эти пока-
затели можно отнести к гемостазиологическим
факторам риска развития тяжелой печеночной
недостаточности. Результаты признаки печеночной недостаточности – сла-
бость, сонливость, легкие формы желтухи и энце-
фалопатии, возникшие вследствие недостаточно-
го объема оставшейся части печени. Активность
патологического процесса в печени характери-
зовалась выраженным цитолитическим син-
дромом:
при
биохимическом
исследовании
крови (табл. 3) выявлено повышение активно-
сти аланинаминотрансферазы (АЛТ) в среднем
в 8,6 ± 1,5 раза и аспартатаминотрансферазы
(АСТ) – в 8,8 ± 1,9 раза. Снижение соотношения
АСТ/АЛТ до 0,65 ± 0,13 на 3-и и 5-е сутки (в нор-
ме коэффициент де Ритиса равен 1,33) может
говорить о тяжести поражения клеток печени. Информативным показателем степени поврежде-
ния паренхимы печени было повышение билиру-
бина в 2 раза на 1–5-е сутки послеоперационного
периода. После расширенных резекций печени
достоверно снизилось содержание в крови обще-
го белка и альбумина (в 1,5 раза на 1–5-е сутки по-
сле операции) (см. табл. 3). Сомонова О.В., Елизарова А.Л., Матвеева И.И.
Синдром диссеминированного внутрисосудистого свертывания при хирургическом лечении больных злокачественными опухолями печен Сомонова О.В., Елизарова А.Л., Матвеева И.И. Заключение Показатели системы гемостаза у больных злокачественными опухолями печени с тяжелой печеночной недостаточностью (n = 9)
Показатель
До операции
Послеоперационный период, сутки
1-е
3–4-е
5–6-е
Концентрация фибриногена, мг/дл
422 ± 8
177 ± 12*, †
140 ± 15,1*
144 ± 10,2*
Протромбиновая активность по Квику, %
82 ± 4
49 ± 1*, †
45 ± 2,1*, †
47 ± 2,9*
Антитромбин III, %
78 ± 2,3
49 ± 2,3*, †
44 ± 1,8*, †
48 ± 2,6*
Плазминоген, %
85 ± 3
39 ± 2,7*, †
46 ± 2,6*, †
52 ± 3,1*
D-димер, мкг/мл
2,1 ± 0,9
20 ± 1,8*, †
20 ± 2,8*, †
20 ± 3*, †
Фактор Виллебранда, %
240 ± 6,9
425 ± 8,1*, †
442 ± 18,2*
540 ± 14,2*, †
* Различия статистически значимы (p < 0,05) по сравнению с дооперационным периодом
† Различия статистически значимы (p < 0,05) по сравнению с показателями пациентов без тяжелой печеночной недостаточности Таблица 3. Показатели биохимического исследования крови у больных злокачественными опухолями печени с подострым синдромом диссеминированного
внутрисосудистого свертывания (n = 34) после операции
Показатель
Норма
До операции
Послеоперационный период, сутки
1-е
3–4-е
5–6-е
АЛТ, ед/мл
< 40
22,9 ± 2,1
344,7 ± 62,2*
198,6 ± 39,8*
88,2 ± 22,5*
ACT, ед/мл
< 37
27,1 ± 2,07
358 ± 67*
109,1 ± 12,9*
50,8 ± 9,3*
АСТ/АЛТ
1,33
0,87 ± 0,07
1,06 ± 0,06
0,67 ± 0,09
0,65 ± 0,13
Общий билирубин, мкмоль/л
0–20,5
20,3 ± 1,8
49,8 ± 8*
42 ± 10,5*
39,1 ± 4,08*
Общий белок, г/л
60–80
73,8 ± 1,8
49,9 ± 1,8*
51,2 ± 1,02*
52,1 ± 3*
Альбумин, г/л
35–50
41,2 ± 1,1
31,3 ± 1,97*
31 ± 1,3*
27,6 ± 2,9*
АЛТ – аланинаминотрансфераза, АСТ – аспартатаминотрансфераза
Данные представлены в виде среднего арифметического значения и стандартного отклонения (M ± SD)
* Различия статистически значимы (p < 0,05) по сравнению с дооперационным периодом Таблица 3. Показатели биохимического исследования крови у больных злокачественными опухолями печени с подострым синдромом диссеминированного
нутрисосудистого свертывания (n = 34) после операции Таблица 4. Конфликт интересов Конфликт интересов
Авторы сообщают об
отсутствии конфликта
интересов и финансовой
заинтересованности
в ходе написания
данной статьи и не несут
ответственности за
наличие и содержание
рекламных материалов,
размещенных на страни-
цах журнала. кова ОВ, Шишкина НА. Резекция печени:
современные технологии при опухолевом
поражении. Анналы хирургической гепато-
логии. 2010;15(2):9–17. рака. Онкологическая колопроктология.
2014;(1):14–20.
2. Патютко ЮИ, Сагайдак ИВ, Котельников АГ,
Поляков АН, Чучуев ЕС, Пылев АЛ, Чистя- рака. Онкологическая колопроктология.
2014;(1):14–20.
2. Патютко ЮИ, Сагайдак ИВ, Котельников АГ,
Поляков АН, Чучуев ЕС, Пылев АЛ, Чистя- Заключение Показатели системы гемостаза у больных злокачественными опухолями печени с тяжелой печеночной недостаточностью (n = 9)
Показатель
До операции
Послеоперационный период, сутки
1-е
3–4-е
5–6-е
Концентрация фибриногена, мг/дл
422 ± 8
177 ± 12*, †
140 ± 15,1*
144 ± 10,2*
Протромбиновая активность по Квику, %
82 ± 4
49 ± 1*, †
45 ± 2,1*, †
47 ± 2,9*
Антитромбин III, %
78 ± 2,3
49 ± 2,3*, †
44 ± 1,8*, †
48 ± 2,6*
Плазминоген, %
85 ± 3
39 ± 2,7*, †
46 ± 2,6*, †
52 ± 3,1*
D-димер, мкг/мл
2,1 ± 0,9
20 ± 1,8*, †
20 ± 2,8*, †
20 ± 3*, †
Фактор Виллебранда, %
240 ± 6,9
425 ± 8,1*, †
442 ± 18,2*
540 ± 14,2*, †
* Различия статистически значимы (p < 0,05) по сравнению с дооперационным периодом
† Различия статистически значимы (p < 0,05) по сравнению с показателями пациентов без тяжелой печеночной недостаточности таза у больных злокачественными опухолями печени с тяжелой печеночной недостаточностью (n = 9) Различия статистически значимы (p < 0,05) по сравнению с дооперационным периодом
† Различия статистически значимы (p < 0,05) по сравнению с показателями пациентов без тяжелой печеночной недостаточност эффективным и адекватным. К гемостазиологи-
ческим факторам риска развития тяжелой пече-
ночной недостаточности можно отнести повы-
шение уровня D-димера, фактора Виллебранда
на фоне резкого снижения протромбиновой ак-
тивности, содержания антитромбина III и уров-
ня плазминогена. Их своевременная диагностика
и коррекция позволяют улучшить результаты хи-
рургического лечения больных вторичными зло-
качественными опухолями печени. защищающих организм от тромбообразования,
что согласуется с данными литературы [19, 20]. Поскольку применение низкомолекулярных ге-
паринов в сочетании с концентратом антитром-
бина III и свежезамороженной плазмой у боль-
ных с тромбозами вен снижает интенсивность
внутрисосудистого свертывания крови, приво-
дит к восстановлению факторов свертывания,
этот метод лечения тромботических осложне-
ний у онкологических больных представляется Заключение Проведенные исследования показали, что хирур-
гические вмешательства у больных злокачествен-
ными опухолями печени вызывают развитие
различных форм синдрома ДВС со значитель-
ным повышением маркеров внутрисосудистого
свертывания крови на фоне резкого снижения
активности факторов протромбинового ком-
плекса, уровня естественных антикоагулянтов
и компонентов фибринолитической системы, Выделена группа больных (9 человек) с раз-
витием тяжелой печеночной недостаточности
в послеоперационном периоде. При изучении си-
стемы гемостаза у этих пациентов было установ-
лено, что уровень D-димера (20 мкг/мл) и фактор
Виллебранда повышались в большей степени, 643 иссеминированного внутрисосудистого свертывания при хирургическом лечении больных злокачественными опухолями печени Альманах клинической медицины. 2016 Июнь-июль; 44 (5): 640–646 защищающих организм от тромбообразования,
[19 20]
эффективным и адекватным. К гемостазиологи-
ф
й
Конфликт интересов
Таблица 3. Показатели биохимического исследования крови у больных злокачественными опухолями печени с подострым синдромом диссеминированного
внутрисосудистого свертывания (n = 34) после операции
Показатель
Норма
До операции
Послеоперационный период, сутки
1-е
3–4-е
5–6-е
АЛТ, ед/мл
< 40
22,9 ± 2,1
344,7 ± 62,2*
198,6 ± 39,8*
88,2 ± 22,5*
ACT, ед/мл
< 37
27,1 ± 2,07
358 ± 67*
109,1 ± 12,9*
50,8 ± 9,3*
АСТ/АЛТ
1,33
0,87 ± 0,07
1,06 ± 0,06
0,67 ± 0,09
0,65 ± 0,13
Общий билирубин, мкмоль/л
0–20,5
20,3 ± 1,8
49,8 ± 8*
42 ± 10,5*
39,1 ± 4,08*
Общий белок, г/л
60–80
73,8 ± 1,8
49,9 ± 1,8*
51,2 ± 1,02*
52,1 ± 3*
Альбумин, г/л
35–50
41,2 ± 1,1
31,3 ± 1,97*
31 ± 1,3*
27,6 ± 2,9*
АЛТ – аланинаминотрансфераза, АСТ – аспартатаминотрансфераза
Данные представлены в виде среднего арифметического значения и стандартного отклонения (M ± SD)
* Различия статистически значимы (p < 0,05) по сравнению с дооперационным периодом
Таблица 4. Литература кова ОВ, Шишкина НА. Резекция печени:
современные технологии при опухолевом
поражении. Анналы хирургической гепато-
логии. 2010;15(2):9–17. 1. Патютко ЮИ, Котельников АГ, Мамонтов КГ,
Пономаренко АА, Лазарев АФ. Непосред-
ственные результаты резекций печени
по поводу метастазов колоректального 1. Патютко ЮИ, Котельников АГ, Мамонтов КГ,
Пономаренко АА, Лазарев АФ. Непосред-
ственные результаты резекций печени
по поводу метастазов колоректального 644 Оригинальные статьи Альманах клинической медицины. 2016 Июнь-июль; 44 (5): 640–646 treatment. Liver Int. 2008;28(6):767–80. doi:
10.1111/j.1478-3231.2008.01777.x. 3. Kanas GP, Taylor A, Primrose JN, Langeberg WJ,
Kelsh MA, Mowat FS, Alexander DD, Choti MA,
Poston G. Survival after liver resection in meta-
static colorectal cancer: review and meta-anal-
ysis of prognostic factors. Clin Epidemiol. 2012;4:283–301. doi: 10.2147/CLEP.S34285. 9. Huang ZQ, Xu LN, Yang T, Zhang WZ, Huang XQ,
Cai SW, Zhang AQ, Feng YQ, Zhou NX, Dong JH. Hepatic resection: an analysis of the impact of
operative and perioperative factors on mor-
bidity and mortality rates in 2008 consecu-
tive hepatectomy cases. Chin Med J (Engl). 2009;122(19):2268–77. 16. Meijer C, Wiezer MJ, Hack CE, Boelens PG,
Wedel NI, Meijer S, Nijveldt RJ, Statius
Muller MG, Wiggers T, Zoetmulder FA, Borel
Rinkes IH, Cuesta MA, Gouma DJ, van de Vel-
de CJ, Tilanus HW, Scotté M, Thijs LG, van Leeu-
wen PA. Coagulopathy following major liver
resection: the effect of rBPI21 and the role of
decreased synthesis of regulating proteins by
the liver. Shock. 2001;15(4):261–71. 4. Патютко ЮИ, Пылев АЛ, Сагайдак ИВ, Ко-
тельников АГ, Подлужный ДВ, Поляков АН,
Абгарян МГ, Чучуев ЕС, Гахраманов АД,
Шишкина НА, Агафонова МГ. Десятилетняя
выживаемость больных злокачественными
опухолями печени после хирургического
лечения. Анналы хирургической гепатоло-
гии. 2010;15(3):39–47. 10. Вишневский ВА, Кубышкин ВА, Чжао АВ,
Икрамов
РЗ. Операции
на
печени. М.: Миклош; 2003. 157 с. 11. Патютко ЮИ. Хирургическое лечение злока-
чественных опухолей печени. М.: Практиче-
ская медицина; 2005. 312 c. 17. Amin C, Mackman N, Key NS. Microparticles
and cancer. Pathophysiol Haemost Thromb. 2008;36(3–4):177–83. doi: 10.1159/000175155. 12. Jarnagin WR, Gonen M, Fong Y, DeMat-
teo RP, Ben-Porat L, Little S, Corvera C, Weber S,
Blumgart LH. Improvement in perioperative
outcome after hepatic resection: analysis of
1,803 consecutive cases over the past de-
cade. Ann Surg. 2002;236(4):397–406. doi:
10.1097/01.SLA.0000029003.66466.B3. 5. Чиссов ВИ, Бутенко АВ, Вашакмадзе ЛА, Си-
доров ДВ, Гришин НА, Ложкин МВ, Степа-
нов СО, Хомяков ВМ, Швейкин АО, Гуц ОВ. Хирургическое лечение первичного и ме-
тастатического рака печени. Российский
онкологический журнал. 2010;(5):8–12. 18. References Shishkina NA, Agafonova MG. Desyatiletnyaya
vyzhivaemost' bol'nykh zlokachestvennymi
opukholyami pecheni posle khirurgichesko-
go lecheniya [Ten-years survival after surgical
management of the liver malignancies]. Annaly
khirurgicheskoy gepatologii [Annals of Surgical
Hepatology]. 2010;15(3):39–47 (in Russian). Shishkina NA, Agafonova MG. Desyatiletnyaya
vyzhivaemost' bol'nykh zlokachestvennymi
opukholyami pecheni posle khirurgichesko-
go lecheniya [Ten-years survival after surgical
management of the liver malignancies]. Annaly
khirurgicheskoy gepatologii [Annals of Surgical
Hepatology]. 2010;15(3):39–47 (in Russian). 8. Kin Y, Nimura Y, Hayakawa N, Kamiya J, Kondo S,
Nagino M, Miyachi M, Kanai M. Doppler analysis
of hepatic blood flow predicts liver dysfunc-
tion after major hepatectomy. World J Surg. 1994;18(1):143–9. 1. Patyutko YuI, Kotel'nikov AG, Mamontov KG,
Ponomarenko AA, Lazarev AF. Neposredstven-
nye rezul'taty rezektsiy pecheni po povodu
metastazov kolorektal'nogo raka [Immediate
results of hepatectomy for metastatic colorec-
tal cancer]. Onkologicheskaya koloproktologiya
[Oncological Coloproctology]. 2014;(1):14–20
(in Russian). 9. Huang ZQ, Xu LN, Yang T, Zhang WZ, Huang XQ,
Cai SW, Zhang AQ, Feng YQ, Zhou NX, Dong JH. Hepatic resection: an analysis of the impact of
operative and perioperative factors on mor-
bidity and mortality rates in 2008 consecu-
tive hepatectomy cases. Chin Med J (Engl). 2009;122(19):2268–77. 5. C 5. Chissov VI, Butenko AV, Vashakmadze LA, Sidor-
ov DV, Grishin NA, Lozhkin MV, Stepanov SO,
Khomyakov VM, Shveykin AO, Guts OV. Khiru-
rgicheskoe lechenie pervichnogo i metasta-
ticheskogo raka pecheni [Surgical treatment
for primary and metastatic tumors of the liver]. Rossiyskiy Onkologicheskiy Zhurnal [Russian
Journal of Oncology]. 2010;(5):8–12 (in Russian). 2. Patyutko YuI, Sagaydak IV, Kotel'nikov AG, Polya-
kov AN, Chuchuev ES, Pylev AL, Chistyakova OV,
Shishkina NA. Rezektsiya pecheni: sovremennye
tekhnologii pri opukholevom porazhenii [Liver
resection: current technologies for cancer]. An-
naly khirurgicheskoy gepatologii [Annals of Sur-
gical Hepatology]. 2010;15(2):9–17 (in Russian). 10. Vishnevskiy VA, Kubyshkin VA, Chzhao AV,
Ikramov RZ. Operatsii na pecheni [Liver sur-
gery]. Moscow: Miklosh; 2003. 157 p. (in Rus-
sian). 3. Kanas GP, Taylor A, Primrose JN, Langeberg WJ,
Kelsh MA, Mowat FS, Alexander DD, Choti MA,
Poston G. Survival after liver resection in meta-
static colorectal cancer: review and meta-anal-
ysis of prognostic factors. Clin Epidemiol. 2012;4:283–301. doi: 10.2147/CLEP.S34285. 6. Sato T, Yasui A, Kondo S, Kato M, Kameoka N,
Nimura Y. Changes in liver blood flow after
hepatectomy in conscious dogs. Surg Today. 1996;26(9):688–93. 11. Patyutko YuI. Khirurgicheskoe lechenie zlok-
achestvennykh opukholey pecheni [Surgical
treatment for hepatic malignancies]. Moscow:
Prakticheskaya meditsina; 2005. 312 p. (in Rus-
sian). 7. Литература Magnus N, D'Asti E, Meehan B, Garnier D,
Rak J. Oncogenes and the coagulation sys-
tem – forces that modulate dormant and
aggressive states in cancer. Thromb Res. 2014;133 Suppl 2:S1–9. doi: 10.1016/S0049-
3848(14)50001-1. 6. Sato T, Yasui A, Kondo S, Kato M, Kameoka N,
Nimura Y. Changes in liver blood flow after
hepatectomy in conscious dogs. Surg Today. 1996;26(9):688–93. 19. Umemura Y, Yamakawa K, Kiguchi T, Yoshi-
kawa Y, Ogura H, Shimazu T, Hamasaki T,
Fujimi S. Design and evaluation of New Uni-
fied Criteria for Disseminated Intravascular
Coagulation based on the Japanese Associ-
ation for Acute Medicine Criteria. Clin Appl
Thromb Hemost. 2016;22(2):153–60. doi:
10.1177/1076029615591169. 13. Герок В, Блюм ХЕ. Заболевания печени
и желчевыводящей системы. Пер. с нем. М.: МЕДпресс-информ; 2009. 200 с. 14. Balzan S, Belghiti J, Farges O, Ogata S, Sau-
vanet A, Delefosse D, Durand F. The "50-50
criteria" on postoperative day 5: an accurate
predictor of liver failure and death after hepa-
tectomy. Ann Surg. 2005;242(6):824–8. doi:
10.1097/01.sla.0000189131.90876.9e. 7. Sato Y, Koyama S, Tsukada K, Hatakeyama K. Acute portal hypertension reflecting shear
stress as a trigger of liver regeneration fol-
lowing partial hepatectomy. Surg Today. 1997;27(6):518–26. 20. Wada H, Matsumoto T, Yamashita Y. Diagnosis
and treatment of disseminated intravascu-
lar coagulation (DIC) according to four DIC
guidelines. J Intensive Care. 2014;2(1):15. doi:
10.1186/2052-0492-2-15. 8. Kin Y, Nimura Y, Hayakawa N, Kamiya J, Kon-
do S, Nagino M, Miyachi M, Kanai M. Doppler
analysis of hepatic blood flow predicts liver
dysfunction after major hepatectomy. World
J Surg. 1994;18(1):143–9. 15. van den Broek MA, Olde Damink SW, De-
jong CH, Lang H, Malagó M, Jalan R, Saner FH. Liver failure after partial hepatic resection:
definition, pathophysiology, risk factors and References Sato Y, Koyama S, Tsukada K, Hatakeyama K. Acute portal hypertension reflecting shear
stress as a trigger of liver regeneration fol-
lowing partial hepatectomy. Surg Today. 1997;27(6):518–26. 12. Jarnagin WR, Gonen M, Fong Y, DeMat-
teo RP, Ben-Porat L, Little S, Corvera C, Weber S,
Blumgart LH. Improvement in perioperative 4. Patyutko YuI, Pylev AL, Sagaydak IV, Ko-
tel'nikov AG, Podluzhnyy DV, Polyakov AN, Ab-
garyan MG, Chuchuev ES, Gakhramanov AD, 645 р
еминированного внутрисосудистого свертывания при хирургическом лечении больных злокачественными опухолями печени Almanac of Clinical Medicine. 2016 June-July; 44 (5): 640–646 treatment. Liver Int. 2008;28(6):767–80. doi:
10.1111/j.1478-3231.2008.01777.x. outcome after hepatic resection: analysis of
1,803 consecutive cases over the past de-
cade. Ann Surg. 2002;236(4):397–406. doi:
10.1097/01.SLA.0000029003.66466.B3. that modulate dormant and aggressive states in
cancer. Thromb Res. 2014;133 Suppl 2:S1–9. doi:
10.1016/S0049-3848(14)50001-1. outcome after hepatic resection: analysis of
1,803 consecutive cases over the past de-
cade. Ann Surg. 2002;236(4):397–406. doi:
10.1097/01.SLA.0000029003.66466.B3. 16. Meijer C, Wiezer MJ, Hack CE, Boelens PG,
Wedel NI, Meijer S, Nijveldt RJ, Statius
Muller MG, Wiggers T, Zoetmulder FA, Borel
Rinkes IH, Cuesta MA, Gouma DJ, van de Vel-
de CJ, Tilanus HW, Scotté M, Thijs LG, van Leeu-
wen PA. Coagulopathy following major liver
resection: the effect of rBPI21 and the role of
decreased synthesis of regulating proteins by
the liver. Shock. 2001;15(4):261–71. 19. Umemura Y, Yamakawa K, Kiguchi T, Yoshi-
kawa Y, Ogura H, Shimazu T, Hamasaki T,
Fujimi S. Design and evaluation of New Uni-
fied Criteria for Disseminated Intravascular
Coagulation based on the Japanese Associ-
ation for Acute Medicine Criteria. Clin Appl
Thromb
Hemost. 2016;22(2):153–60. doi:
10.1177/1076029615591169. 13. Gerok W, Blum HE. Krankheiten der Leber und
des biliären Systems. Stuttgart – New York:
Schattauer Verlag; 2007 (in German). 14. Balzan S, Belghiti J, Farges O, Ogata S, Sau-
vanet A, Delefosse D, Durand F. The "50-50 crite-
ria" on postoperative day 5: an accurate predic-
tor of liver failure and death after hepatectomy. Ann Surg. 2005;242(6):824–8. doi: 10.1097/01. sla.0000189131.90876.9e. 17. Amin C, Mackman N, Key NS. Microparticles
and cancer. Pathophysiol Haemost Thromb. 2008;36(3–4):177–83. doi: 10.1159/000175155. 20. Wada H, Matsumoto T, Yamashita Y. Diagno-
sis and treatment of disseminated intravas-
cular coagulation (DIC) according to four DIC
guidelines. J Intensive Care. 2014;2(1):15. doi:
10.1186/2052-0492-2-15. 15. van den Broek MA, Olde Damink SW, De-
jong CH, Lang H, Malagó M, Jalan R, Saner FH. 1 N.N. Blokhin Russian Cancer Research Center;
24 Kashirskoe shosse, Moscow, 115478, Russian
Federation References Liver failure after partial hepatic resection:
definition, pathophysiology, risk factors and 18. Magnus N, D'Asti E, Meehan B, Garnier D, Rak J. Oncogenes and the coagulation system – forces Somonova O.V.1 • Elizarova A.L.1 • Matveeva I.I.1 Somonova O.V.1 • Elizarova A.L.1 • Matveeva I.I.1 Rationale: Extended resections or extended lo-
bectomies are the most common types of surgical
interventions in patients with liver malignancies,
and they are associated with serious post-opera-
tive complications. Aim: To characterize the role
of hemostasis abnormalities in the pathophys-
iology of post-operative hepatic insufficiency,
as well as that of thrombotic and hemorrhagic
complications in patients with liver malignancies. Materials and methods: One hundred and twen-
ty patients with liver malignancies were recruited
into the study (20 patients with primary hepatic
tumors and 100 with colorectal cancer and liver
metastases). Extended liver resections (right and
left simple and extended lobectomies, both sim-
ple and extended) were performed in 100 (84%)
of patients; multi-segmental liver resections, in
20 (16%). Assessment of hemostasis was done
pre-operatively and at days 1 to 20 after surgery
(hemostasis analyzer system STA-R Evolution and
Chrono-log aggregometer). Results: After surgi-
cal intervention in the liver, subacute disseminat-
ed intravascular coagulation (DIC) was found in
34 patients. It was most common (65%) after the right lobectomy and was associated with a de-
crease in fibrinogen levels to 121 mg/dL (p < 0.01),
prothrombin complex factors, to 45% (р < 0.05),
antithrombin III to 48% (р < 0.05), with a sig-
nificant increase in D dimmer levels of up to
14.5 mcg/mL (р < 0.05). Twelve patients with sub-
acute DIC developed deep venous thrombosis of
the lower extremities, and 9 patients had severe
hepatic insufficiency. Patients with severe hepat-
ic insufficiency had a statistically significant de-
crease in prothrombin activity to 45% (p < 0.05),
antithrombin III to 44%, plasminogen < 50%, with
high D dimer (> 20 mcl/mL) and von Willebrand
factor levels. Conclusion: Surgical interventions
in patients with liver malignancy may lead to the
development of DIC. Early diagnosis and correc-
tion of hemostasis-related risk factors of hepatic
insufficiency allows for improvement of the results
of surgery in patients with secondary hepatic ma-
lignancies. Somonova Oksana V. – MD, PhD, Leading Research
Fellow, Clinical Diagnostic Laboratory1
*
* 24 Kashirskoe shosse, Moscow, 115478, Russian
Federation. Tel.: +7 (499) 324 28 90. E-mail: somonova@mail.ru
Elizarova Anna L. – PhD (in Biol.), Senior Research
Fellow, Clinical Diagnostic Laboratory1
Matveeva Irina I. – MD, PhD, Head of Clinical Diag-
nostic Laboratory1 Matveeva Irina I. Somonova O.V.1 • Elizarova A.L.1 • Matveeva I.I.1 – MD, PhD, Head of Clinical Diag-
nostic Laboratory1 Key words: liver resection, hemostasis system, he-
patic insufficiency Key words: liver resection, hemostasis system, he-
patic insufficiency doi: 10.18786/2072-0505-2016-44-5-640-646 doi: 10.18786/2072-0505-2016-44-5-640-646 646
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https://refubium.fu-berlin.de/bitstream/fub188/27764/1/2020_Hanitsch_etal.pdf
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English
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Outpatient decolonization after recurrent skin infection with Panton-Valentine leukocidin (PVL)-producing S. aureus—The importance of treatment repetition
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PloS one
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PLOS ONE PLOS ONE RESEARCH ARTICLE OPEN ACCESS Citation: Hanitsch LG, Kru¨ger R, Hoppe P-A,
Humme D, Pokrywka A, Niebank M, et al. (2020)
Outpatient decolonization after recurrent skin
infection with Panton-Valentine leukocidin (PVL)-
producing S. aureus—The importance of treatment
repetition. PLoS ONE 15(4): e0231772. https://doi. org/10.1371/journal.pone.0231772 Editor: Bok-Luel Lee, Pusan National University,
REPUBLIC OF KOREA Editor: Bok-Luel Lee, Pusan National University,
REPUBLIC OF KOREA Editor: Bok-Luel Lee, Pusan National University,
REPUBLIC OF KOREA Received: November 1, 2019
Accepted: March 31, 2020
Published: April 21, 2020 Received: November 1, 2019
Accepted: March 31, 2020
Published: April 21, 2020
Copyright: © 2020 Hanitsch et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. Background Recurrent skin abscesses are often associated with Panton-Valentine leukocidin-producing
strains of S. aureus (PVL-SA). Decolonization measures are required along with treatment
of active infections to prevent re-infection and spreading. Even though most PVL-SA
patients are treated as outpatients, there are few studies that assess the effectiveness of
outpatient topical decolonization in PVL-SA patients. Outpatient decolonization after recurrent skin
infection with Panton-Valentine leukocidin
(PVL)-producing S. aureus—The importance of
treatment repetition Leif G. Hanitsch1,2, Renate Kru¨ger2,3, Pia-Alice Hoppe2,3, Daniel Humme2,4,
Anna Pokrywka2,4, Michaela Niebank2,5, Miriam Stegemann2,5, Axel Kola6,
Rasmus LeistnerID2,6* a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 1 Institute of Medical Immunology, Charite´ Universita¨tsmedizin Berlin, Berlin, Germany, 2 Interdisciplinary
workgroup on PVL-positive S. aureus, Charite´ Universita¨tsmedizin Berlin, Berlin, Germany, 3 Department
of Pediatric Pulmonology and Immunology, Charite´ Universita¨tsmedizin Berlin, Berlin, Germany,
4 Department of Dermatology and Allergy, Charite´ Universita¨tsmedizin Berlin, Berlin, Germany,
5 Department of Internal Medicine, Infectious Diseases and Pulmonary Medicine, Charite´
Universita¨tsmedizin, Berlin, Germany, 6 Institute of Hygiene and Environmental Medicine, Charite´
Universita¨tsmedizin Berlin, Berlin, Germany * rasmus.leistner@charite.de Methods Copyright: © 2020 Hanitsch 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. We assessed the results of topical decolonization of PVL-SA in a retrospective review of
patient files and personal interviews. Successful decolonization was defined as the absence
of any skin abscesses for at least 6 months after completion of the final decolonization treat-
ment. Clinical and demographic data was assessed. An intention-to-treat protocol was used. Conclusion In our cohort, topical decolonization was a successful preventive measure for reducing the
risk of PVL-SA skin abscesses in the outpatient setting. Special attention should be given to
patients living in multiple person households because these settings could confer a risk that
decolonization will not be successful. Results Our cohort consisted of 115 symptomatic patients, 66% from PVL-positive MSSA and 19%
from PVL-positive MRSA. The remaining 16% consisted of symptomatic patients with close
contact to PVL-SA positive index patients but without detection of PVL-SA. The majority of
patients were female (66%). The median age was 29.87% of the patients lived in multiple
person households. Our results showed a 48% reduction in symptomatic PVL-SA cases
after the first decolonization treatment. The results also showed that the decrease continued
with each repeated decolonization treatment and reached 89% following the 5th treatment. Funding: The author(s) received no specific
funding for this work. Competing interests: The authors have declared
that no competing interests exist. Competing interests: The authors have declared
that no competing interests exist. Abbreviations: IQR, interquartile range; MRSA,
Methicillin resistant Staphylococcus aureus; PLOS ONE | https://doi.org/10.1371/journal.pone.0231772
April 21, 2020 1 / 9 PLOS ONE (PVL)-producing S. aureus—The importance of repeting decolonization A built multivariable Cox proportional-hazards model showed that the absence of PVL-SA
detection (OR 2.0) and living in single person households (OR 2.4) were associated with an
independently increased chance of successful decolonization. A built multivariable Cox proportional-hazards model showed that the absence of PVL-SA
detection (OR 2.0) and living in single person households (OR 2.4) were associated with an
independently increased chance of successful decolonization. MSSA, Methicillin susceptible Staphylococcus
aureus; PCR, polymerase chain reaction; PVL,
Panton-Valentine leucocidin; SA, Staphylococcus
aureus. Introduction Recurrent skin abscesses in patients who do not have a predisposing condition are often
associated with Panton-Valentine leukocidin-producing strains of S. aureus (PVL-SA) [1–3]. Although in North America PVL-SA is predominantly associated with community-acquired
MRSA related primarily to the clone USA 300 [3, 4], in Europe the vast majority of PVL-posi-
tive S. aureus (PVL-SA) strains are methicillin-susceptible (MSSA) [5]. The verification of
PVL-SA colonization or infection requires microbiological screening for S. aureus and addi-
tional PCR testing for the production of Panton-Valentine leukocidin by amplifying the
encoding genes LukS/LukF [1]. There can be a considerable delay in diagnosis despite its typical clinical presentation with
non-immunocompromised patients who suffer from recurrent skin abscesses. This results in
multiple episodes of skin infections and often leads to the transmission of pathogens to close
contacts [1, 2, 6, 7]. The diagnostic confirmation of a PVL-SA skin infection can be challenging
for several reasons. Although microbiological screening has a high sensitivity for monocloncal
PVL-positive MRSA, it is not clear how well nasal screening performs in cases of polyclonal
MSSA colonization [8–10]. Moreover, often many members of a family or individuals in the
same household can be affected. But the ping-pong-like transmission and retransmission
events of S. aureus can make PVL-SA colonization time-dependent [10, 11]. This makes it
difficult to eradicate PVL-SA colonization in all potential carriers. After the primary treatment of PVL-SA-related infections, secondary prevention requires
topical decolonization [6, 12–14]. Despite the fact that many patients show relevant skin infec-
tions, most cases can be treated in an outpatient setting. This includes decolonization proce-
dures. However, there is a dearth of studies regarding outpatient decolonization and its specific
hurdles in the outpatient setting for patients with recurrent PVL-SA skin infections. Hence, in
this article we will focus on factors for the success of PVL-SA topical decolonization that are
relevant to the outpatient setting. Methods Our cohort consisted of patients with recurrent skin infections combined with the detection of
PVL-positive S. aureus (PVL-SA) as well as their close contacts who sometimes also displayed
symptoms. All cohort members were seen in our outpatient clinic between December 2010
and August 2017. Recurrence of an abscess was defined as more than one abscess that required
surgical drainage within a two-month period. Patients with a (predisposing) chronic skin con-
dition, e.g. Acne inversa or severe atopic dermatitis, were excluded from the present study. In 2 / 9 PLOS ONE | https://doi.org/10.1371/journal.pone.0231772
April 21, 2020 PLOS ONE (PVL)-producing S. aureus—The importance of repeting decolonization clusters with more than one symptomatic and / or PVL-SA-positive patient, the topical decolo-
nization protocol was followed for all household members. In case of active PVL-SA skin
infections, patients received a rifampicin-based dual antibiotic regime combined with TMP/
SMX or as determined by antibiogram results. In addition, patients were advised to start a
decolonization treatment at home after the resolution of the infection. Decolonization
involved a 5-day standard decolonization protocol that included daily antiseptic washing of
the whole body and hair with a commercially available octenidin-based product, antiseptic
treatment of the oral cavity (octenidin-based), intranasal application of mupirocin, and
extended hygiene measures including the washing of clothes at more than 60˚C and disposing
of personal care products [2]. Success of the decolonization procedure was defined as a period of at least six months
without a skin abscess or other SSTI following completion of the decolonization treatment. Decolonization was repeated in cases when skin abscesses recurred. The following variables
were systematically evaluated in interviews to assess the factors that reduced the success of
decolonization: age, gender, number of decolonization treatments, household size, number
of close contacts to the index case, abscess localization, and phenotype of PVL-SA (MRSA or
MSSA). A cluster was defined as individuals in the index patient’s immediate environment
(e.g. family or friends with frequent close physical contact) who were also colonized or infected
with PVL-SA. Household size was defined as the number of individuals living together in a
household with the index patient. All patients were screened for PVL-SA in nares, throat and wounds, if applicable. Swabs
were cultivated on Columbia Agar with 5% sheep blood. Species identification and antimicro-
bial susceptibility testing were performed using a Vitek 2 system and applying EUCAST break-
points. Methods The detection of PVL LukS/LukF was performed using PCR [15]. In order to assess parameters influencing the length of time needed for successful decoloni-
zation, we performed a univariate Kaplan-Meier analysis and a multivariable Cox regression
of all symptomatic patients, applying an intention-to-treat protocol. All analyses were per-
formed using SPSS (IBM SPSS statistics, Somer, NY, USA). Ethics approval and consent to participate: The study was conducted in accordance with
the Declaration of Helsinki, national, and institutional standards. The study was approved by
the local Ethics Committee (Charite´, Berlin, Germany, EA2/190/17). The study is based on sec-
ondary data that was generated for routine clinical care. Within this scope, written consent for
treatment purposes and secondary analysis was provided by each patient. Consent for minors
under age 18 was obtained from parents or guardians. The Ethics Committee waived the
requirement for additional consent for this study. PLOS ONE | https://doi.org/10.1371/journal.pone.0231772
April 21, 2020 Results Sixty seven index patients with PVL-SA-positive recurrent skin abscesses presented to our out-
patient clinics between December 2010 and August 2017. Altogether, they reported 128 close
contacts (accounting for n: 195). 48 of 128 contacts reported recurrent skin abscesses. Of the
remaining 80 asymptomatic contacts, 40 contacts participated in PVL-SA screening, revealing
an additional 21 PVL-SA positive cases. The remaining 40 asymptomatic contact patients were
not available for examination of PVL-SA (Fig 1). Taken together, of 155 patients and contacts, PVL-SA was verified in 118 cases (76%). One
patient was colonized with both phenotypes, hence 27 MRSA (23%), 92 MSSA (78%) were
detected. By the time of diagnosis (skin abscess due to PVL-SA), the patients’ medical histories
showed a median of 4 episodes of skin abscesses (IQR 2–10) and a median delay of 5 months PLOS ONE | https://doi.org/10.1371/journal.pone.0231772
April 21, 2020 3 / 9 PLOS ONE (PVL)-producing S. aureus—The importance of repeting decolonization Fig 1. Flow chart of all enrolled patients. https://doi.org/10.1371/journal.pone.0231772.g001 Fig 1. Flow chart of all enrolled patients. htt
//d i
/10 1371/j
l
0231772 001 Fig 1. Flow chart of all enrolled patients. Fig 1. Flow chart of all enrolled patients. https://doi.org/10.1371/journal.pone.0231772.g001 between first episode and final diagnosis (IQR 1–14). We found it helpful to calculate the aver-
age number of abscesses per month as an estimator of the severity of the infection and the psy-
chological strain on patients. The result was a median of 1.0 (IQR 0.3–2.8). Common abscess
sites included extremities (50%), trunk (47%), and head or face (42%), without any specific dis-
tribution pattern. Few patients (4%) showed invasive infections, such as necrotizing fasciitis,
necrotizing pneumonia or bloodstream infection. Based on the patients’ past medical histories, many reported that they had undergone multi-
ple antibiotic treatments and multiple episodes of surgical intervention before being diagnosed
with skin abscesses from PVL-SA. 38 (33%) patients reported hospital stays for the treatment
of their recurrent abscesses with a length of stay between 1 and 42 days (median 3 days). Table 1 provides an overview of all assessed parameters. Following an intention-to-treat protocol, we analyzed all 115 symptomatic patients for suc-
cessful topical decolonization. 77 (67%) received systemic antimicrobial treatment beforehand
without relevant reduction in infection incidence. The median follow-up time was 15 months
after the final decolonization treatment. Results After the first decolonization treatment, the number
of symptomatic patients was reduced by 48% (56 patients) (Fig 2). 13 patients were lost to fol-
low up (11%). Altogether, this amounted to a success rate of 89% after 5 decolonization
treatments. The following parameters were considered in the Cox regression analysis: age, gender,
number of decolonization treatments, single vs. multiple person household and phenotype of
PVL-SA (MSSA/MRSA). Factors independently associated with a successful decolonization
were living in a single household (in contrast to a multiple person household) and undetected
PVL-SA in symptomatic patients (Table 2 and Fig 3). Discussion Skin and soft tissue infection caused by PVL-positive S. aureus are frequently associated with
recurrent episodes of infection, delayed diagnoses, and eventually an increased risk of trans-
mission often resulting in clusters of affected patients in close personal proximity [1–3, 6, 16]. Although these infections are often community-acquired, there is a lack of literature that ana-
lyzes the effectiveness of outpatient decolonization [17, 18]. PLOS ONE | https://doi.org/10.1371/journal.pone.0231772
April 21, 2020 4 / 9 (PVL)-producing S. aureus—The importance of repeting decolonization MRSA, methicillin-resistant S. aureus. MSSA, methicillin-susceptible S. aureus. PVL, Panton-Valentine leukodicin. Successful decolonization was defined as remaining
clinically asymptomatic for at least 6 months after final decolonization treatment. PLOS ONE PLOS ONE PLOS ONE Table 1. Overview on the analyzed PVL-SA patients. Parameter
Total cohort (n = 155)
Clinically symptomatic (n = 115)
Clinically asymptomatic (n = 40)
Median (range) / % (number)
Median (range) / % (number)
Median (range) / % (number)
Age (years)
29 (0–75)
28 (0–65)
31 (0–75)
Male sex
44% (68)
43% (49)
48% (19)
Months before diagnosis
not applicable
5 (0–158)
not applicable
Number of abscesses before diagnosis
not applicable
4 (1–100)
-
Number of antimicrobial treatments before diagnosis
1 (0–15)
1 (1–15)
0 (0–3)
Number of surgical treatments before diagnosis
not applicable
1 (1–13)
-
Detected pathogen PVL-positive S. aureus
MRSA
17% (27)
19% (22)
13% (5)
MSSA
59% (92)
66% (76)
40% (16)
Not detected
24% (37)
16% (18)
48% (19)
Successful decolonization
Yes
88% (137)
89% (102)
not applicable
Lost to follow up
8% (13)
11% (13)
-
Number of decolonization treatments
1 (1–5)
1 (1–5)
1 (1–3)
Household size
Single person
10% (15)
13% (15)
-
Multiple persons
90% (140)
87% (100)
100% (40)
Abscess Localization
Extremities
37% (58)
50% (58)
-
Trunk
35% (54)
47% (54)
-
Head/Face
31% (48)
42% (48)
-
Gluteal
22% (34)
30% (34)
-
Axilla
13% (20)
17% (20)
-
Genital
11% (17)
15% (17)
-
Inguinal
5% (8)
7% (8)
-
Invasive
3% (4)
4% (4)
-
MRSA, methicillin-resistant S. aureus. MSSA, methicillin-susceptible S. aureus. PVL, Panton-Valentine leukodicin. Successful decolonization was defined as remaining
clinically asymptomatic for at least 6 months after final decolonization treatment. MRSA, methicillin-resistant S. aureus. MSSA, methicillin-susceptible S. aureus. PVL, Panton-Valentine leukodicin. Successful decolonization was defined as remaining
clinically asymptomatic for at least 6 months after final decolonization treatment. MRSA, methicillin-resistant S. aureus. MSSA, methicillin-susceptible S. aureus. PVL, Panton-Valentine leukodicin. Successful decolonization was defined as remaining
clinically asymptomatic for at least 6 months after final decolonization treatment. https://doi.org/10.1371/journal.pone.0231772.t001
Fig 2. Kaplan-Meier curve of PVL-SA decolonization in 115 symptomatic patients. PVL-SA, Panton-Valentine
leukocidin-positive Staphylococcus aureus. https://doi.org/10.1371/journal.pone.0231772.g002 Fig 2. Kaplan-Meier curve of PVL-SA decolonization in 115 symptomatic patients. PVL-SA, Panton-Valentine
leukocidin-positive Staphylococcus aureus. Fig 2. Kaplan-Meier curve of PVL-SA decolonization in 115 symptomatic patients. PVL-SA, Panton-Valentine
leukocidin-positive Staphylococcus aureus. 5 / 9 PLOS ONE | https://doi.org/10.1371/journal.pone.0231772
April 21, 2020 PLOS ONE (PVL)-producing S. aureus—The importance of repeting decolonization Table 2. Multivariable Cox regression analysis of parameters associated with successful decolonization. Parameter
P-value
OR
95% Confidence Interval
Lower
Upper
PVL-S. PLOS ONE | https://doi.org/10.1371/journal.pone.0231772
April 21, 2020 PLOS ONE aureus phenotype
MSSA
Reference
MRSA
0.615
0.873
0.516
1.479
Undetected
0.012
2.030
1.170
3.523
Single household
0.006
2.372
1.285
4.381
PVL, pantone-valentine leukocidine. MSSA, Methicillin-susceptible S. aureus. MRSA, methicillin-resistant S. aureus. OR, odds ratio. https://doi.org/10.1371/journal.pone.0231772.t002 Table 2. Multivariable Cox regression analysis of parameters associated with successful decolonization. Pathogen transfer within households has been described repeatedly as the dominant trans-
mission route for community-acquired S. aureus in the past [7, 11, 19, 20]. Our data under-
scores this observation for PVL-SA, as we found that 69/88 (78%) contacts screened were also
clinically symptomatic and / or PVL-positive (Fig 1). The clinical relevance of household con-
tact is further emphasized by our observation that living in a multiple person household was a
relevant risk factor for requiring repeated decolonization as compared to living alone. Knox et al. stated that the degree of physical contact among household members and the
amount of time spent at home determine the risk of transmission [11]. This could result in
epidemic S. aureus clones ‘ping-ponging’ between family members [19–22]. These observa-
tions support the need for the simultaneous decolonization of close physical contacts, at least
in cases where patients remain PVL-SA colonized despite decolonization treatment. Fig 3. Multivariable Cox regression curve of PVL-SA decolonization in 115 symptomatic patients. PVL-SA,
Panton-Valentine leukocidin-positive Staphylococcus aureus. https://doi org/10 1371/journal pone 0231772 g003 Fig 3. Multivariable Cox regression curve of PVL-SA decolonization in 115 symptomatic patients. PVL-SA,
Panton-Valentine leukocidin-positive Staphylococcus aureus. https://doi.org/10.1371/journal.pone.0231772.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0231772
April 21, 2020 6 / 9 PLOS ONE (PVL)-producing S. aureus—The importance of repeting decolonization Our results show that symptomatic patients in which PVL-SA could not be detected have a
higher chance of successful decolonization. Hence, this observation could be an indication
that a low-level colonization with PVL-SA might facilitate the eradication process. Earlier stud-
ies of MRSA decolonization have shown that the success of eradication can be dependent on
the localization of the colonization [23–25]. In our cohort, testing for PVL-SA was not per-
formed separately, using different swabs for nose and throat. In the present study many patients reported a past medical history typical of PVL-SA
patients–recurrent skin abscess that often required incision and drainage and reporting other
affected patients in their immediate environment. PVL-SA-positive patients are often symp-
tomatic for several months, before they are finally diagnosed and treated for PVL-SA [16, 26–
29]. PLOS ONE Our results thus underscore earlier work that shows that a diagnosis can be challenging in
areas with low PVL-SA prevalence [6, 30]. The patients in our cohort were on average 29 years old, confirming earlier studies on
patients with community-onset PVL-SA-associated skin abscess [30, 31]. Whether there are
socio-cultural or medico-biological factors driving this observation is unclear. Our study has several limitations. This is a retrospective study on PVL-SA patients from a
single institution. It represents the patient population from our area and mirrors the results of
our locally established treatment procedures. All patients with an active infection were treated
with an oral antimicrobial agent—at least once—parallel to topical treatment. This makes it
impossible to determine the effect of the antimicrobial therapy on the decolonization results. However, considering the fact that 2/3 of our patients had received antibiotics without any
topical decolonization and continued to suffer from recurrent skin abscesses argues against a
major protective role of isolated antibiotic use in these patients. Patients in our cohort had a
mean total of four skin abscesses in the five months prior to topical decolonization; after suc-
cessful decolonization, all patients were asymptomatic for at least 6 months with a mean fol-
low-up of 15.6 months. Conclusion Our data shows that patients with skin abscesses associated with PVL-SA can be successfully
treated with decolonization as outpatients, but they often require multiple attempts at decolo-
nization. The delayed success of treatment of patients living in multiple person households
suggests that decolonization treatment should be conducted simultaneously on all close
contacts. PLOS ONE | https://doi.org/10.1371/journal.pone.0231772
April 21, 2020 References 1. Shallcross LJ, Fragaszy E, Johnson AM, Hayward AC. The role of the Panton-Valentine leucocidin
toxin in staphylococcal disease: a systematic review and meta-analysis. The Lancet Infectious dis-
eases. 2013; 13:43–54. https://doi.org/10.1016/S1473-3099(12)70238-4 PMID: 23103172 2. Saeed K, Gould I, Esposito S, Ahmad-Saeed N, Ahmed SS, Alp E, et al. Panton-Valentine leukocidin-
positive Staphylococcus aureus: a position statement from the International Society of Chemotherapy. International journal of antimicrobial agents. 2018; 51:16–25. https://doi.org/10.1016/j.ijantimicag.2017
11.002 PMID: 29174420 3. Planet PJ. Life after USA300: the rise and fall of a superbug. The Journal of infectious diseases. 2017;
215:S71–S7. https://doi.org/10.1093/infdis/jiw444 PMID: 28375517 4. Skov R, Christiansen K, Dancer SJ, Daum RS, Dryden M, Huang Y-C, et al. Update on the prevention
and control of community-acquired meticillin-resistant Staphylococcus aureus (CA-MRSA). Interna-
tional journal of antimicrobial agents. 2012; 39:193–200. https://doi.org/10.1016/j.ijantimicag.2011.09. 029 PMID: 22226649 5. Rasigade JP, Laurent F, Lina G, Meugnier H, Bes M, Vandenesch F, et al. Global distribution and evolu-
tion of Panton-Valentine leukocidin-positive methicillin-susceptible Staphylococcus aureus, 1981–
2007. The Journal of infectious diseases. 2010; 201:1589–97. https://doi.org/10.1086/652008 PMID:
20367458 6. Gillet Y, Dumitrescu O, Tristan A, Dauwalder O, Javouhey E, Floret D, et al. Pragmatic management of
Panton-Valentine leukocidin-associated staphylococcal diseases. International journal of antimicrobial
agents. 2011; 38:457–64. https://doi.org/10.1016/j.ijantimicag.2011.05.003 PMID: 21733661 7. Coll F, Harrison EM, Toleman MS, Reuter S, Raven KE, Blane B, et al. Longitudinal genomic surveil-
lance of MRSA in the UK reveals transmission patterns in hospitals and the community. Science trans-
lational medicine. 2017; 9:eaak9745. 8. Warren DK, Liao RS, Merz LR, Eveland M, Dunne WM. Detection of methicillin-resistant Staphylococ-
cus aureus directly from nasal swab specimens by a real-time PCR assay. Journal of clinical microbiol-
ogy. 2004; 42:5578–81. https://doi.org/10.1128/JCM.42.12.5578-5581.2004 PMID: 15583284 9. Cespedes C, Saïd-Salim B, Miller M, Lo S-H, Kreiswirth BN, Gordon RJ, et al. The clonality of Staphylo-
coccus aureus nasal carriage. The Journal of infectious diseases. 2005; 191:444–52. https://doi.org/10. 1086/427240 PMID: 15633104 10. Votintseva A, Miller R, Fung R, Knox K, Godwin H, Peto T, et al. Multiple-strain colonization in nasal car-
riers of Staphylococcus aureus. Journal of clinical microbiology. 2014; 52:1192–200. https://doi.org/10. 1128/JCM.03254-13 PMID: 24501033 11. Knox J, Uhlemann A-C, Lowy FD. Staphylococcus aureus infections: transmission within households
and the community. Trends in microbiology. 2015; 23:437–44. https://doi.org/10.1016/j.tim.2015.03. 007 PMID: 25864883 12. Stevens DL, Bisno AL, Chambers HF, Dellinger EP, Goldstein EJ, Gorbach SL, et al. Writing – original draft: Leif G. Hanitsch, Rasmus Leistner. Writing – original draft: Leif G. Hanitsch, Rasmus Leistner. Writing – review & editing: Leif G. Hanitsch, Renate Kru¨ger, Pia-Alice Hoppe, Daniel
Humme, Anna Pokrywka, Michaela Niebank, Miriam Stegemann, Axel Kola, Rasmus
Leistner. Resources: Axel Kola, Rasmus Leistner. Supervision: Renate Kru¨ger, Pia-Alice Hoppe, Daniel Humme, Anna Pokrywka, Michaela
Niebank, Miriam Stegemann, Axel Kola. Validation: Leif G. Hanitsch, Daniel Humme, Axel Kola, Rasmus Leistner. Visualization: Rasmus Leistner. Visualization: Rasmus Leistner. PLOS ONE | https://doi.org/10.1371/journal.pone.0231772
April 21, 2020 Author Contributions Conceptualization: Leif G. Hanitsch, Pia-Alice Hoppe, Anna Pokrywka, Rasmus Leistner. Data curation: Leif G. Hanitsch, Rasmus Leistner. Formal analysis: Axel Kola, Rasmus Leistner. Investigation: Leif G. Hanitsch, Rasmus Leistner. Methodology: Leif G. Hanitsch, Rasmus Leistner. Project administration: Leif G. Hanitsch, Rasmus Leistner. Conceptualization: Leif G. Hanitsch, Pia-Alice Hoppe, Anna Pokrywka, Rasmus Leistner. Data curation: Leif G. Hanitsch, Rasmus Leistner. Project administration: Leif G. Hanitsch, Rasmus Leistner. 7 / 9 PLOS ONE | https://doi.org/10.1371/journal.pone.0231772
April 21, 2020 PLOS ONE (PVL)-producing S. aureus—The importance of repeting decolonization Resources: Axel Kola, Rasmus Leistner. References Practice guide-
lines for the diagnosis and management of skin and soft tissue infections: 2014 update by the Infectious
Diseases Society of America. Clinical infectious diseases. 2014; 59:e10–e52. https://doi.org/10.1093/
cid/ciu444 PMID: 24973422 13. Daum RS, Miller LG, Immergluck L, Fritz S, Creech CB, Young D, et al. A Placebo-Controlled Trial of
Antibiotics for Smaller Skin Abscesses. The New England journal of medicine. 2017; 376:2545–55. https://doi.org/10.1056/NEJMoa1607033 PMID: 28657870 8 / 9 PLOS ONE | https://doi.org/10.1371/journal.pone.0231772
April 21, 2020 PLOS ONE (PVL)-producing S. aureus—The importance of repeting decolonization 14. Septimus EJ, Schweizer ML. Decolonization in Prevention of Health Care-Associated Infections. Clini-
cal microbiology reviews. 2016; 29:201–22. https://doi.org/10.1128/CMR.00049-15 PMID: 26817630 15. Lina G, Piemont Y, Godail-Gamot F, Bes M, Peter MO, Gauduchon V, et al. Involvement of Panton-Val-
entine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clinical
infectious diseases: an official publication of the Infectious Diseases Society of America. 1999;
29:1128–32. 16. Fogo A, Kemp N, Morris-Jones R. PVL positive Staphylococcus aureus skin infections. Bmj. 2011; 343:
d5343. https://doi.org/10.1136/bmj.d5343 PMID: 21908525 17. Kapadia S, Bazzali J, Leung W, Guth H, Millership S. Panton-Valentine producing Staphylococcus
aureus (PVL-SA)—what happens to patients after skin decolonisation in the community? The Journal of
hospital infection. 2018. 18. Shallcross L, Williams K, Hopkins S, Aldridge R, Johnson A, Hayward A. Panton-Valentine leukocidin
associated staphylococcal disease: a cross-sectional study at a London hospital, England. Clinical
Microbiology and Infection. 2010; 16:1644–8. https://doi.org/10.1111/j.1469-0691.2010.03153.x PMID:
20969671 19. Davis MF, Iverson SA, Baron P, Vasse A, Silbergeld EK, Lautenbach E, et al. Household transmission
of meticillin-resistant Staphylococcus aureus and other staphylococci. The Lancet infectious diseases. 2012; 12:703–16. https://doi.org/10.1016/S1473-3099(12)70156-1 PMID: 22917102 20. Uhlemann A-C, Dordel J, Knox JR, Raven KE, Parkhill J, Holden MT, et al. Molecular tracing of the
emergence, diversification, and transmission of S. aureus sequence type 8 in a New York community. Proceedings of the National Academy of Sciences. 2014; 111:6738–43. 21. Calfee DP, Durbin LJ, Germanson TP, Toney DM, Smith EB, Farr BM. Spread of methicillin-resistant
Staphylococcus aureus (MRSA) among household contacts of individuals with nosocomially acquired
MRSA. Infection control and hospital epidemiology. 2003; 24:422–6. https://doi.org/10.1086/502225
PMID: 12828318 22. Mollema FP, Richardus JH, Behrendt M, Vaessen N, Lodder W, Hendriks W, et al. Transmission of
methicillin-resistant Staphylococcus aureus to household contacts. Journal of clinical microbiology. 2010; 48:202–7. https://doi.org/10.1128/JCM.01499-09 PMID: 19923490 23. Bagge K, Benfield T, Westh H, Bartels MD. PLOS ONE | https://doi.org/10.1371/journal.pone.0231772
April 21, 2020 References Eradicating MRSA carriage: the impact of throat carriage
and Panton-Valentine leukocidin genes on success rates. European Journal of Clinical Microbiology &
Infectious Diseases. 2019; 38:683–8. 24. Buehlmann M, Frei R, Fenner L, Dangel M, Fluckiger U, Widmer A. Highly effective regimen for decolo-
nization of methicillin-resistant Staphylococcus aureus carriers. Infection Control & Hospital Epidemiol-
ogy. 2008; 29:510–6. 25. Petersen IS, Christensen JM, Zeuthen AB, Madsen PB. Danish experience of meticillin-resistant Staph-
ylococcus aureus eradication with emphasis on nose-throat colonization and supplementary systemic
antibiotic treatment. Journal of Hospital Infection. 2019. 26. Hoppe PA, Hanitsch LG, Leistner R, Niebank M, Buhrer C, von Bernuth H, et al. Periorbital infections
and conjunctivitis due to Panton-Valentine Leukocidin (PVL) positive Staphylococcus aureus in chil-
dren. BMC infectious diseases. 2018; 18:371. https://doi.org/10.1186/s12879-018-3281-8 PMID:
30081842 27. Jaton L, Pillonel T, Jaton K, Dory E, Prod’hom G, Blanc DS, et al. Common skin infection due to Panton-
Valentine leucocidin-producing Staphylococcus aureus strains in asylum seekers from Eritrea: a
genome-based investigation of a suspected outbreak. Clinical microbiology and infection: the official pub-
lication of the European Society of Clinical Microbiology and Infectious Diseases. 2016; 22:739.e5–8. 28. Zanger P, Nurjadi D, Schleucher R, Scherbaum H, Wolz C, Kremsner PG, et al. Import and spread of
Panton-Valentine Leukocidin-positive Staphylococcus aureus through nasal carriage and skin infec-
tions in travelers returning from the tropics and subtropics. Clinical infectious diseases: an official publi-
cation of the Infectious Diseases Society of America. 2012; 54:483–92. 29. Wiese-Posselt M, Heuck D, Draeger A, Mielke M, Witte W, Ammon A, et al. Successful termination of a
furunculosis outbreak due to lukS-lukF-positive, methicillin-susceptible Staphylococcus aureus in a
German village by stringent decolonization, 2002–2005. Clinical infectious diseases: an official publica-
tion of the Infectious Diseases Society of America. 2007; 44:e88–95. 30. Edelstein M, Kearns A, Cordery R. Panton-Valentine Leukocidin associated Staphylococcus aureus
infections in London, England: clinical and socio-demographic characterisation, management, burden
of disease and associated costs. Journal of infection and public health. 2011; 4:145–53. https://doi.org/
10.1016/j.jiph.2011.04.001 PMID: 21843861 31. Ellington M, Perry C, Ganner M, Warner M, Smith IM, Hill R, et al. Clinical and molecular epidemiology
of ciprofloxacin-susceptible MRSA encoding PVL in England and Wales. European journal of clinical
microbiology & infectious diseases. 2009; 28:1113. PLOS ONE | https://doi.org/10.1371/journal.pone.0231772
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Pulmonary Mucormycosis as the Leading Clinical Type of Mucormycosis in Western China
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Frontiers in cellular and infection microbiology
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ORIGINAL RESEARCH
published: 22 November 2021
doi: 10.3389/fcimb.2021.770551
Pulmonary Mucormycosis
as the Leading Clinical Type of
Mucormycosis in Western China
Junyan Qu 1, Xijiao Liu 2 and Xiaoju Lv 1*
1 Center of Infectious Disease, West China Hospital, Sichuan University, Chengdu, China, 2 Radiology Department, West
China Hospital, Sichuan University, Chengdu, China
Edited by:
Kai Huang,
University of Texas Medical Branch at
Galveston, United States
Reviewed by:
Muhammad Usman Tariq,
Abu Dhabi School of Management,
United Arab Emirates
Valentina Giudice,
University of Salerno, Italy
Dora Edith Corzo-Leon,
University of Aberdeen,
United Kingdom
*Correspondence:
Xiaoju Lv
lvxj33966@126.com
Specialty section:
This article was submitted to
Clinical Microbiology,
a section of the journal
Frontiers in Cellular and
Infection Microbiology
Received: 04 September 2021
Accepted: 04 November 2021
Published: 22 November 2021
Citation:
Qu J, Liu X and Lv X (2021)
Pulmonary Mucormycosis as
the Leading Clinical Type of
Mucormycosis in Western China.
Front. Cell. Infect. Microbiol. 11:770551.
doi: 10.3389/fcimb.2021.770551
The aim was to better understand the clinical characteristics of patients with
mucormycosis in western China. We retrospectively investigated the clinical, laboratory,
radiological and treatment profiles of mucormycosis patients during a 10-year period
(2010–2019). As a result, 59 proven mucormycosis were enrolled in this study. It was
found that 52.5% of patients had worse clinical outcomes. Pulmonary mucormycosis (PM)
was the most common clinical manifestation. The most frequent risk factor was diabetes
mellitus (38, 64.4%) for mucormycosis patients. Cough (43, 93.5%), fever (24, 52.2%) and
hemoptysis/bloody phlegm (21, 45.7%) were the most common manifestations of PM.
There were no differences in clinical manifestations, risk factors and laboratory tests
between different clinical outcome groups (P>0.05). Lymph node enlargement (30,
65.2%), patchy shadows (28, 60.9%), cavitation (25, 53.3%) and bilateral lobe
involvement (39, 84.8%) were the most common on chest CT. Nodule was more
common in good outcome group (P <0.05). A total of 48 cases (81.4%) were
confirmed by histopathological examination, 22 cases (37.3%) were confirmed by direct
microscopy. PM patients were treated with amphotericin B/amphotericin B liposome or
posaconazale had better clinical outcomes (P <0.05). In conclusion, PM was the most
common clinical type of mucormycosis in China. Diabetes mellitus was the most common
risk factor. PM has diverse imaging manifestations and was prone to bilateral lobes
involvement. Early diagnosis and effective anti-mucor treatment contribute to
successful treatment.
Keywords: mucormycosis, pulmonary mucormycosis, diabetes, clinical analysis, clinical outcome
INTRODUCTION
Mucormycosis is a rare, emerging and opportunistic fungal infection with high morbidity and
mortality caused by filamentous fungi of the Mucoraceae family, order Mucorales. Mucorales fungi
are ubiquitous in nature. Humans are infected mainly by inhaling sporangiospores, occasionally
through the ingestion of contaminated food or traumatic inoculation (Prakash and Chakrabarti,
2019; Shariati et al., 2020). The prevalence of mucormycosis varies between developing and
developed countries, ranging from 0.01 to 14 per 100 000 population in Europe and India (Bitar
et al., 2009; Chakrabarti and Singh, 2014; Ruhnke et al., 2015). With the growth of the number of
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org
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November 2021 | Volume 11 | Article 770551
Qu et al.
Clinical Analysis of Mucormycosis
information, underlying diseases, use of corticosteroid or
immunosuppressive agent, clinical manifestations, laboratory
data, radiologic findings, diagnostic procedures, therapeutic
strategies and clinical outcomes at 90 days were collected.
The study was approved by the Ethics Committee of West
China Hospital, Sichuan University. Because all the data in this
study were routinely obtained, written informed consent
was waived.
immunocompromised patients, increased awareness and
development of diagnostic techniques, the incidence of
mucormycosis is rising (Prakash et al., 2019). According to
clinical presentations, mucormycosis is mainly classified as
rhino-orbito-cerebral, pulmonary, cutaneous, gastrointestinal
and disseminated types (Skiada et al., 2020). The common risk
factors of mucormycosis are diabetes, hematological malignancy,
use of corticosteroids or immunosuppressants, and trauma
(Petrikkos et al., 2014; Jeong et al., 2019). However, the main
cause of mucormycosis also varies in different countries.
Hematological malignancies are the main cause in countries
with high income, while diabetes mellitus (DM) or trauma are
the main cause in developing countries (Chakrabarti and Singh,
2014). Diagnosis of mucormycosis is challenging because of the
low sensitivity and specificity of clinical diagnostic methods
(Skiada et al., 2018).
The mortality of mucormycosis remains high, it may be
related to delayed diagnosis, high cost of managing
mucormycosis and limited treatment options (Skiada et al.,
2018). Previous studies on the characteristics of mucormycosis
have been conducted mainly in America, Europe and India
(Prakash and Chakrabarti, 2019). Data about mucormycosis
from China is sparse. The causative agents of mucormycosis
vary with different geographical locations (Prakash and
Chakrabarti, 2019), and the epidemiology, the clinical disease
pattern of mucormycosis vary from country to country.
To better understand the clinical characteristics of patients
with mucormycosis in China, in this retrospective study, we
compared the demographic features, clinical presentations,
laboratory data, radiographic findings and therapeutic
strategies in mucormycosis patients with different clinical
outcomes who were admitted to a university hospital from Jan
2010 to Dec 2019 in western China.
Laboratory Studies
Laboratory tests including complete blood count, blood
biochemistry, procalcitionin (PCT), C-reactive protein, T
lymphocyte subset, HIV testing, serum (1,3)-beta-D-glucan test
(BDG test), and galactomannan test (GM test) were performed.
Clinical samples (including blood, sputum, secretions, urine)
were aseptically collected and cultured under aerobic or
anaerobic conditions. Bacterial species were isolated and
identified using MicroScan WalkAway-96 System (Siemens,
USA). Fungal culture was performed on Sabouraud dextrose
agar (SDA) and incubated at 30°C. All the items were performed
in the Department of Laboratory Medicine of our hospital.
Radiological Assessment
Imaging examinations such as chest computed tomography
(CT), abdominal CT, fibroptic bronchoscopy were performed
at the discretion of the treating physicians. The CT scans were
performed using 64-row multi-slice spiral CT scanner
(SOMATOM definition AS+, Siemens) in our hospital. All
images were reviewed independently by two experienced
radiologists. For patients with PM, chest CT findings including
nodule, mass, cavity, patchy consolidation, ground glass opacity,
reversed halo sign, lymph node enlargement, pleural effusion and
the distribution of the lesion in the lungs were recorded.
Treatment Strategies and Clinical
Outcomes
The patients with mucormycosis were treated with amphotericin
B (AmB, 0.5-1 mg/kg per day) (North China Pharmaceutical Co.,
Ltd., China) or amphotericin B liposome (LAmB, 3-6 mg/kg per
day) in accordance with the drug instructions, guidelines (Skiada
et al., 2013) and patient tolerance. Posaconazole oral suspension
(40mg/ml, 10ml, twice daily) (Patheon Inc, Whitby, ON,
Canada) was used in patients who have contraindications to
amphotericin B or who cannot tolerate the side effects of
amphotericin B like reduced kidney function, electrolyte
imbalances, nausea and vomiting. The treatment duration was
determined by the treating physicians according to the patient’s
treatment response and the size of the focus. Management of
patients with diabetes included dietary guidance, tight glucose
control with insulin and/or hypoglycemic drugs, multiple flash
glucose monitoring daily and treatment of complications. A
multidisciplinary consulting team, including diabetes,
infectious disease, nutrition, wound therapy, surgery, clinical
microbiology, and clinical pharmacy, that offered specialist
advice, on-going management. Other underlying diseases of
these patients such as immune system disease, chronic lung
disease were treated normally and systematically. For bacterial
MATERIALS AND METHODS
Patients
From Jan 2010 to Dec 2019, the patients with a diagnosis of
mucormycosis at hospital discharge were retrospectively reviewed
in West China Hospital, Sichuan University, a 4,300-bed academic
tertiary hospital in Chengdu, China. According to the European
Organization for Research and Treatment of Cancer/Mycoses
Study Group (EORTC/MSG) criteria and previous references
(De Pauw et al., 2008; Petrikkos et al., 2014; Skiada et al., 2018),
inclusion in the final study group required the diagnosis of proven
mucormycosis as defined as follows: (1) age ≥ 14 years; (2) clinical
manifestations and radiographic findings consistent with
mucormycosis; and (3) histological presence of mucormycosis in
tissue specimens, and/or broad-based, ribbon-like, non-septate
hyphae with right-angle branching filamentous fungi on direct
microscopy of clinical specimens, and/or Mucorales species
cultured from clinical specimens. Based on clinical presentation
and the involvement of the body sites, rhino-orbital-cerebral,
pulmonary, cutaneous and disseminated mucormycosis were
classified (Jeong et al., 2019). The following data of demographic
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November 2021 | Volume 11 | Article 770551
Qu et al.
Clinical Analysis of Mucormycosis
These patients were classified as pulmonary type and other type
mucormycosis based on clinical type. The demographic and
clinical outcomes of these patients were summarized in
Table 1. The ages of patients with PM was higher than that of
patients with non-pulmonary mucormycosis (P=0.015). The
most frequent underlying disease was diabetes mellitus (38,
64.4%) for mucormycosis patients. More patients with PM had
diabetes mellitus than patients with non-pulmonary
mucormycosis (P=0.027). A total of 31 mucormycosis patients
(52.5%) had worse clinical outcomes, clinical outcome was not
obviously different between the two groups (P>0.05).
co-infection patients, empirical or targeted antimicrobial therapy
were given. Clinical outcomes of patients with mucormycosis
were evaluated at 90 days after diagnosis. According to clinical
manifestations, laboratory findings and image changes, their
clinical outcomes were divided into good and worse outcome.
Death or disease progression or persistence were classified as
worse outcome. The continuous improvement of clinical
symptoms and imaging findings were classified as good outcome.
Statistical Analysis
Statistical analyses were performed using IBM SPSS Statistics for
Windows v.26.0 (IBM Corp., Armonk, NY, USA). The ShapiroWilk normality test was used to test the normality of all
quantitative variables. Continuous variables with normal
distribution were presented as mean± standard deviation (SD)
and compared by Student’s t-test. The relationship between
categorical variables was assessed using Chi-square test or
Fisher’s exact test. A two-tailed P value lower than 0.05 was
considered statistically significant.
Demographic and Clinical Characteristics
in Patients With PM
Demographic and clinical features of PM patients with different
clinical outcomes were shown in Table 2. The most common
clinical symptoms of PM patients were cough (43, 93.5%), fever
(24, 52.2%) and hemoptysis/bloody phlegm (21, 45.7%). Diabetes
mellitus (33, 71.7%) was the most frequent underlying disease.
There were no significant difference in the gender, age, clinical
symptoms and underlying diseases beween good and worse
outcome group (P>0.05). For laboratory results, there was no
significant difference between the two groups (P > 0.05).
RESULTS
Demographic Characteristics and Clinical
Outcomes of Mucormycosis Patients
Findings of CT Scan in Patients With PM
A total of 68 patients with mucormycosis were admitted to our
hospital from January 2010 to December 2019, 59 of whom with
proven mucormycosis (mean age 54.75 ± 14.72 years; 44 males)
were enrolled in this retrospective study. Pulmonary
mucormycosis (PM) was the most commonly observed
manifestation (46/59, 78.0%), followed by rhino-orbitalcerebral (9/59, 15.3%) and disseminated mucormycosis (3/59,
5.0%). There was only one case of cutaneous mucormycosis.
The chest CT features of patients with PM were diverse, as shown
in Figure 1. The detail of chest CT findings in these patients were
shown in Table 3. The most common manifestations were
lymph node enlargement (30, 65.2%), patchy shadows (28,
60.9%), cavitation (25, 53.3%) and nodules (24, 52.2%). Nodule
was more common in good outcome group than in worse
outcome group (P <0.05). Lymph node enlargement were more
common in patients with worse outcome (P<0.05). Bilateral
TABLE 1 | Demographic and clinical outcomes in patients with mucormycosis.
Variables
Male
Age (years) (mean ± SD)
Underlying diseases or risk factors
Diabetes mellitus
Immune system disease
Corticosteroid medication or immunosuppressive drugs
Hematological malignancy
Solid tumor
Chronic kidney diseases
Chronic lung disease
Chronic liver disease
Truma
None
Co-infection of other pathogens
Bacteria
Other fungi
Clinical outcome
Good outcome
Worse outcome
Clinical type
P-value
All (N = 59)
Pulmonary type (N = 46)
Other type (N = 13)
44 (74.6)
54.75 ± 14.72
35 (76.1)
57.41 ± 12.40
9 (69.2)
45.31 ± 18.64
0.616
0.015*
38 (64.4)
2 (3.4)
4 (6.8)
3 (5.1)
2 (3.4)
3 (5.1)
8 (13.6)
2 (1.7)
2 (3.4)
8 (13.6)
33 (71.7)
1 (2.2)
3 (6.5)
1 (2.2)
2 (4.3)
3 (6.5)
7 (15.2)
1 (2.2)
1 (2.2)
6 (13.0)
5 (38.5)
1 (7.7)
1 (7.7)
2 (15.4)
0 (0.0)
0 (0.0)
1 (7.7)
1 (7.7)
1 (7.7)
2 (15.4)
0.027*
0.395
1.000
0.119
1.000
1.000
0.671
0.395
0.395
1.000
42 (71.2)
16 (23.7)
32 (69.6)
14 (30.4)
10 (76.9)
2 (15.4)
0.738
0.481
28 (47.5)
31 (52.5)
23 (50.0)
23 (50.0)
5 (38.5)
8 (61.5)
0.462
*P < 0.05.
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TABLE 2 | Demographic and clinical characteristics in patients with pulmonary mucormycosis.
Variables
Male
Age
Presenting symptoms and signs
Fever
Cough
hemoptysis or bloody phlegm
Chest pain
Shortness of breath
Underlying diseases or risk factors
Diabetes mellitus
Immune system disease
Corticosteroid medication or immunosuppressive drugs
Hematological malignancy
Solid tumor
Chronic kidney diseases
Chronic lung disease
Chronic liver disease
Truma
None
Laboratory data
White blood cell (× 109/L)
Neutrophil (%)
Lymphocyte (× 109/L)
Eosinophil (× 109/L)
Mononuclear cell (× 109/L)
Procalcitionin (ng/mL)
C-reactive protein (mg/L)
Eerythrocyte sedimentation rate (mm/h)
CD4+ T lymphocyte (%)
CD8+ T lymphocyte (%)
CD4/CD8 ratio
CD4+ T lymphocyte (cells/ml)
CD8+ T lymphocyte (cells/ml)
Co-infecting agent
Bacteria
Virus
Fungi
All (N = 46) (n,%)
Good outcome (N = 23) (n,%)
Worse outcome (N = 23) (n,%)
P-value
35 (76.1)
57.41 ± 12.40
5 (21.7)
56.00 ± 13.44
6 (26.1)
58.82 ± 11.38
0.288
0.446
24 (52.2)
43 (93.5)
21 (45.7)
7 (15.2)
14 (30.4)
13 (56.5)
21 (91.3)
9 (39.1)
2 (8.7)
9 (39.1)
11 (47.8)
22 (95.7)
12 (52.2)
5 (21.7)
5 (21.7)
0.768
1.000
0.554
0.414
0.200
33 (71.7)
1 (2.2)
3 (6.5)
1 (2.2)
2 (4.3)
3 (6.5)
7 (15.2)
1 (2.2)
1 (2.2)
6 (13.0)
16 (69.6)
0 (0.0)
2 (8.7)
1 (4.3)
0 (0.0)
2 (8.7)
4 (17.4)
1 (4.3)
1 (4.3)
4 (17.4)
17 (73.9)
1 (4.3)
1 (4.3)
0 (0.0)
2 (8.7)
1 (4.3)
3 (13.0)
0 (0.0)
0 (0.0)
2 (8.7)
1.000
1.000
1.000
1.000
0.489
1.000
1.000
1.000
1.000
0.665
8.69 ± 3.76
69.38 ± 12.10
1.62 ± 0.73
0.19 ± 0.26
0.50 ± 0.20
0.12 ± 0.13
73.17 ± 82.08
60.42 ± 23.31
36.55 ± 12.54
30.30 ± 10.80
1.48 ± 1.01
740.2 ± 357.47
466.80 ± 253.69
0.665
0.170
0.180
0.397
0.539
0.289
0.831
0.278
0.771
0.475
0.409
0.062
0.250
13 (56.5)
1 (4.3)
8 (34.8)
1.000
1.000
0.763
9.13 ± 6.84
71.96 ± 12.24
1.48 ± 0.67
0.33 ± 1.17
0.49 ± 0.23
0.21 ± 0.49
70.31 ± 87.94
55.23 ± 24.06
36.94 ± 10.33
31.61 ± 9.49
1.36 ± 0.77
565.45 ± 288.69
396.82 ± 176.78
9.58 ±
74.42 ±
1.35 ±
0.48 ±
0.47 ±
0.29 ±
66.75 ±
49.00 ±
37.32 ±
32.92 ±
1.23 ±
419.83 ±
338.50 ±
25 (54.3)
2 (4.3)
18 (39.1)
12 (52.2)
1 (4.3)
10 (43.5)
were treated with amphotericin B/LAmB. More patients with PM
were treated with AmB/LAmB (P=0.006) or posaconazale
(P=0.004) in good outcome group than in worse outcome group.
For patients with PM, only 3 patients were treated with
surgery and LAmB, but there was no statistical difference in
clinical outcome (P>0.05). The most common adverse events of
AmB/LAmB included renal insufficiency (16/42, 38.1%) and
hypokalemia (9/42, 21.4%) in this study. The severity of all
adverse events were mild to moderate (grade 1-2) according to
the US National Cancer Institute Common Toxicity Criteria
(NCI-CTC) (US, National Cancer Institute, 2017). The adverse
reaction ratio of AmB/LAmB has not statistical difference
between the two groups (P>0.05). As shown in Table 4.
lungs were involved in 84.8% (39/46) of patients, with the left
lower lobe (20, 43.5%) and right lower lobe (18, 39.1%) most
commonly involved. There was no significant difference in the
lobe of lesion distribution between the two groups (P>0.05).
There were 13 patients with Mucor and Aspergillus co-infection
and 1 patient with lung cancer. Based on imaging findings, 12
patients were suspected to be tumors, and 2 patients were
suspected to be aspergillosis in the absence of concomitant
Aspergillus. Figure 2 showed the changes of chest CT lesions
during the follow-up of a patient with pulmonary fungal
infection (Mucor with Aspergillus).
Diagnostic Procedures and
Treatment Strategies
Of the 59 proven patients with mucormycosis, 48 cases (81.4%)
were confirmed by histopathological examination, 22 cases (37.3%)
were confirmed by direct microscopy. There was no significant
difference in the diagnostic procedures between the two groups
(P>0.05). A total of 47 patients (79.7%) received treatment.
Treatment strategies for these patients with mucormycosis include
antifungal drugs, surgery, or both. Most patients (38/59, 64.4%)
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9.02
12.11
0.60
1.46
0.26
0.66
97.40
24.64
8.00
8.17
0.40
92.04
45.12
DISCUSSION
Mucormycosis is rare, neglected, and associated with high
mortality rates. The study found that 52.5% of patients with
mucormycosis had poor clinical outcomes. Which is similar to
previous research results (Jeong et al., 2019). PM was the most
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A
B
C
D
E
F
G
H
FIGURE 1 | The findings of chest computed tomography of pulmonary mucormycosis (patients with good outcome: A, B; patients with worse outcome: C–H).
(A) Multiple pleomorphic lesions in both lungs; (B) cavity with muralnodule (Mucor co-infecting with Aspergillus); (C) cavity in left lung; (D) thick-walled cavity shadow
with gas-fluid level (Mucor co-infecting with Klebsiella pneumoniae); (E) mass shadow in the upper lobe of the left lung; (F) patchy shadow, nodules and
consolidation in right lung; (G) cavity and ground-glass opacity; (H) reversed halo sign.
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TABLE 3 | Chest CT findings of proven patients with pulmonary mucormycosis .
Morphology
Consolidation
Nodule
Ground-glass opacity
Mass
Cavitation
Patchy shadow
Fibrosis
Pleural effusion
Pleural thickening
Lymph node enlargement
Reversed halo sign
Lobe of lesion distribution
Left upper lobe
Left lower lobe
Right upper lobe
Right middle lobe
Right lower lobe
Bilateral involvement
All (N = 46) (n,%)
Good outcome (N = 23) (n,%)
Worse outcome (N = 23) (n,%)
P-value
20 (43.5)
24 (52.2)
7 (15.2)
15 (32.6)
25 (53.3)
28 (60.9)
17 (37.0)
22 (47.8)
17 (37.0)
30 (65.2)
5 (10.9)
9 (39.1)
16 (69.6)
5 (21.7)
8 (34.8)
10 (43.5)
14 (60.9)
8 (34.8)
11 (47.8)
6 (26.1)
11 (47.8)
2 (8.7)
11 (47.8)
8 (34.8)
2 (8.7)
7 (30.4)
15 (65.2)
14 (60.9)
9 (39.1)
10 (43.5)
11 (47.8)
19 (82.6)
3 (13.0)
0.767
0.038*
0.414
1.000
0.139
1.000
1.000
1.000
0.221
0.029*
1.000
12 (26.1)
20 (43.5)
12 (26.1)
11 (23.9)
18 (39.1)
39 (84.8)
5 (21.7)
11 (47.8)
7 (30.4)
8 (34.8)
8 (34.8)
20 (87.0)
7 (30.4)
9 (39.1)
5 (21.7)
3 (13.0)
10 (43.5)
19 (82.6)
0.738
0.767
0.738
0.165
0.763
1.000
*P < 0.05.
increased mortality (Spellberg et al., 2012). Neutropenia was not
found in our patients with PM, it might not be an important risk
factor for mortality in these patients.
The most frequent imaging findings of PM were patchy
shadows, cavitation and pulmonary nodules in this study.
According to the imaging findings, PM might be misdiagnosed as
tumor or pulmonary aspergillosis. Nodule was more common in
good outcome group. Lymph node enlargement were more
common in patients with worse outcome. Many previous studies
have found multiple nodules, reversed halo sign, and cavities
associated with PM (McAdams et al., 1997; Marom and
Kontoyiannis, 2011). Pulmonary nodules, halo sign and cavitation
was also found in patients pulmonary invasive aspergillosis (Althoff
Souza et al., 2006; Muldoon et al., 2016). The reversed halo sign has
been considered an important clue to the diagnosis of PM, however,
it has also been described in other pulmonary diseases, including
invasive pulmonary aspergillosis, tuberculosis, organising
pneumonia and malignancy (Sullivan and Rana, 2019). Therefore,
early biopsy to establish the underlying cause is very important in
patients with suspected pulmonary fungal infections.
Diagnosis of mucormycosis is challenging. In clinical practice,
laboratory diagnosis of mucormycosis includes histopathology,
direct examination and cultures (Skiada et al., 2020). Most
patients in this study were diagnosed by histopathology. Many
previous studies have also reported that histopathology was the
main diagnostic method of mucormycosis (Prakash and
Chakrabarti, 2019). In this study, Mucorales culture positive
was found only in 13 patients. It was previously reported that the
positive rate of Mucorales culture could reach 79% (Prakash and
Chakrabarti, 2019). Which may be related to the low vigilance of
doctors to invasive pulmonary mycosis and the absence of fungal
culture for puncture specimens. More needs to be done to raise
awareness of pulmonary mycosis among doctors.
In this study, twelve patients did not receive treatment and left
the hospital against medical advice after diagnosis because of their
critical condition and financial constraints. PM patients were treated
common form of mucormycosis, accounting for 78.0% of all the
patients with mucormycosis. Although previous studies have
reported that rhino-orbital mucormycosis was the most common
clinical type (Patel et al., 2020), PM maybe more common in
China (Peng et al., 2019). This study also found the most
common underlying disease was diabetes mellitus. Previous
studies have shown that PM mainly occurred in patients with
hematological malignancies, while rhino-orbital mucormycosis
mostly occurred in patients with diabetes mellitus (Skiada et al.,
2011; Chakrabarti and Singh, 2014). However, our results were
consistent with those from China (Peng et al., 2019). Perhaps
because the number of diabetes patients in China is much larger
than that of hematological malignancies (Liu et al., 2019; Khan
et al., 2020), the underlying disease of more mucormycosis
patients is diabetes. The high prevalence of diabetes in China
and endemic mucor species different from other countries may
also be the reasons for the high incidence of PM in diabetic
patients in China. More detailed etiological studies of
mucormycosis need to be done. We found the incidence of
mucormycosis was tended to rise, which may be owing to the
improvement of fungal diagnostic technology, health awareness
and the visiting rate. In addition, diabetes incidence is on the rise
worldwide, especially in China and India (Khan et al., 2020),
which may be the reason for the increase in the incidence of
mucormycosis in China.
For PM, the most common clinical symptoms were cough,
fever, and hemoptysis/bloody phlegm. This result was similar to
some previous studies (Skiada et al., 2011; Peng et al., 2019).
Molecular-based assays can help to identify different fungal species
and it can be used as a supplement to conventional diagnostic
methods (Machouart et al., 2006; Springer et al., 2016). The study
also found that laboratory results were not associated with patient
outcomes. Most previous studies have not found a correlation
between laboratory results and clinical outcomes in patients with
mucormycosis (Patel et al., 2020; Son et al., 2020), and only a few
studies have found that neutropenia may be associated with
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A
B
C
D
E
F
FIGURE 2 | Pulmonary fungal infection (Mucor with Aspergillus) in a 69-year-old man with uncontrolled diabetes, chronic obstructive pulmonary disease and liver cirrhosis.
CT imaging showed thick-walled cavity shadow with gas-fluid level in the right upper lobe and left lower lobe of the lung (A). The lesion was significantly reduced after half a
month of treatment with posaconazole (B). The lesion continued to shrink after 40 days (C), three months (D), five months (E) and 1 year (F) of treatment.
alone was observed. Bilateral lobe involvement occurred in 84.8%
of patients in this study, which was similar to previous studies (Peng
et al., 2019). Multifocal involvement limited the surgical options,
early and effective antifungal therapy may be more important for
patients with PM. The recommended combination of antifungal
agents and surgical treatment as a treatment option for
mucormycosis may be based on the presence of more rhinoorbito-cerebral mucormycosis in other countries. More researches
may be needed to optimize treatment options for different types of
mucormycosis. New treatment drugs or methods, such as
combination of lipid amphotericin B and caspofungin or
posaconazole, VT-1161, deferasirox in combination with a
polyene, hyperbaric oxygen and so on, still deserve to be expected
to improve clinical outcome (Skiada et al., 2018).
with amphotericin B or posaconazole had better clinical outcomes,
while untreated patients had poor outcomes. For rhino-orbitocerebral and disseminated mucormycosis patients, there was no
statistically significant difference in clinical outcome between
different treatment strategies, which maybe related to the small
number of cases. In addition, patients with rhino-orbito-cerebral
mucormycosis who underwent surgery and antifungal therapy had
better clinical outcomes. At present, treatment options for
mucormycosis remain very limited. LAmB combined with
surgery are strongly recommend as first-line therapy.
Isavuconazole and posaconazole are also options as second-line
agents (Cornely et al., 2019). For patients with PM, only 3 patients
in this study were received pulmonary lobectomy, so no benefit of
antifungal combined surgical treatment over antifungal therapy
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TABLE 4 | Diagnostic procedures and treatment strategies for proven patients with mucormycosis .
Diagnostic method
Cluture
Direct microscopy
Histology
Cluture positive only
Direct microscopy positive only
Histology positive only
Cluture+histology
Direct microscopy+histology
Cluture+Direct microscopy
BDG test
GM test
Therapeutic strategies
Pulmonary
AmB/LAmB
Posaconazale
LAmB+Surgery
untreated
Rhino-orbito-cerebral
AmB/LAmB
Posaconazale
AmB+Surgery
Disseminated
AmB
untreated
Cutaneous
AmB+Surgery
Adverse reactions of AmB/LAmB
Pulmonary
Hypokalemia
Renal insufficiency
Phlebophlogosis
Allergic reaction
Gastrointestinal toxicity
Cardiotoxicity
Rhino-orbito-cerebral
Hypokalemia
Renal insufficiency
Disseminated
Hypokalemia
Renal insufficiency
Gastrointestinal toxicity
Agranulocytosis
All (N = 59) (n,%)
Good outcome (N = 28) (n,%)
Worse outcome (N = 31) (n,%)
P-value
13 (22.0)
22 (37.3)
48 (81.4)
8 (13.6)
2 (3.4)
25 (52.4)
4 (6.8)
19 (32.2)
1 (1.7)
3 (5.1)
3 (5.1)
7 (25.0)
7 (25.0)
23 (82.1)
5 (17.9)
1 (3.6)
14 (50.1)
2 (7.1)
6 (21.4)
0 (0.0)
1/24 (4.2)
2/20 (10.0)
6 (19.4)
15 (48.4)
25 (80.6)
3 (9.7)
1 (3.2)
11 (35.5)
2 (6.5)
13 (41.9)
1 (3.2)
2/25 (8.0)
1/20 (5.0)
0.755
0.105
1.000
0.458
1.000
0.260
1.000
0.105
1.000
1.000
1.000
All
(N=46) (n,%)
28 (60.9)
11 (23.9)
3 (6.5)
11 (23.9)
All
(N=9) (n,%)
7 (77.8)
1 (11.1)
1 (22.2)
All
(N=3) (n,%)
2 (66.7)
1 (33.3)
All
(N=1) (n,%)
1 (100.0)
Good outcome
(N=23) (n,%)
19 (82.6)
10 (43.5)
1 (4.3)
0 (0.0)
Good outcome
(N=3) (n,%)
2 (66.7)
0 (0.0)
1 (33.3)
Good outcome
(N=1) (n,%)
1 (100.0)
0 (0.0)
Good outcome
(N=1) (n,%)
0 (100.0)
Worse outcome
(N=23) (n,%)
9 (39.1)
1 (4.3)
2 (8.7)
11 (47.8)
Worse outcome
(N=6) (n,%)
5 (83.3)
1 (16.7)
0 (0.0)
Worse outcome
(N=2) (n,%)
1 (50.0)
1 (50.0)
Worse outcome
(N=0) (n,%)
0 (0.0)
P-value
All
(N=31) (n,%)
7 (22.6)
11 (35.5)
1 (3.2)
1 (3.2)
4 (12.9)
3 (10.7)
All
(N=8) (n,%)
1 (1.3)
4 (50.0)
All
(N=2) (n,%)
1 (50.0)
1 (50.0)
1 (50.0)
1 (50.0)
Good outcome
(N=20) (n,%)
5 (25.0)
8 (40.0)
1 (5.0)
1 (5.0)
2 (10.0)
2 (10.0)
Good outcome
(N=3) (n,%)
0 (0.0)
3 (100.0)
Good outcome
(N=1) (n,%)
0 (0.0)
1 (100.0)
0 (0.0)
0 (0.0)
Worse outcome
(N=11) (n,%)
2 (18.2)
3 (27.3)
0 (0.0)
0 (0.0)
2 (18.2)
1 (9.1)
Worse outcome
(N=5) (n,%)
1 (20.0)
1 (20.0)
Worse outcome
(N=1) (n,%)
1 (100.0)
0 (0.0)
1 (100.0)
1 (100.0)
P-value
0.006*
0.004*
1.000
0.000*
P-value
1.000
1.000
0.333
P-value
NA
NA
P-value
NA
1.000
0.698
1.000
1.000
0.601
1.000
P-value
NA
NA
P-value
NA
NA
NA
NA
AmB, amphotericin B; BDG test, (1,3)-beta-D-glucan test; GM test, galactomannan test; LAmB, liposomal amphotericin B.
*P < 0.05; NA, Not Applicable.
study was limited. More larger-scale, multicenter studies of
mucormycosis in the real world should be done.
This study has some limitations. First, it was a retrospective
observational study. Only some patients have complete followup data in our hospital, especially imaging data, and the followup of some patients could only be completed by telephone.
Second, the detection rate of tissue culture was low, the
pathological diagnosis was determined mainly by morphology
and special staining. It is important to raise the awareness of
doctors about fungal disease and the importance of tissue
culture. Third, because mucormycosis is a relatively rare fungal
disease in China (Liao et al., 2013), the number of cases in this
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CONCLUSIONS
The most common clinical type of mucormycosis in China was
PM. The most common risk factor was diabetes mellitus.
Diabetic patients with clinical manifestations of febrile and
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Clinical Analysis of Mucormycosis
hemoptysis, CT findings of nodules, cavities and bilateral lung
involvement should be vigilant against PM. Early diagnosis and
effective anti-mucor treatment are very important to improve the
prognosis of patients with mucormycosis.
required for this study in accordance with the national legislation
and the institutional requirements.
AUTHOR CONTRIBUTIONS
XL and JQ conceived of and designed the study. JQ and XL
collected, analysed or interpreted data. JQ wrote the draft. All
authors read, revised and approved the final manuscript.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in
the article. Further inquiries can be directed to the
corresponding author.
FUNDING
This study was supported by Sichuan Province Science and
Technology Support Program of China (grant number:
2021YFS0170), 1•3•5 project for disciplines of excellenceClinical Research Incubation Project, West China Hospital,
Sichuan University (grant number: 2021HXFH032).
ETHICS STATEMENT
The studies involving human participants were reviewed and
approved by Ethics Committee of West China Hospital, Sichuan
University. Written informed consent for participation was not
Marom, E. M., and Kontoyiannis, D. P. (2011). Imaging Studies for Diagnosing
Invasive Fungal Pneumonia in Immunocompromised Patients. Curr. Opin.
Infect. Dis. 24 (4), 309–314. doi: 10.1097/QCO.0b013e328348b2e1
McAdams, H. P., Rosado de Christenson, M., Strollo, D. C., and Patz, E. F. Jr.
(1997). Pulmonary Mucormycosis: Radiologic Findings in 32 Cases. AJR Am. J.
Roentgenol 168 (6), 1541–1548. doi: 10.2214/ajr.168.6.9168721
Muldoon, E. G., Sharman, A., Page, I., Bishop, P., and Denning, D. W. (2016).
Aspergillus Nodules; Another Presentation of Chronic Pulmonary
Aspergillosis. BMC Pulm Med. 16 (1), 123. doi: 10.1186/s12890-0160276-3
Patel, A., Kaur, H., Xess, I., Michael, J. S., Savio, J., Rudramurthy, S., et al. (2020). A
Multicentre Observational Study on the Epidemiology, Risk Factors,
Management and Outcomes of Mucormycosis in India. Clin. Microbiol.
Infect. 26 (7), 944 e949–944.e915. doi: 10.1016/j.cmi.2019.11.021
Peng, M., Meng, H., Sun, Y., Xiao, Y., Zhang, H., Lv, K., et al. (2019). Clinical
Features of Pulmonary Mucormycosis in Patients With Different Immune
Status. J. Thorac. Dis. 11 (12), 5042–5052. doi: 10.21037/jtd.2019.12.53
Petrikkos, G., Skiada, A., and Drogari-Apiranthitou, M. (2014). Epidemiology of
Mucormycosis in Europe. Clin. Microbiol. Infect. 20 (Suppl 6), 67–73. doi:
10.1111/1469-0691.12563
Prakash, H., and Chakrabarti, A. (2019). Global Epidemiology of
Mucormycosis. J. Fungi (Basel) 5 (1), 26. doi: 10.3390/jof5010026
Prakash, H., Ghosh, A. K., Rudramurthy, S. M., Singh, P., Xess, I., Savio, J., et al.
(2019). A Prospective Multicenter Study on Mucormycosis in India:
Epidemiology, Diagnosis, and Treatment. Med. Mycol 57 (4), 395–402. doi:
10.1093/mmy/myy060
Ruhnke, M., Groll, A. H., Mayser, P., Ullmann, A. J., Mendling, W., Hof, H., et al.
(2015). University of Manchester in Association With the Lp: Estimated Burden of
Fungal Infections in Germany. Mycoses 58 (Suppl 5), 22–28. doi: 10.1111/
myc.12392
Shariati, A., Moradabadi, A., Chegini, Z., Khoshbayan, A., and Didehdar, M.
(2020). An Overview of the Management of the Most Important Invasive
Fungal Infections in Patients With Blood Malignancies. Infect. Drug Resist. 13,
2329–2354. doi: 10.2147/IDR.S254478
Skiada, A., Lanternier, F., Groll, A. H., Pagano, L., Zimmerli, S., Herbrecht, R., et al.
(2013). Diagnosis and Treatment of Mucormycosis in Patients With
Hematological Malignancies: Guidelines From the 3rd European Conference
on Infections in Leukemia (ECIL 3). Haematologica 98 (4), 492–504. doi:
10.3324/haematol.2012.065110
Skiada, A., Lass-Floerl, C., Klimko, N., Ibrahim, A., Roilides, E., and Petrikkos, G.
(2018). Challenges in the Diagnosis and Treatment of Mucormycosis. Med.
Mycol 56 (suppl_1), 93–101. doi: 10.1093/mmy/myx101
REFERENCES
Althoff Souza, C., Muller, N. L., Marchiori, E., Escuissato, D. L., and Franquet, T.
( 2006 ). Pulmon ary In vasiv e Asperg illosi s an d Can di diasis in
Immunocompromised Patients: A Comparative Study of the HighResolution CT Findings. J. Thorac. Imaging 21 (3), 184–189. doi: 10.1097/
01.rti.0000213552.16011.ad
Bitar, D., Van Cauteren, D., Lanternier, F., Dannaoui, E., Che, D., Dromer, F., et al.
(2009). Increasing Incidence of Zygomycosis (Mucormycosis), France, 19972006. Emerg. Infect. Dis. 15 (9), 1395–1401. doi: 10.3201/eid1509.090334
Chakrabarti, A., and Singh, R. (2014). Mucormycosis in India: Unique Features.
Mycoses 57 (Suppl 3), 85–90. doi: 10.1111/myc.12243
Cornely, O. A., Alastruey-Izquierdo, A., Arenz, D., Chen, S. C. A., Dannaoui, E.,
Hochhegger, B., et al. (2019). Global Guideline for the Diagnosis and
Management of Mucormycosis: An Initiative of the European Confederation
of Medical Mycology in Cooperation With the Mycoses Study Group
Education and Research Consortium. Lancet Infect. Dis. 19 (12), e405–e421.
doi: 10.1016/S1473-3099(19)30312-3
De Pauw, B., Walsh, T. J., Donnelly, J. P., Stevens, D. A., Edwards, J. E., Calandra,
T., et al. (2008). Revised Definitions of Invasive Fungal Disease From the
European Organization for Research and Treatment of Cancer/Invasive Fungal
Infections Cooperative Group and the National Institute of Allergy and
Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group.
Clin. Infect. Dis. 46 (12), 1813–1821. doi: 10.1086/588660
Jeong, W., Keighley, C., Wolfe, R., Lee, W. L., Slavin, M. A., Kong, D. C. M., et al.
(2019). The Epidemiology and Clinical Manifestations of Mucormycosis: A
Systematic Review and Meta-Analysis of Case Reports. Clin. Microbiol. Infect.
25 (1), 26–34. doi: 10.1016/j.cmi.2018.07.011
Khan, M. A. B., Hashim, M. J., King, J. K., Govender, R. D., Mustafa, H., and Al
Kaabi, J. (2020). Epidemiology of Type 2 Diabetes - Global Burden of Disease
and Forecasted Trends. J. Epidemiol. Glob Health 10 (1), 107–111. doi: 10.2991/
jegh.k.191028.001
Liao, Y., Chen, M., Hartmann, T., Yang, R. Y., and Liao, W. Q. (2013).
Epidemiology of Opportunistic Invasive Fungal Infections in China: Review
of Literature. Chin. Med. J. (Engl) 126 (2), 361–368.
Liu, W., Liu, J., Song, Y., Zeng, X., Wang, X., Mi, L., et al. (2019). Burden of
Lymphoma in China, 2006-2016: An Analysis of the Global Burden of Disease
Study 2016. J. Hematol. Oncol. 12 (1), 115. doi: 10.1186/s13045-019-0785-7
Machouart, M., Larché , J., Burton, K., Collomb, J., Maurer, P., Cintrat, A., et al.
(2006). Genetic Identification of the Main Opportunistic Mucorales by PCRRestriction Fragment Length Polymorphism. J. Clin. Microbiol. 44, 805–810.
doi: 10.1128/JCM.44.3.805-810.2006
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org
9
November 2021 | Volume 11 | Article 770551
Qu et al.
Clinical Analysis of Mucormycosis
US, National Cancer Institute (2017). Common Terminology Criteria for Adverse
Events (CTCAE): Version 5.0. Available from: https://ctep.cancer.gov/
protocoldevelopment/electronic_applications/docs/CTCAE_v5_Quick_
Reference_8.5x11.pdf. (Accessed December13, 2021).
Skiada, A., Pagano, L., Groll, A., Zimmerli, S., Dupont, B., Lagrou, K., et al. (2011).
Gaustad P Et Al: Zygomycosis in Europe: Analysis of 230 Cases Accrued by the
Registry of the European Confederation of Medical Mycology (ECMM)
Working Group on Zygomycosis Between 2005 and 2007. Clin. Microbiol.
Infect. 17 (12), 1859–1867. doi: 10.1111/j.1469-0691.2010.03456.x
Skiada, A., Pavleas, I., and Drogari-Apiranthitou, M. (2020). Epidemiology and
Diagnosis of Mucormycosis: An Update. J. Fungi (Basel) 6 (4), 265. doi:
10.3390/jof6040265
Son, H. J., Song, J. S., Choi, S., Jung, J., Kim, M. J., Chong, Y. P., et al. (2020). Risk
Factors for Mortality in Patients With Pulmonary Mucormycosis. Mycoses 63
(7), 729–736. doi: 10.1111/myc.13092
Spellberg, B., Kontoyiannis, D. P., Fredricks, D., Morris, M. I., Perfect, J. R., ChinHong, P. V., et al. (2012). Risk Factors for Mortality in Patients With
Mucormycosis. Med. Mycol 50 (6), 611–618. doi: 10.3109/13693786.2012.
669502
Springer, J., Lackner, M., Ensinger, C., Risslegger, B., Morton, CO., Nachbaur, D.,
et al. (2016). Clinical Evaluation of a Mucorales-Specific Real-Time PCR Assay
in Tissue and Serum Samples. J. Med. Microbiol. 65, 1414–1421. doi: 10.1099/
jmm.0.000375
Sullivan, T., and Rana, M. (2019). The Reversed Halo Sign and Mucormycosis.
Lancet Infect. Dis. 19 (12), 1379. doi: 10.1016/S1473-3099(19)30548-1
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org
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Financial Statements Are About To Get A New Look
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Financial Statements are about to get a New Look
Financial Statements are about to get a New Look Clemense Ehoff Jr. Kean University, ehoffc@cwu.edu Clemense Ehoff Jr. Kean University, ehoffc@cwu.edu Follow this and additional works at: https://digitalcommons.cwu.edu/cobfac
Part of the Corporate Finance Commons Follow this and additional works at: https://digitalcommons.cwu.edu/cobfac Follow this and additional works at: https://digitalcommons.cwu.edu/cobfac
Part of the Corporate Finance Commons College of Business College of Business College of Business Recommended Citation
Recommended Citation Recommended Citation
Recommended Citation Ehoff, Jr., C. (2010). Financial statements are about to get a new look. Journal of Business and
Economics Research 8(12), 69-76. DOI: 10.19030/jber.v8i12.783 This Article is brought to you for free and open access by the College of Business at ScholarWorks@CWU. It has
been accepted for inclusion in All Faculty Scholarship for the College of Business by an authorized administrator of
ScholarWorks@CWU. For more information, please contact scholarworks@cwu.edu. Volume 8, Number 12 Journal of Business & Economics Research – December, 2010 ABSTRACT In October 2008, The FASB and the IASB issued a discussion letter for comment proposing a
complete redesign of financial statements. This was the result of a project that began for both
boards in 2001. More than 200 comment letters were received from individuals, accounting firms,
professional societies, corporations, and others from the business community. The FASB and
IASB have analyzed the input, and are currently preparing an exposure draft scheduled for
release in early 2011. This paper reviews the proposed changes to the financial statements,
summarizes the favorable and unfavorable responses contained in the comment letters, and
examines the implications that the changes will have on the business community, the accounting
profession, educators, and investors. Keywords: Financial Statements; Comment letters; FASB; IASB Keywords: Financial Statements; Comment letters; FASB; IASB Keywords: Financial Statements; Comment letters; FASB; IASB INTRODUCTION n October 16, 2008, the Financial Accounting Standards Board (FASB) and the International
Accounting Standards Board (IASB) issued a discussion paper “Preliminary Views on Financial
Statement Presentation,” the result of a project that began for both boards in 2001. The paper sought
public comment, to be used as input in the development of an exposure draft. The comment period ended on April
14, 2009. 227 comment letters were received from individuals, accounting firms, professional societies,
corporations, academics, and others from the business community. A comment letter summary was presented at the
July 14, 2009 IASB/FASB meeting. In July 2010, the IASB and the FASB posted a draft of the Exposure Draft, a
working document that expresses the current and tentative decision made so far on this project. O bliO The changes to financial statement presentation proposed by the IASB and FASB are far reaching and will
have an enormous impact on the entire business community. This paper reviews the proposed changes to the
financial statements, summarizes the favorable and unfavorable responses contained in the comment letters, and
examines the implications that the changes will have on the business community, the accounting profession,
educators, and investors. Financial Statements
Are About To Get A New Look
Clemense Ehoff Jr., Kean University, USA Clemense Ehoff Jr., Kean University, USA THE DISCUSSION PAPER The October 16, 2008 Discussion Paper (Preliminary Views, 2008) was the result of a project that began in
2001. In undertaking the financial statements presentation project, the FASB and IASB „s intention was to establish
a new standard for presenting information in the financial statements. In doing so, they set a goal of improving the
usefulness of financial statement information to help users make more informed decisions as capital providers. Three broad objectives for financial statement presentation were developed: 1. Cohesiveness: There is a clear relationship between items across financial statements. 1. Cohesiveness: There is a clear relationship between items across financial statements. regation of Information: Financial information should be disaggregated into reasonably
nous groups of items so that it is useful in predicting an entity‟s future cash flows. 2. Disaggregation of Information: Financial information should be disaggregated into reasonably
homogenous groups of items so that it is useful in predicting an entity‟s future cash flows. 2. Disaggregation of Information: Financial information should be disaggregat
homogenous groups of items so that it is useful in predicting an entity‟s future cash flow g
g
p
p
g
y
3. Liquidity and Flexibility: Liquidity information helps users asses the entity‟s ability to meet financial
commitments as they become due. Information flexibility helps users assess the entity‟s ability to invest in
opportunities and react to unexpected situations. g
g
p
p
g
y
3. Liquidity and Flexibility: Liquidity information helps users asses the entity‟s ability to meet financial
commitments as they become due. Information flexibility helps users assess the entity‟s ability to invest in
opportunities and react to unexpected situations. 69 Journal of Business & Economics Research – December, 2010 Volume 8, Number 12 In comparing these structures, several significant changes are noticed: 1. Assets and liabilities are still divided into current and long-term categories, but are further divided into
business (and divided again into operating and investing components) and financing categories. 2. In the new Statement of Financial Position, subtotals for current assets, current liabilities, total assets and
total liabilities have been included and placed at the bottom of the statement. 3. The new Statement of Income goes beyond traditional Net Income and includes components of other
comprehensive income in arriving at total comprehensive income. 4. The new Statement of Income does not contain a separate line item for extraordinary items. 5. The new Statement of Cash Flows must use the direct method. 1. Assets and liabilities are still divided into current and long-term categories, but are further divided into
business (and divided again into operating and investing components) and financing categories. g
p
g
g
p
g
g
2. In the new Statement of Financial Position, subtotals for current assets, current liabilities, total assets and
total liabilities have been included and placed at the bottom of the statement. 3. The new Statement of Income goes beyond traditional Net Income and includes components of other
comprehensive income in arriving at total comprehensive income. p
g
p
The new Statement of Income does not contain a separate line item for extraordinary items. g
p
w Statement of Income does not contain a separate line item for extraordinary items. The new Statement of Cash Flows must use the direct method. The fourth statement in the proposed model is the statement of changes in equity. The structure proposed for this
statement resembles a reconciliation of beginning balances, ending balances, and how each amount changed during
the period. Each component of equity is presented in the statement. Currently, a reconciliation of only Retained
Earnings (or Owners‟ Equity) is shown. The fourth statement in the proposed model is the statement of changes in equity. The structure proposed for this
statement resembles a reconciliation of beginning balances, ending balances, and how each amount changed during
the period. Each component of equity is presented in the statement. Currently, a reconciliation of only Retained
Earnings (or Owners‟ Equity) is shown. The statement of financial position, the income statement, the statement of cash flows, and the statement of
changes in equity comprise a complete set of financial statements. Samples of each financial statement are
presented in the Appendix. In comparing these structures, several significant changes are noticed: There are several additional important aspects of the proposed presentation model worth mentioning. First,
the model relies on a management approach to classify assets and liabilities in the business and financing sections in
a manner that best reflects the way the asset or liability is used within the entity. Second, the Boards have
concluded that use of the direct method for the statement of cash flows is more consistent with the objectives of
coherency and disaggregation of information. Finally, the proposed presentation model includes a new schedule that
reconciles cash flows to comprehensive income. This schedule will be included in the notes to the financial
statements. Table 3 illustrates the structure of the schedule: Table 3: Reconciliation Schedule
Statement of
Cash Flows
Cash
Flows
Accruals,
Allocations
and Other
Recurring
Valuation
Adjustment
All
Other
Comprehensive
Income
Statement of
Comprehensive Income
Cash from
wholesale
Customers
1,928,798
662,602
2,991,400
Sales-Wholesale
Cash from
retail
customers
643,275
4,575
647,850
Sales-retail
Total cash
from
customers
2,572,073
667,177
3,219,250
Total Revenue
Note: Adapted from Discussion Paper (Preliminary Views, 2008). The discussion paper included an invitation for comments on the proposals included in the paper. Twenty-
six questions were included in the paper. Respondents were asked to respond in writing by April 14, 2009. The discussion paper included an invitation for comments on the proposals included in the paper. Twenty-
six questions were included in the paper. Respondents were asked to respond in writing by April 14, 2009. Journal of Business & Economics Research – December, 2010 The proposed financial statement model divides the statement of financial position (balance sheet), the
income statement, and the statement of cash flows into two sections: business activities, and financing activities. The business section is further subdivided into operating activities and investing activities. The financing section
describes how the entity‟s business activities are financed, segregating owner and non-owner sources. Discontinued
operations are shown separately from the entity‟s business and financing activities. Income taxes are shown
separately in the statement of financial position and the statement of cash flows. In the income statement, income
tax information is shown separately for 1) income from continuing operations, 2) discontinued operations, and 3)
other comprehensive income items. Table 1 illustrates the proposed classification scheme. Table 1: Proposed Financial Statement Classification
Statement of Financial Position
Statement of Comprehensive Income
Statement of Cash Flows
Business
Operating assets and liabilities
Investing assets and liabilities
Business
Operating income and expenses
Investment income and expenses
Business
Operating cash flows
Investing cash flows
Financing
Financing assets
Financing liabilities
Financing
Financing asset income
Financing
Financing asset cash flows
Financing liability cash
flows
Income taxes
Income taxes on continuing operations
(business and financing)
Income taxes
Discontinued operations
Discontinued operations net of tax
Discontinued operations
Other comprehensive income, net of tax
Equity
Equity
Note: Adapted from Discussion Paper (Preliminary Views, 2008). This structure is substantially different from the current financial statement structure as shown in Table 2: structure is substantially different from the current financial statement structure as shown in Table 2 Table 2: Current Financial Statement Classification
Balance Sheet
Income Statement
Statement of Cash Flows
(Direct or Indirect Method)
Assets
Current Assets
Property, Plant & Equipment
Other Assets
Operating Income
Sales
Cost of Goods Sold
Gross Profit
Operation Expenses
Operating Income
Operating Activities
Operating cash flows
Investing cash flows
Liabilities
Current Liabilities
Long-term Liabilities
Other
Other Revenue
Other Expense
Investing Activities
Equity
Continuing Operations
Income Before Taxes
Income Taxes
Income from Continuing
Operations
Financing Activities
Discontinued operations net of tax
Change in Cash
Extraordinary Item (net of tax)
Net Income
Note: Adapted from Intermediate Accounting (6th ed.) by D. Spiceland, J. Sepe, & M. Nelson. New York: McGraw-Hill Irwin. (2011). 70 Journal of Business & Economics Research – December, 2010 Volume 8, Number 12 THE COMMENT LETTERS Respondents who opposed
the direct method claimed that management was not currently using operating cash receipts and payments
information to run its business and financial statement users were not asking for it. These respondents also
pointed to a complete retooling of their accounting and financial reporting systems, suggesting that the
costs of such an endeavor would far outweigh the benefits. 7. Direct method statement of cash flows – Two-thirds of the respondents did not agree that the direct
method would provide more decision-useful information than the indirect method, and a majority of the
respondents did not favor requiring all entities to use only the direct method. Respondents who opposed
the direct method claimed that management was not currently using operating cash receipts and payments
information to run its business and financial statement users were not asking for it. These respondents also
pointed to a complete retooling of their accounting and financial reporting systems, suggesting that the
costs of such an endeavor would far outweigh the benefits. 8. g
Reconciliation schedule – Most respondents did not favor the proposed schedule that reconciles the
statement of comprehensive income with the cash flows statement. They thought that 1) the reconciliation
schedule was rather long and might be too complex for most financial statement users to grasp, and 2) the
cost of preparing the schedule by far outweighed the benefits. Reconciliation schedule – Most respondents did not favor the proposed schedule that reconciles the
statement of comprehensive income with the cash flows statement. They thought that 1) the reconciliation
schedule was rather long and might be too complex for most financial statement users to grasp, and 2) the
cost of preparing the schedule by far outweighed the benefits. 9. Application to nonpublic entities – Although the board had not considered whether the proposed financial
statement presentation model should apply to nonpublic companies, respondents were asked for their views
on this issue. Respondents‟ views were mixed. Those that favored inclusion cited two reasons: 1) if the
proposed financial statement presentation model proved useful for public entities, then it should prove
useful for nonpublic entities, and 2) one presentation model eliminates possible confusion among users. THE COMMENT LETTERS g
3. A separate equity section - There was majority support for a separate equity section. 4. A separate discontinued operations section – There was nearly unanimous support for a separate
discontinued operations section. 3. A separate equity section - There was majority support for a separate equity section. 3. A separate equity section - There was majority support for a separate equity section. 4. A separate discontinued operations section – There was nearly unanimous support for a separate
discontinued operations section. 5. Management approach to classification – Respondents‟ views were mixed on this issue. Respondents
who favored the management approach think that classifying assets and liabilities in a manner that reflects
their use provides relevant information for financial statement users. Respondents who did not support this
approach pointed to reduced comparability among entities. 5. Management approach to classification – Respondents‟ views were mixed on this issue. Respondents
who favored the management approach think that classifying assets and liabilities in a manner that reflects
their use provides relevant information for financial statement users. Respondents who did not support this
approach pointed to reduced comparability among entities. 6. Statement of comprehensive income – Respondents were split on whether an entity should include all
components of comprehensive income in a single statement or two separate statements. Respondents who
favored the approach pointed to greater transparency, consistency and comparability. Respondents who
opposed the single statement approach argued that operating income and net income were the primary
focus of most investors, and that the inclusion of other comprehensive items within a single statement
might lead to confusion. 6. Statement of comprehensive income – Respondents were split on whether an entity should include all
components of comprehensive income in a single statement or two separate statements. Respondents who
favored the approach pointed to greater transparency, consistency and comparability. Respondents who
opposed the single statement approach argued that operating income and net income were the primary
focus of most investors, and that the inclusion of other comprehensive items within a single statement
might lead to confusion. 7. g t ead to co
us o . Direct method statement of cash flows – Two-thirds of the respondents did not agree that the direct
method would provide more decision-useful information than the indirect method, and a majority of the
respondents did not favor requiring all entities to use only the direct method. THE COMMENT LETTERS The IASB/FASB boards received a total of 227 comment letters. The comments were reviewed and
summarized by the technical staff. A comment letter summary report was presented for discussion at the
IASB/FASB July 14, 2009 meeting. A summary of the respondents is presented below: A summary of the respondents is presented below: 71 Journal of Business & Economics Research – December, 2010 Volume 8, Number 12 Table 4: Comment Letter Summary
Total
IFRS
U.S. GAAP
Preparers
98
73
25
Auditors
38
29
9
Users
24
17
7
Standard setters/Regulators
22
22
0
Academics
18
7
11
Others
27
22
5
Total
227
170
57
Note: Adapted from Comment Letter Summary (2009) Some of the main points highlighted in the technical staff summary are as follows: Some of the main points highlighted in the technical staff summary are as follow 1. Objectives - There was general support for the Boards‟ objectives of cohesiveness, disaggregation,
liquidity, and financial flexibility. However, most respondents took issue with the concept of line-item
cohesiveness, and felt that cohesiveness might be more appropriate if applied at a higher level. Some
respondents felt that presenting disaggregated information in the notes to financial statements might be
more appropriate than on the face of the financial statements. Lastly, many respondents suggested that the
proposed financial statement changes were not designed to meet the needs of a broad range of users, but
seemed to be designed more for analysts who use financial statement information for valuation purposes. 1. Objectives - There was general support for the Boards‟ objectives of cohesiveness, disaggregation,
liquidity, and financial flexibility. However, most respondents took issue with the concept of line-item
cohesiveness, and felt that cohesiveness might be more appropriate if applied at a higher level. Some
respondents felt that presenting disaggregated information in the notes to financial statements might be
more appropriate than on the face of the financial statements. Lastly, many respondents suggested that the
proposed financial statement changes were not designed to meet the needs of a broad range of users, but
seemed to be designed more for analysts who use financial statement information for valuation purposes. 2. Separation of business activities from financing activities – Most respondents supported the separation
of business activities from financing activities, although several respondents pointed out that separation
might prove to be difficult in actual practice, suggesting that the distinction might be arbitrary, thereby
reducing the information‟s usefulness. Journal of Business & Economics Research – December, 2010 Volume 8, Number 12 ies, and 2) the proposed presentation model might be too complex for small nonpublic companies. companies, and 2) the proposed presentation model might be too complex for small nonpublic com Clearly, the most significant area of concern from the respondents was the mandate requiring companies to use the
direct method in presenting the statement of cash flows. Over two-thirds of the respondents (including such
companies as McDonald Corporation (Comment Letter # 120), Intel Corporation (Comment Letter # 25), Microsoft
Corporation (Comment Letter # 134), and Bayer (Comment Letter #57)) have raised serious objections, claiming
that the cost far outweighs the benefits. Intel‟s controller estimated that implementation costs for his company
would exceed $5 million, and ongoing costs would approximate $2million per year (Comment Letter #25). THE STAFF DRAFT OF THE EXPOSURE DRAFT On July 1, 2010, the FASB and the IASB posted a draft copy of an exposure draft on financial statement
presentation (Staff Draft, 2010). Although similar to the Discussion Paper issued in October 2008, this 151- page
document takes on the style and structure of a typical FASB statement, focusing more on the “nuts and bolts” of
implementation and less on justification. The basic financial statement presentation model has changed little
between the two documents. The significant changes are as follows: 1. The Boards have decided that the proposed financial statement presentation standard should apply to all
business entities, public and private. 1. The Boards have decided that the proposed financial statement presentation standard should apply to all
business entities, public and private. 1. The Boards have decided that the proposed financial statement presentation standard sh
b
i
titi
bli
d
i
t business entities, public and private. p
p
2. The Boards have dropped the reconciliation statement (reconciling the statement of comprehensive income
with the statement of cash flows on a line-by-line basis. Instead, a reconciliation of significant asset and
liability accounts will be included in the notes to the financial statements. Despite the opposition heard from more than two-thirds of the respondents, the Boards have remained steadfast in
requiring the companies use the direct method in presenting the statement of cash flows. The exposure draft of the financial statement presentation model is scheduled for release during 1st quarter
of 2011. Given the similarity in scope between the Discussion Paper and the Staff draft, it is reasonable to assume
that little will change between the Staff Draft and the final Exposure Draft. THE COMMENT LETTERS Those who opposed inclusion cited two reasons: 1) the cost doesn‟t justify the benefit for nonpublic Application to nonpublic entities – Although the board had not considered whether the proposed financial
statement presentation model should apply to nonpublic companies, respondents were asked for their views
on this issue. Respondents‟ views were mixed. Those that favored inclusion cited two reasons: 1) if the
proposed financial statement presentation model proved useful for public entities, then it should prove
useful for nonpublic entities, and 2) one presentation model eliminates possible confusion among users. Those who opposed inclusion cited two reasons: 1) the cost doesn‟t justify the benefit for nonpublic 72 Journal of Business & Economics Research – December, 2010 IMPLICATIONS The proposed financial statement presentation model has enormous and far- reaching implications. It is
worthwhile to examine the implications that the changes are likely to have on the business community, the
accounting profession, educators, and investors. First, there is the cost associated with implementing the new financial statement presentation model. As
already mentioned, Intel‟s controller had estimated implementation costs of more than $5 million, with ongoing
costs approximating $2million per year. Even assuming that his estimate is somewhat exaggerated, it is safe to
conclude that the cost to Corporate America for retooling its accounting software to accommodate this new financial
statement presentation model is enormous. Every accounting system, every software package (both custom and off-
the-shelf), and nearly every financial model will require a significant update. Given the increase in financial
information, audit fees will undoubtedly rise. Corporate America (and corporate stockholders indirectly) will be
paying the bill for the new financial statement presentation model. In terms of winners and losers, the accounting
and finance software producers are clear winners, while financial statement preparers (the companies and their
stockholders) are the losers. It only takes a quick perusal of Table 1 and Table 2 to conclude that financial statements and the
accompanying notes to the financial statements will be more complex and contain more information. Accounting
firms will be the winners, increase their fess to cover the additional work. The financial statement preparers (the
companies and their stockholders) are the losers, paying a higher price to the public accounting firms. Educators should fare nicely as a result of the new financial statement presentation model, especially in the
continuing professional education segment. Much of Corporate America has not even seen the proposed financial 73 Journal of Business & Economics Research – December, 2010 Volume 8, Number 12 statement presentation model; it may come as quite an initial shock. Educators come across as winners, with
potentially plenty to do for a long while. Every accounting and finance book and profession publication will require
an update. Book sales should soar. There are no losers here. statement presentation model; it may come as quite an initial shock. Educators come across as winners, with
potentially plenty to do for a long while. Every accounting and finance book and profession publication will require
an update. Book sales should soar. There are no losers here. CONCLUSION The IASB/FASB boards have spent nearly 10 years on designing a new financial statement presentation
model. Having gone through countless meetings and absorbing an enormous amount of commentary from
accountants, educators, financial analysts, businessmen and investors, the boards are close to bringing this project to
its conclusion. This project and its implications are too big to escape controversy. The objection by many respondents to
elimination of the indirect method approach to the statement of cash flows appears lost for now, but the new
financial statement presentation model must go through the exposure draft phase before becoming “cast in stone.”
Since December 2009, an 18-member panel formed by the AICPA, the Financial Accounting Foundation (the
FASB‟s parent organization), and the National Association of State Boards of Accountancy have been working on
models that are based on current U.S. GAAP that would result in different standards for private companies (Private
Company Financial Reporting, September 2010). So, as the IASB/FASB financial statement presentation model
project turns the corner and heads “into the home stretch,” a “relative” group puts forth an effort suggesting that
financial standards (including financial statement presentation models) be split into two groups: one for public
companies, and one for private companies. What effect the work of this group will have on the IASB/FASB
financial statement presentation model is not clear at this time. What is clear is that financial statements, as we
know them, are about to change. A working draft of the financial statement presentation model can be viewed on the
FASB‟s website. And although significant changes to the model are still possible, it is now time to start preparing
for a new set of financial statements. IMPLICATIONS Finally, investors and analysts will have a new set of financial statements and notes to analyze, containing
substantially more data. For the sophisticated investor and analyst, the new financial statement model should satisfy
their seemingly inexhaustible appetite for more data. For the unsophisticated investor, the new financial statement
presentation model might give them heartburn. The level of sophistical seems to be the deciding factor as to which
investor or analyst is a winner or a loser. AUTHOR INFORMATION Dr. Clemense Ehoff Jr., CPA is Assistant Professor of Accounting, Kean University, Union New Jersey. He holds
a Ph. D in Business Administration from San Francisco‟s Golden Gate University. He has more than 30 years
professional business experience and has held full-time faculty and adjunct positions at universities predominantly
in the Eastern United States. Over the last ten years, Dr. Ehoff has been involved in teaching accounting and tax
courses in an online platform. He operates a consulting and tax practice. He has published articles in Elevator
World, and other journals. 1.
DeFelice, A. (2010, September). Blue-Ribbon Panel Narrows Field for Private Company Financial
Reporting. Journal of Accountancy, 24-28.
2.
Financial Accounting Standards Board (FASB) (2008) Discussion Paper: Preliminary Views on Financial
Statement Presentation. Retrieved February 15, 2009 from
http://www.fasb.org/cs/ContentServer?c=Document_C&pagename=FASB%2FDocument_C%2FDocument
Page&cid=1175801986226http://www.fasb.org/cs/ContentServer?c=Document_C&pagename=FASB%2F
Document_C%2FDocumentPage&cid=1175801986226.
3.
Financial Accounting Standards Board (FASB)(2009) Comment Letters. Retrieved October 12, 2009 from
http://www.fasb.org/jsp/FASB/CommentLetter_C/CommentLetterPage&cid=1218220137090&project_id=
1630-100. 4.
International Accounting Standards Board (IASB), & Financial Accounting Standards Board (FASB)
(2009) Comment Letter Summary presented at the July 14, 2009 Boards meeting. Retrieved August 5,
2010 from http://www.iasb.org/NR/rdonlyres/791DE489-E887-4652-9DD1-
79789C36F032/0/FSP0907b17Cobs.pdf. p
5.
Financial Accounting Standards Board (FASB)(2010) Staff Draft of an Exposure Draft on Financial
Statement Presentation. Retrieved July 20, 2010 from
http://www.fasb.org/cs/BlobServer?blobcol=urldata&blobtable=MungoBlobs&blobkey=id&blobwhere=11
75820952978&blobheader=application%2Fpdf.
6.
Spiceland, D., Sepe, J., & Nelson, M. (2011). Intermediate Accounting (6th ed.). New York: McGraw-Hill
Irwin. g
g
(2009) Comment Letter Summary presented at the July 14, 2009 Boards meeting. Retrieved August 5,
2010 from http://www.iasb.org/NR/rdonlyres/791DE489-E887-4652-9DD1-
79789C36F032/0/FSP0907b17Cobs.pdf.
5.
Financial Accounting Standards Board (FASB)(2010) Staff Draft of an Exposure Draft on Financial
Statement Presentation. Retrieved July 20, 2010 from
http://www.fasb.org/cs/BlobServer?blobcol=urldata&blobtable=MungoBlobs&blobkey=id&blobwhere=11
75820952978&blobheader=application%2Fpdf.
6.
Spiceland, D., Sepe, J., & Nelson, M. (2011). Intermediate Accounting (6th ed.). New York: McGraw-Hill
Irwin. REFERENCES 1. DeFelice, A. (2010, September). Blue-Ribbon Panel Narrows Field for Private Company Financial
Reporting. Journal of Accountancy, 24-28. 2. Financial Accounting Standards Board (FASB) (2008) Discussion Paper: Preliminary Views on Financial
Statement Presentation. Retrieved February 15, 2009 from
http://www.fasb.org/cs/ContentServer?c=Document_C&pagename=FASB%2FDocument_C%2FDocument
Page&cid=1175801986226http://www.fasb.org/cs/ContentServer?c=Document_C&pagename=FASB%2F
Document_C%2FDocumentPage&cid=1175801986226. _
g
3. Financial Accounting Standards Board (FASB)(2009) Comment Letters. Retrieved October 12, 2009 from
http://www.fasb.org/jsp/FASB/CommentLetter_C/CommentLetterPage&cid=1218220137090&project_id=
1630-100. g
3. Financial Accounting Standards Board (FASB)(2009) Comment Letters. Retrieved October 12, 2009 from
http://www.fasb.org/jsp/FASB/CommentLetter_C/CommentLetterPage&cid=1218220137090&project_id=
1630-100. 74 Journal of Business & Economics Research – December, 2010 Volume 8, Number 12 4. International Accounting Standards Board (IASB), & Financial Accounting Standards Board (FASB)
(2009) Comment Letter Summary presented at the July 14, 2009 Boards meeting. Retrieved August 5,
2010 from http://www.iasb.org/NR/rdonlyres/791DE489-E887-4652-9DD1-
79789C36F032/0/FSP0907b17Cobs.pdf. 5. Financial Accounting Standards Board (FASB)(2010) Staff Draft of an Exposure Draft on Financial
Statement Presentation. Retrieved July 20, 2010 from
http://www.fasb.org/cs/BlobServer?blobcol=urldata&blobtable=MungoBlobs&blobkey=id&blobwhere=11
75820952978&blobheader=application%2Fpdf. 6. Spiceland, D., Sepe, J., & Nelson, M. (2011). Intermediate Accounting (6th ed.). New York: McGraw-Hill
Irwin. pp
p
6. Spiceland, D., Sepe, J., & Nelson, M. (2011). Intermediate Accounting (6th ed.). New York: McGraw-Hill
Irwin. 75 75 Journal of Business & Economics Research – December, 2010 Journal of Business & Economics Research – December, 2010
Volume 8, Number 12
NOTES Volume 8, Number 12 Volume 8, Number 12 Volume 8, Number 12 76
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Pharmacokinetic modelling of N-(4-[18F]fluorobenzoyl)interleukin-2 binding to activated lymphocytes in an xenograft model of inflammation
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European journal of nuclear medicine and molecular imaging
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Eur J Nucl Med Mol Imaging (2012) 39:1551–1560
DOI 10.1007/s00259-012-2176-y Eur J Nucl Med Mol Imaging (2012) 39:1551–1560
DOI 10.1007/s00259-012-2176-y ORIGINAL ARTICLE ORIGINAL ARTICLE Abstract de Vries
Department of Nuclear Medicine & Molecular Imaging,
University Medical Center Groningen,
Groningen, The Netherlands V. Di Gialleonardo: A. Signore: A. T. M. Willemsen:
J. W. A. Sijbesma: R. A. J. O. Dierckx: E. F. J. de Vries
Department of Nuclear Medicine & Molecular Imaging,
University Medical Center Groningen,
Groningen, The Netherlands V. Di Gialleonardo: A. Signore: A. T. M. Willemsen:
J. W. A. Sijbesma: R. A. J. O. Dierckx: E. F. J. de Vries
Department of Nuclear Medicine & Molecular Imaging,
University Medical Center Groningen,
Groningen, The Netherlands Keywords Pharmacokinetics . [18F]FB-IL2 . Molecular
imaging . Inflammation V. Di Gialleonardo: A. Signore
Nuclear Medicine Unit, Faculty of Medicine and Psychology,
“Sapienza” University,
Rome, Italy Pharmacokinetic modelling of N-(4-[18F]fluorobenzoyl)
interleukin-2 binding to activated lymphocytes
in an xenograft model of inflammation Valentina Di Gialleonardo & Alberto Signore &
Antoon T. M. Willemsen & Jurgen W. A. Sijbesma &
Rudi A. J. O. Dierckx & Erik F. J. de Vries Received: 30 January 2012 /Accepted: 6 June 2012 /Published online: 10 July 2012
# The Author(s) 2012. This article is published with open access at Springerlink.com Abstract the Akaike information criterion (AIC) and correlation
between PET measurements and the number of CD25-
positive cells were evaluated. Purpose N-(4-[18F]Fluorobenzoyl)interleukin-2 ([18F]FB-
IL2) specifically binds to interleukin-2 receptors (IL-2R)
and thus may be used to detect inflammation processes
using positron emission tomography (PET). We now vali-
dated whether [18F]FB-IL2 can be used to quantify activated
human peripheral blood mononuclear cells (hPBMC) in rats
by pharmacokinetic modelling. p
Results A high correlation between ex vivo tracer uptake
(standardized uptake value) in the xenograft and the number
of inoculated CD25-positive cells was observed (R200.90). Plasma time-activity curves showed a rapid washout of the
radiopharmaceutical from blood, while the time-activity
curves of the inflammatory lesions showed slower washout. Time-activity curves could be fitted well by the Logan
analysis method, indicating that the binding between [18F]
FB-IL2 and CD25 is reversible. AIC indicated that data
could be modelled best by a two-tissue reversible compart-
ment model. A high correlation was observed between the
binding potential and the number of CD25-positive cells
(R200.876, p<0.0001). Based on binding potential mea-
sured by PET, the limit of detection was about 160,000
CD25-positive cells per 200 μl lesion (95 % confidence). Conclusion [18F]FB-IL2 kinetics in this animal model of
inflammation could be best described by a reversible two-
tissue compartment model. The [18F]FB-IL2 binding poten-
tial is a suitable measure for accurate quantification of lym-
phocytic infiltration in pathological conditions with PET. Methods Eleven Wistar rats were subcutaneously inoculated
in the shoulder with different amounts of phytohaemaggluti-
nin (PHA) activated hPBMC 15 min before i.v. injection of
[18F]FB-IL2. A 60-min dynamic PET scan was acquired and
arterial blood sampling and metabolite analysis were per-
formed. At the end of the scan, animals were terminated and
the inflammatory lesion dissected. PET data were analysed
using Logan and Patlak analysis as well as one-tissue and two-
tissue compartment models. Model preferences according to Conclusion [18F]FB-IL2 kinetics in this animal model of
inflammation could be best described by a reversible two-
tissue compartment model. The [18F]FB-IL2 binding poten-
tial is a suitable measure for accurate quantification of lym-
phocytic infiltration in pathological conditions with PET. Electronic supplementary material The online version of this article
(doi:10.1007/s00259-012-2176-y) contains supplementary material,
which is available to authorized users. V. Di Gialleonardo: A. Signore: A. T. M. Willemsen:
J. W. A. Sijbesma: R. A. J. O. Dierckx: E. F. J. hPBMC activation and FACS analysis Human blood was obtained from the local blood bank. hPBMC were isolated from human peripheral blood by
density gradient medium centrifugation (Lymphoprep,
Axis-Shield) using the rapid centrifugation procedure devel-
oped by Bøyum [23, 24]. Cells were kept in RPMI 1640
supplemented with L-glutamine, 10 % fetal calf serum
(FCS), 100 IU penicillin/ml and 100 μg streptomycin/ml
(all from GIBCO). Isolated hPBMC were incubated for 48 h
with 5 μg/ml of phytohaemagglutinin (PHA-P, Sigma-
Aldrich) at 37 °C and 5 % CO2 for cell activation. At the
day of the microPET scan, overexpression of CD25 was
determined by fluorescence-activated cell sorting (FACS)
analysis. For this purpose, the cultured activated hPBMC
were washed once with 3 ml of cold phosphate-buffered
saline (PBS) and resuspended in PBS at a concentration of
106 cells/ml. Aliquots (0.1 ml) of the cell suspension were
incubated for 45 min in ice with PE-CD25 antibody or PE-
IgG antibody (E-Bioscience) as controls. After being
washed twice in 3 ml of cold PBS, the cells were
resuspended in 0.5 ml of FACS solution (PBS supplemented
with 5 % FCS) immediately prior to analysis using a FACS
Calibur (BD Biosciences). FACS data were analysed using
Winlist 5.0 software (Verity Software House) in order to
determine the percentage of CD25-expressing cells in each
sample. Since IL-2 is the natural ligand of the IL-2R, this
glycoprotein was selected as a potential radiopharma-
ceutical for imaging activated T lymphocytes [16]. In
the past, IL-2 was labelled with various isotopes for
single proton emission computed tomography (SPECT)
imaging [17–21]. The labelled IL-2 derivatives showed
good results in terms of localization of the inflammation
within the limitations of gamma camera imaging. Label-
ling with a suitable positron emission tomography (PET)
isotope may improve sensitivity of this radiopharmaceu-
tical and therefore N-(4-[18F]fluorobenzoyl)interleukin-2
([18F]FB-IL2) was developed by our group. In our pre-
vious studies, we demonstrated the ability of [18F]FB-
IL2 to selectively detect activated human peripheral
blood mononuclear cells (hPBMC) in rodent models of
inflammation by noninvasive PET imaging [22]. In ad-
dition, [18F]FB-IL2 was able to detect insulitis, a chron-
ic inflammatory process that precedes the clinical onset
of type 1 diabetes, in diabetes-prone rats and mice (data
submitted for publication). The detection of chronic inflammation is important
for diagnostic, prognostic purposes and for monitoring
the disease progression and the efficacy of treatment. hPBMC activation and FACS analysis To
this end, quantitative measurement of the extent of
lymphocytic infiltration is required. In the present study
we aimed to investigate whether [18F]FB-IL2 PET is
able to quantify the amount of activated lymphocytes
in infiltrated tissue. For this purpose, pharmacokinetic
modelling was performed. Wistar rats were subcutane-
ously inoculated with activated hPBMC and subsequent-
ly studied with [18F]FB-IL2 microPET. Dynamic PET
data were analysed using various kinetics modelling
approaches and correlated to the number of CD25-
positive cells administered. Synthesis of [18F]FB-IL2 [18F]FB-IL2 was synthesized as described elsewhere [22]. [18F]FB-IL2 was obtained in 25 % radiochemical yield
based on succinimidyl 4-[18F]fluorobenzoic acid ([18F]
SFB) (corrected for the decay), with a specific activity of
117±6 GBq/μmol at the end of synthesis and with a radio-
chemical purity >95 %. Introduction A. Signore (*)
Medicina Nucleare, Ospedale S.Andrea, “Sapienza” University,
Via di Grottarossa 1035,
00189 Rome, Italy
e-mail: alberto.signore@uniroma1.it High levels of interleukin-2 receptors (IL-2R) can be found
mainly on the surface of activated T lymphocytes (both CD4+
and CD8+, mainly Th1 lymphocytes) [1–3] after endogenous
stimulation. T lymphocytes are activated by inflammatory 1552 Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 Materials and methods processes, such as tissue degeneration [4–6], autoimmune
diseases [7, 8], viral, fungal and mycobacterial infections,
graft rejection and in tumour infiltrates [9–15]. These disor-
ders are characterized by the activation of the immune
system and slow recruitment of immune cells (peripher-
al blood mononuclear cell, PBMC) in the lesion. The
infiltration of PBMC in the inflammatory lesion is the
histopathological hallmark of chronic inflammation, in
contrast to the granulocyte infiltration found in acute
inflammatory conditions. During activation, immune
cells overexpress multimeric IL-2R, which are able to
sustain immune cell proliferation and cell survival. The
IL-2R consists of three subunits (α, β and γ chains,
called CD25, CD122 and CD132, respectively). CD25
contains the main binding site for IL-2 and can be
present as a transmembrane or soluble receptor. Because
IL-2R expression is low in resting immune cells, the
receptor might be a suitable biomarker to study active
inflammation in chronic inflammatory diseases. PET acquisition protocol and arterial blood sampling Rats were anaesthetized with 2 % isoflurane in medical air and
a cannula for blood sampling was surgically inserted in the
femoral artery. The animals were placed in a transaxial posi-
tion in the PET camera (microPET Focus 220, Siemens/Con-
corde) with the shoulders of the rats in the field of view. Rats
were injected trough the penile vein with 17.1±1.2 MBq of
[18F]FB-IL2 and a dynamic PET scan was immediately ac-
quired for 60 min. After the emission scan, a transmission
scan of 515 s with a 57Co point source was obtained for the
correction of tissue attenuation. Two animals were scanned
simultaneously in each scan session. Arterial blood was
sampled during the course of the scan at 15, 30, 45, 60, 75,
90, 120, 180, 300, 450, 600, 900, 1,800 and 3,600 s after
tracer injection. When a blood sample was collected, 0.1 ml of
heparinized saline was injected via the artery cannula to
prevent large changes in blood pressure. Blood samples were
used to measure the plasma radioactivity concentration. Blood
was centrifuged (5 min at 13,000 rpm) to separate cells from
plasma. Then, 50 μl of each plasma sample was collected and
the activity in these plasma samples was measured with a
gamma counter (LKB Wallac, Turku, Finland). Plasma activ-
ity was corrected for decay. Animals All animal experiments were carried out according to the
Dutch Regulations for Animal Welfare. The protocol was
approved by the Ethics Committee of the University of
Groningen (protocol number: 5705 C). After arrival, 11 Wistar rats (Charles River, The Nether-
lands) were acclimatized for at least 7 days. The animals
were housed in Makrolon cages on a layer of wood shavings
in a room with constant temperature (21±2 °C) and 12-h
light-dark regime. Commercial chow and tap water were
available ad libitum. All rats were subcutaneously implanted
with an increasing number of activated hPBMC in 100 μl
PBS mixed with 100 μl of Matrigel (Becton Dickinson, The 1553 Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 identification of the radioactive metabolites. HPLC analyses
were carried out with Elite LaChrom Merck Hitachi L-7100
pump system using Luna C18-column (5 μm, 4.6×250 mm)
equipped with both UV (Elite LaChrom VWR Hitachi L-2400
UV detector set at 254 nm) and a Bicron radioactivity monitor. Gradient elution was performed using a mixture of 0.1 %
aqueous trifluoroacetic acid (solvent A) and 0.1 % trifluoro-
acetic acid in acetonitrile (solvent B). The following gradient
profile was used: 0–5 min 0 % B, 5–10 min 40 % B, 10–
35 min 65 % B, 35–45 min 100 % B and 45–47 min 0 % B at
flow rate of 1 ml/min. Retention times were: 24 min for [18F]
FB-IL2, 26 min for [18F]FBA and 30 min for [18F]SFB. The
same HPLC procedure was applied to analyse urine samples
for the formation of radioactive metabolites, using samples
collected 60 min after tracer injection. Netherlands). The activated cells were inoculated in the
right shoulder of the animal 15 min before the PET
experiment. PET acquisition protocol and arterial blood sampling Image reconstruction All emission scans were normalized and corrected for random
coincidences, dead time, scatter, attenuation and decay. Emis-
sion sinograms were reconstructed using an ordered subset
expectation maximization (OSEM) algorithm with 4 iterations
and 16 subsets. Three-dimensional regions of interest (3-D
ROIs) were generated automatically based on an intensity
threshold method using Inveon Research Workplace software
(Inveon, Siemens, USA). Briefly, all frames (0-60 min) were
summed and an ROI of the inflammatory region was gener-
ated automatically with a 50 % threshold using a region
growing method, i.e. only pixels were included with tracer
uptake greater than 50 % of the maximum value within the
lesion. The resulting ROIs were used on the original data set to
create the corresponding time-activity curves (TACs) using
standard software (Inveon, Siemens, USA). Radiometabolite analysis of rat plasma was performed in a
separate study in rats without hPBMC lesion. Arterial blood
samples were collected at 1, 10, 20, 30, 40 and 60 min after
administration of 32.1±9.8 MBq [18F]FB-IL2. Blood was
centrifuged for 5 min at 13,000 rpm to separate the cells from
the plasma fraction. After centrifugation, 5 μl of each plasma
sample was spotted onto a thin-layer chromatography (TLC)
plate (Merck F-254 silica gel strip). The strip was eluted at
room temperature with ethyl acetate/n-hexane (3:1). [18F]SFB
migrated with the solvent front (Rf01), and [18F]FB-IL2
remained at the origin (Rf00). The degradation product 4-
[18F]fluorobenzoic acid ([18F]FBA) has an Rf00.7. Detection
of the reference compounds on the TLC plates was performed
by UV light (254 nm). For radiolabelled compounds, the
detection on the TLC was performed by phosphor storage
imaging (multisensitive screens, Packard). These screens were
exposed to the TLC strips for a few minutes and subsequently
read out using a Cyclone phosphor storage imager
(PerkinElmer, USA) and analysed with OptiQuant software. Ex vivo biodistribution After the PET scan, Wistar rats were sacrificed by extirpa-
tion of the heart while under deep anaesthesia. The xeno-
graft consisting of Matrigel mixed with inoculated cells was
dissected and weighed. As a reference tissue muscle from
the contralateral shoulder was isolated. Radioactivity in
each sample was measured by an automated gamma counter
(LKB Wallac, Turku, Finland). Radioactivity accumulation
in the target lesion was expressed as standardized uptake
value (SUV), using the formula: [(tissue activity concentra-
tion (MBq/g)]/[(injected dose (MBq)/body weight (g)]. Ex vivo measurement of tracer accumulation For comparison with the PET studies, ex vivo measurement
of radioactivity accumulated in the inflammatory lesion was
performed. The radiopharmaceutical uptake of the dissected
Matrigel + hPBMC was calculated in ten animals. In one
animal, the ex vivo tracer accumulation could not be deter-
mined due to an experimental error during dissection and
only kinetic modeling was performed in this animal. Muscle
in the shoulder contralateral with respect to the site of
hPBMC inoculation was used as a control tissue. As shown
in Fig. 1, there is a strong correlation between [18F]FB-IL2
uptake and the number of inoculated activated CD25+ cells
(R200.90, p value <0.0001). As expected, no correlation
was found between muscle uptake and number of inoculated
cells (R200.13, p value00.31). [18F]FB-IL2 kinetics Figure 4a shows the blood clearance of [18F]FB-IL2. Biex-
ponential curve-fitting analysis of [18F]FB-IL2 blood Statistical analysis All data are expressed as mean ± standard deviation. Corre-
lations were calculated with the linear regression algorithm
in Sigma Plot and were considered statistically significant
whenever R2>0.5 and p<0.05. Fig. 1 Correlation between [18F]FB-IL2 uptake (ex vivo SUV) and the
number of CD25+ cells in the inflammatory lesion (closed symbols)
and in muscle as a reference tissue (open symbols). R200.90,
p<0.0001, y00.14x−0.006 and R200.13, p00.31, y00.005x+0.013
for the inflammatory lesion and muscle, respectively Kinetic analysis Pharmacokinetic modelling of the tissue TACs was performed
using standard software (Inveon, Siemens, USA). Graphical
Logan analysis and Patlak analysis were used to determine the
volume of distribution (Vd). Two compartmental models were An additional 0.2 ml plasma sample obtained at 60 min
was filtered through a Vivaspin filter with a 30 kDa cutoff;
0.1 ml of this filtrate was analysed by analytical HPLC for the Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 1554 used to fit ROI data. These were a reversible one-tissue com-
partment model (1TCMR, one tissue compartment with two
kinetic rate constants) and a reversible two-tissue compartment
model (2TCMR, two tissue compartments with four kinetic
rate constants). The blood pool compartment was considered
as the input function using the present terminology [25]. Best-
fit analysis was used to calculate the Vd and with the latter
model also the binding potential (BP). Vd is defined as K1/k2
for the 1TCMR and (K1/k2)(1+BP) for the 2TCMR, with the
BP being equal to k3/k4. The optimal model was selected based
on the Akaike information criterion (AIC) values generated by
the analysis software. AIC selects the model with the best fit of
the data, taking into account the number of fitted data points
and the number of fitted parameters [26]. Small animal PET imaging At 60 min after intravenous injection of [18F]FB-IL2, acti-
vated hPBMC could be clearly visualized in the right shoul-
der of all animals (Fig. 2). By applying 50 % of SUVmax as
the threshold, an ROI was obtained for the inflammatory
lesions. SUV values of [18F]FB-IL2 were calculated for
each animal and were correlated with the number of
CD25-positive cells in the inflamed region. As shown in
Fig. 3, there was a moderate correlation between the cell
number and the SUV of [18F]FB-IL2 PET (R200.56,
p00.008). Limit of detection To assess the sensitivity of the PET method, the limit of
detection (LOD) for CD25-positive cells was determined by
measuring the BP of [18F]FB-IL2 in the contralateral shoul-
der (control). For this purpose, the ROI that was generated
for the inflammatory lesion at the injection site was copied
to the contralateral unaffected shoulder. The LOD was
defined as the mean of the BP in the control tissue + 2 times
the standard deviation in order to assure with 95 % confi-
dence that a signal at the inflammatory lesion was not due to
statistical variability. Subsequently, the LOD was applied to
calculate the minimum number of CD25-positive cells that
can be detected with [18F]FB-IL2, using the correlation
between the number of CD25-positive cells and the BP
(Fig. 7). FACS analysis FACS analysis FACS analysis The percentage of CD25-positive cells was calculated on the
day of the microPET experiment. PHA-P caused variable
stimulation of overexpression of CD25 on the cell surface of
hPBMC. The fraction of activated cells ranged between 5.5
and 9.9 %. The number of the inoculated hPBMC was
corrected for the fraction of CD25-positive cells before
correlation with the PET results. The number of CD25-
positive cells injected in the shoulder of the rats ranged from
0.17×106 to 1.9×106. Fig. 1 Correlation between [18F]FB-IL2 uptake (ex vivo SUV) and the
number of CD25+ cells in the inflammatory lesion (closed symbols)
and in muscle as a reference tissue (open symbols). R200.90,
p<0.0001, y00.14x−0.006 and R200.13, p00.31, y00.005x+0.013
for the inflammatory lesion and muscle, respectively Pharmacokinetic modelling Pharmacokinetic modelling using the [18F]FB-IL2 TACs at
the inoculation site from 0 to 60 min after tracer injection
showed a significantly better fit (p<0.0001) using Logan
graphical analysis (R200.97±0.02) than with Patlak analysis
(R200.81±0.12). In addition, Logan analysis required a delay
time of only 3 min, whereas for Patlak analysis a delay time of
15 min had to be applied to obtain a reasonable fit (Fig. 5). This suggests that the binding between the fluorinated IL-2
and CD25 is better described by a reversible model. 1555 Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 Fig. 2 Small animal PET images of [18F]FB-IL2 in the inflammatory
lesion of a rat inoculated with 0.99×106 CD25+ cells. Coronal (a),
sagittal (b) and transaxial (c) views of the thorax/abdomen of the rat. The inflammatory lesion is indicated by a red arrow. The image represents uptake in the inflammatory lesion from 0 to 60 min after
injection of [18F]FB-IL2. In the transverse image, the submandibular
glands are also visible Fig. 2 Small animal PET images of [18F]FB-IL2 in the inflammatory
lesion of a rat inoculated with 0.99×106 CD25+ cells. Coronal (a),
sagittal (b) and transaxial (c) views of the thorax/abdomen of the rat. The inflammatory lesion is indicated by a red arrow. The image represents uptake in the inflammatory lesion from 0 to 60 min after
injection of [18F]FB-IL2. In the transverse image, the submandibular
glands are also visible represents uptake in the inflammatory lesion from 0 to 60 min after
injection of [18F]FB-IL2. In the transverse image, the submandibular
glands are also visible FB-IL2 with a retention time of 22 min. [18F]FBA and [18F]
FB-IL2 (26 and 24 min, respectively) were not found in the
urine. Because of the high stability of the tracer in plasma,
the total activity in plasma without correction for metabo-
lites was used as input function for the pharmacokinetic
modelling study. clearance showed a two-phase blood clearance with 25±
11 % of the injected activity having a half-clearance time of
0.71±0.29 min and 75±11 % of the injected activity having
a half-clearance time of 8.4±2.6 min. Figure 4b shows the
average TAC for the inflammatory lesion in the animals
inoculated with approximately 106 CD25-positive hPBMC. In contrast to plasma, the clearance from the cell inoculation
site is slower and it can be described by a one-phase curve
(0–60 min) with a half-clearance time of 37±4 min. clearance showed a two-phase blood clearance with 25±
11 % of the injected activity having a half-clearance time of
0.71±0.29 min and 75±11 % of the injected activity having
a half-clearance time of 8.4±2.6 min. Figure 4b shows the
average TAC for the inflammatory lesion in the animals
inoculated with approximately 106 CD25-positive hPBMC. In contrast to plasma, the clearance from the cell inoculation
site is slower and it can be described by a one-phase curve
(0–60 min) with a half-clearance time of 37±4 min. Metabolite analysis Metabolites in plasma were analysed by TLC and by HPLC. TLC analysis did not show any formation of hydrophobic
metabolites or formation of [18F]FBA (hydrolysis product). HPLC analysis confirmed that at 60 min post-injection
(p.i.), the fraction of intact [18F]FB-IL2 in plasma was still
97.7±1.2 %. Urine samples were analysed as well. After 60 min of tracer distribution, the main excretion prod-
uct is an unknown hydrophilic degradation product of [18F] Next, the distribution volume was calculated by Logan
graphical analysis and compartment analysis using a 1TCMR
and a 2TCMR model. Correlation between the number of
CD25+ cells and the distribution volume are displayed in
Fig. 6. The distribution volume showed a moderate correlation
with the number of CD25+ cells when either Logan analysis
or compartment analysis with a 1TCMR or 2TCMR model
was used (R200.59, 0.59 and 0.52, respectively). Fig. 3 Correlation between PET imaging-derived [18F]FB-IL2 uptake
expressed as SUV and the number of CD25+ cells present in the
inflamed region. (R200.56, p00.008, y00.07x+0.17) The model of preference for compartment modelling was
selected based on AIC values. The AIC values are 273±16
and 241±23 for 1TCMR and 2TCMR, respectively, with a
highly significant difference between those models
(p00.0011). Thus, TACs could be fit best using a 2TCMR
model. In contrast to the distribution volume and the
SUV, the BP is only dependent on the receptor binding
characteristics of the radiopharmaceutical. To exclude
any influence of differences in perfusion between ani-
mals, BP were calculated using the 2TCMR model. The
number of CD25-positive cells and the corresponding
BP values are plotted in Fig. 7. The BP is 0.45 for the Fig. 3 Correlation between PET imaging-derived [18F]FB-IL2 uptake
expressed as SUV and the number of CD25+ cells present in the
inflamed region. (R200.56, p00.008, y00.07x+0.17) Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 1556 Fig. 4 [18F]FB-IL2 time-activity curves a for plasma and b for the inflamed region (mean ± standard deviation of animals injected with 106 CD25+
human activated PBMC) Fig. 4 [18F]FB-IL2 time-activity curves a for plasma and b for the inflamed region (mean ± standard deviation of animals injected with 106 CD25+
human activated PBMC) lesion with 0.17×106 CD25-positive cells and gradually
increases up to 7.44 for the lesion with 1.9×106 CD25+
cells. The BP increases by a factor of 4 for each million cells preset in the inflammatory lesion. Metabolite analysis There is a strong
correlation between the BP and the number of CD25-
positive cells (R2 0.88, p<0.0001). . 5 Representative Logan and Patlak graphical analysis for the inflamed region. a Logan graphical analysis showing a good fit of the inflamed
ion from 3 min after [18F]FB-IL2 injection, whereas b Patlak graphical analysis shows a delay time of 15 min Fig. 5 Representative Logan and Patlak graphical analysis for the inflamed region. a Logan graphical analysis showing a good fit of the inflamed
region from 3 min after [18F]FB-IL2 injection, whereas b Patlak graphical analysis shows a delay time of 15 min Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 1557 Fig. 6 Correlation between the volume of distribution (Vd) determined
by a Logan analysis, b 1TCMR and c 2TCMR and the number of
CD25-positive cells. R2 was 0.59, p00.006, y00.32x+0.49 for Logan
analysis. R2 was 0.59, p00.005, y00.34x+0.48 for 1TCMR, and R2
was 0.52, p00.013, y00.32 x+0.56 for 2TCMR analysis. R2 was 0.59, p00.005, y00.34x+0.48 for 1TCMR, and R2
was 0.52, p00.013, y00.32 x+0.56 for 2TCMR analysis. R2 was 0.59, p00.005, y00.34x+0.48 for 1TCMR, and R2
was 0.52, p00.013, y00.32 x+0.56 for 2TCMR Fig. 6 Correlation between the volume of distribution (Vd) determined
by a Logan analysis, b 1TCMR and c 2TCMR and the number of
CD25-positive cells. R2 was 0.59, p00.006, y00.32x+0.49 for Logan Limit of detection untreated shoulder. The average BP of [18F]FB-IL2 in the
contralateral shoulder was 0.42±0.20. Using a 95 % confi-
dence interval, the LOD can be defined as the average back-
ground signal plus 2 standard deviations. In this model, the
LOD of [18F]FB-IL2 would therefore be equal to 0.82. Using
the equation for the correlation between the BP and the
number of CD25-positive hPBMC (Fig. 7), we calculated that
the lowest number of CD25-positive cells that can be reliably
(95 % confidence) detected by this methods is approximately
160,000 cells in a lesion with a volume of 200 μl. To assess the sensitivity of [18F]FB-IL2, the LOD was deter-
mined by measuring the background signal in the contralateral Fig. 7 Correlation between the binding potential and the number of
CD25-positive cells. R2 0.88, p<0.0001, y04.06x+0.17 Discussion The present investigation aims to demonstrate that [18F]FB-
IL2 PET imaging is a suitable tool to quantify lymphocyte
infiltration in tissues. In this study, pharmacokinetic analysis
of [18F]FB-IL2 was carried out in a rat xenograft model of
inflammation. Different parametric methods were used to
investigate which method was best to quantitatively mea-
sure activated immune cell infiltration. Fig. 7 Correlation between the binding potential and the number of
CD25-positive cells. R2 0.88, p<0.0001, y04.06x+0.17 Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 1558 injected cells (165,000 cells). A moderate correlation was
found between the number of CD25-positive cells and the
SUV determined from PET data (R200.56). This correlation
obtained from the imaging study was substantially lower
than the one from the ex vivo studies. This discrepancy
might be explained by partial volume effects, which can
underestimate the uptake in small or heterogeneous target
regions. In this study, inflammatory lesions were relatively
small (injection volume 0.2 ml), when compared to the
spatial resolution of the PET camera (1.35 mm full-width
at half-maximum). In addition, inflammatory lesions could
be heterogeneous, because of the inhomogeneous presence
of the Matrigel in the subcutaneous layer where the hPBMC
are trapped. Furthermore, the inflammatory lesions are ir-
regularly shaped. The inhomogeneity and irregular shape of
the lesion could lead to differences in influence of the partial
volume effect between lesions. Moreover, inflammation is a
process that involves several changes in the inflammatory
lesion, such as change in tissue perfusion and vessel perme-
ability, which can reduce the usefulness of SUVs. Therefore,
pharmacokinetic modelling might be required for accurate
quantification of lymphocyte infiltration. Wistar rats inoculated with different numbers of human
activated PBMC were selected as the animal model of choice. The big advantage of this animal model is the possibility to
determine the exact number of inoculated CD25-positive tar-
get cells by measuring PHA-P-induced CD25 expression
using FACS analysis. PHA-P is a strong activator of hPBMC. One of the most important markers of this activation process is
the alpha subunit of the IL-2R (CD25). PHA-P-mediated cell
activation depends on several factors such as time of incuba-
tion and amount of hPBMC in culture. Moreover, hPBMC
derived from different healthy subjects show different predis-
position to the activation. In this study, hPBMC were isolated
from blood obtained from the local blood bank. Discussion hPBMC in
these samples are less prone to activation by PHA-P than
freshly isolated hPBMC from peripheral blood. Consequently,
the fraction of CD25-expressing cells in the cell cultures was
relatively low in this study, but the experimental design of this
study allowed us to correct the data for the exact number of
CD25-positive cells inoculated in the inflammatory lesion. Ideally, an internal control could have been added to this
model by injection of an equal number of non-activated
hPBMCs or Matrigel alone in the contralateral shoulder of
the animal. In a previous study in mice, however, we
observed that inoculation of the Matrigel without cells al-
ready induced a strong migration of the activated hPBMC to
the contralateral shoulder [22]. This cell migration caused a
variable decrease of the activated PBMC in the inoculation
site. Consequently, injection of a control xenograft would
not allow us to have an exact estimation of the number of
CD25-positive cells present in the inflammatory lesion and
thus would hamper correlation of tracer accumulation with
the number of CD25-positive cells. For the pharmacokinetic modelling, we used the total [18F]
FB-IL2 plasma TAC without correction for tracer metabolites
as the input function, since [18F]FB-IL2 was highly stable in
plasma. More than 97 % of the tracer remained intact in
plasma 60 min p.i. In contrast, only radioactive metabolites
were found in urine. This suggests that [18F]FB-IL2 is metab-
olized by the kidneys and subsequently secreted into the urine. This observation is in agreement with the fate of intravenously
injected native IL-2, which is also cleared into the urine after
renal tubular catabolism [27]. Although the model used in this study does not represent
all aspects of inflammation, it is an ideal model for the
validation of the quantitative analysis of activated T lym-
phocytes using [18F]FB-IL2 PET. Other animal models of
inflammation may better mimic disease processes, but do
not allow exact quantification of the number of CD25-
positive cells and can only give an indirect and inaccurate
estimation of the number of cells present in the inflamma-
tory lesion. Therefore, these more physiological animal
models do not allow accurate correlation of the PET signal
with the number of CD25 cells. A complicating factor in our
xenograft animal model could be the involvement of host
immune cells. Discussion Quantification of the tracer uptake as
SUVor distribution volume is less accurate, as these parameters
are not only dependent on receptor binding characteristics, but
also on perfusion effects. The results of this study indicate that
[18F]FB-IL2 PET could provide hitherto unavailable opportu-
nities to assess lymphocytic infiltration in inflammatory dis-
eases in a non invasive and quantitative manner. The technique
is likely sensitive enough to detect even low numbers of
infiltrating lymphocytes or small changes in the inflammatory
response. Despite the fact that we did not include a control lesion in
the contralateral side, we were still able to estimate the
amount of nonspecific uptake at the inflammatory lesion
from the correlations of the tracer uptake with the number
of CD25-positive cells. In fact, Figs. 3 and 6 show that both
the curves of the SUV and the distribution volume as func-
tions of the number of CD25-positive cells do not cross the
y-axis at the origin, indicating that an SUVof approximately
0.17 and a distribution volume of 0.5 are due to nonspecific
uptake (i.e. in case the number of CD25-positive cells is 0). However, when the tracer uptake is expressed as the BP the
nonspecific binding is negligible, as the curve in Fig. 7
almost crosses the y-axis at the origin. This again stresses
the importance of pharmacokinetic modelling to compen-
sate for nonspecific effects on tracer uptake. 18 Discussion To avoid interference of the host immune
response, the interval between inoculation and PET imaging
was therefore kept as short as possible. Because the tracer is directly injected into the systemic
circulation, the peak of the activity concentration in plasma
of [18F]FB-IL2 reaches its maximum value within 15 s after
tracer injection and then drops progressively. The plasma
[18F]FB-IL2 concentration initially declines quite rapidly
with an α phase of 0.71±0.29 min due to the elimination
from the plasma and the distribution of the tracer in tissue. The elimination of tracer from plasma is described by a β
phase with a half-life of 8.4±2.6 min. In contrast to the
plasma curves, the TACs of the inflammatory lesion show a
clearance half-life of 37±4 %. These data suggest that after
an initial nonspecific distribution of the tracer to the entire
body of the animal [18F]FB-IL2 is retained in the inflamma-
tory lesion because of the presence of target cells that over-
express the IL-2R. Uptake of [18F]FB-IL2 in the region of inoculated acti-
vated hPBMC, measured ex vivo as SUV, increased with the
number of inoculated CD25-positive cells and an excellent
correlation between these parameters was found (R200.90). Logan and Patlak analysis are simple graphical methods
to estimate reversible and irreversible binding of a tracer to
the target, respectively. We used these methods to study the
binding characteristics of [18F]FB-IL2. The TACs of the
hPBMC xenografts showed a better fit with Logan analysis
than with the Patlak analysis method, indicating that binding MicroPET images showed clear [18F]FB-IL2 uptake at
the cell inoculation site, even for the smallest number of 1559 Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 between [18F]FB-IL2 and the IL-2R could be reversible. Compartment modelling showed that the kinetics of [18F]
FB-IL2 are best described by a 2TCMR. However, the
volume of distribution derived from Logan, 1TCMR or
2TCMR showed only moderate correlations with the cell
number present in the inflammatory lesion. In contrast, BPs
are strongly correlated with the number of CD25-positive
cells present in the inflammatory lesion (R200.88). The BP
increases proportionally with the increase of the number of
activated hPBMC in the inflammatory region, suggesting
that with this method changes in the number of activated
hPBMC should be easily detectable. activated hPBMC can be best quantified by measuring the BP
with [18F]FB-IL2 PET. Conflicts of interest
None. Open Access
This article is distributed under the terms of the Crea-
tive Commons Attribution License which permits any use, distribution,
and reproduction in any medium, provided the original author(s) and
the source are credited. References 1. Benczik M, Gaffen SL. The interleukin (IL)-2 family cytokines:
survival and proliferation signaling pathways in T lymphocytes. Immunol Invest 2004;33(2):109–42. 2. Gaffen SL, Liu KD. Overview of interleukin-2 function, produc-
tion and clinical applications. Cytokine 2004;28(3):109–23. Taken together, our data clearly demonstrate that [18F]
FB-IL2 BP is a better measure of activated hPBMC in the
inflammatory lesion than the distribution volume or SUV. Both the distribution volume and the SUV depend not only
on the binding characteristics of the tracer to IL-2R, but also
on the perfusion of the tracer into the lesion. In contrast, BP
is independent of tracer influx (K1) and efflux (k2), but only
depends on the ratio of receptor binding and release. 3. Malek TR, Castro I. Interleukin-2 receptor signaling: at the
interface between tolerance and immunity. Immunity 2010;33
(2):153–65. 4. Meyer KC. The role of immunity and inflammation in lung senes-
cence and susceptibility to infection in the elderly. Semin Respir
Crit Care Med 2010;31(5):561–74. 4. Meyer KC. The role of immunity and inflammation in lung senes-
cence and susceptibility to infection in the elderly. Semin Respir
Crit Care Med 2010;31(5):561–74. 5. Singh T, Newman AB. Inflammatory markers in population studies
of aging. Ageing Res Rev 2011;10(3):319–29. 6. Vasto S, Candore G, Balistreri CR, Caruso M, Colonna-Romano
G, Grimaldi MP, et al. Inflammatory networks in ageing, age-
related diseases and longevity. Mech Ageing Dev 2007;128
(1):83–91. Besides accuracy, sensitivity is also an important property
of a diagnostic tool. Therefore, we determined the LOD of
[18F]FB-IL2 PET and demonstrated that this technique can
detect reliably as little as 160,000 CD25+ cells, if the radio-
pharmaceutical uptake is determined as BP. Moreover, this
method is also sensitive with respect to detection of differ-
ences in CD25+ cell number. A difference in the number of
CD25+ cells of 1 million leads to a difference in 4 BP units. 7. Marincheva-Savcheva G, Subramanian S, Qadir S, Figueroa A,
Truong Q, Vijayakumar J, et al. Imaging of the aortic valve using
fluorodeoxyglucose positron emission tomography increased val-
vular fluorodeoxyglucose uptake in aortic stenosis. J Am Coll
Cardiol 2011;57(25):2507–15. 8. Yetkin E, Waltenberger J. Molecular and cellular mechanisms of
aortic stenosis. Int J Cardiol 2009;135(1):4–13. 9. Ghiringhelli F, Ménard C, Martin F, Zitvogel L. The role of
regulatory T cells in the control of natural killer cells: relevance
during tumor progression. Immunol Rev 2006;214:229–38. Conclusion 10. Swann JB, Vesely MD, Silva A, Sharkey J, Akira S, Schreiber RD,
et al. Demonstration of inflammation-induced cancer and cancer
immunoediting during primary tumorigenesis. Proc Natl Acad Sci
U S A 2008;105(2):652–6. In conclusion, the kinetics of [18F]FB-IL2 in an inflammatory
lesion is well described by Logan graphical analysis and com-
partment modelling with a 2TCMR. Since [18F]FB-IL2 is
stable in plasma, total plasma radioactivity without correction
for metabolites can be used as the input function. Because of
the high correlation between the BP and the number of CD25-
positive cells in the inflammatory lesion, infiltration of 11. Yoshioka K, Ueno Y, Tanaka S, Nagai K, Onitake T, Hanaoka R, et
al. Role of natural killer T cells in the mouse colitis-associated
colon cancer model. Scand J Immunol 2012;75(1):16–26. 12. Rangwala S, Tsai KY. Roles of the immune system in skin cancer. Br J Dermatol 2011;165(5):953–65. Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 1560 13. Allavena P, Garlanda C, Borrello MG, Sica A, Mantovani A. Pathways connecting inflammation and cancer. Curr Opin Genet
Dev 2008;18(1):3–10. 20. Signore A, Chianelli M, Annovazzi A, Bonanno E, Spagnoli LG,
Pozzilli P, et al. 123I-interleukin-2 scintigraphy for in vivo assess-
ment of intestinal mononuclear cell infiltration in Crohn’s disease. J Nucl Med 2000;41(2):242–9. 14. Mantovani A, Allavena P, Sica A, Balkwill F. Cancer-related
inflammation. Nature 2008;454(7203):436–44. 21. Signore A, Picarelli A, Annovazzi A, Britton KE, Grossman AB,
Bonanno E, et al. 123I-Interleukin-2: biochemical characterization
and in vivo use for imaging autoimmune diseases. Nucl Med
Commun 2003;24(3):305–16. 15. Sica A, Allavena P, Mantovani A. Cancer related inflammation:
the macrophage connection. Cancer Lett 2008;267(2):204–15. 16. Chianelli M, Mather SJ, Martin-Comin J, Signore A. Radiophar-
maceuticals for the study of inflammatory processes: a review. Nucl Med Commun 1997;18(5):437–55. 22. Di Gialleonardo V, Signore A, Glaudemans AWJM, Dierckx RA,
De Vries EF. N-(4-18F-Fluorobenzoyl)interleukin-2 for PET of
human-activated T lymphocytes. J Nucl Med 2012;53:679–86. doi:10.2967. 17. Chianelli M, Signore A, Fritzberg AR, Mather SJ. The devel-
opment of technetium-99m-labelled interleukin-2: a new ra-
diopharmaceutical for the in vivo detection of mononuclear
cell infiltrates in immune-mediated diseases. Nucl Med Biol
1997;24(6):579–86. 23. Bøyum A. Separation of white blood cells. Nature 1964;204:793–4. 24. Bøyum A. Separation of blood leucocytes, granulocytes and lym-
phocytes. Tissue Antigens 1974;4(4):269–74. 18. D’Alessandria C, di Gialleonardo V, Chianelli M, Mather SJ, de Vries
EF, Scopinaro F, et al. Conclusion Synthesis and optimization of the labeling
procedure of 99mTc-HYNIC-interleukin-2 for in vivo imaging of
activated T lymphocytes. Mol Imaging Biol 2010;12(5):539–46. 25. Innis RB, Cunningham VJ, Delforge J, Fujita M, Gjedde A, Gunn
RN, et al. Consensus nomenclature for in vivo imaging of revers-
ibly binding radioligands. J Cereb Blood Flow Metab 2007;27
(9):1533–9. 19. Signore A, Chianelli M, Toscano A, Monetini L, Ronga G, Nimmon
CC, et al. A radiopharmaceutical for imaging areas of lymphocytic
infiltration: 123I-interleukin-2. Labelling procedure and animal stud-
ies. Nucl Med Commun 1992;13(10):713–22. 26. Akaike H. Data analysis by statistical models. No To Hattatsu
1992;24(2):127–33. 27. Donohue JH, Rosenberg SA. The fate of interleukin-2 after in vivo
administration. J Immunol 1983;130(5):2203–8.
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Cancer-associated stroke Dubenko O.Ye.1,2, ORCID: 0000-0002-4911-5613, e-mail: olgadubenko05@gmail.com
1State Organization «Grigoriev Institute for Medical Radiology and Oncology o O.Ye.1,2, ORCID: 0000-0002-4911-5613, e-mail: olgadubenko05@gmail.com
1State Organization «Grigoriev Institute for Medical Radiology and Oncology
of the National Academy of Medical Sciences of Ukraine», Kharkiv, Ukraine
2Kharkiv Medical Academy of Postgraduate Education
of the Ministry of Health of Ukraine, Kharkiv, Ukraine Dubenko O.Ye.1,2, ORCID: 0000-0002-4911-5613, e-mail: olgadubenko05@gmail.com 1State Organization «Grigoriev Institute for Medical Radiology and Oncology
of the National Academy of Medical Sciences of Ukraine», Kharkiv, Ukraine
2Kharkiv Medical Academy of Postgraduate Education
of the Ministry of Health of Ukraine, Kharkiv, Ukraine РЕЗЮМЕ Ключові слова: Актуальність. Системний рак і мозковий інсульт є найбільш розповсюдженими
захворюваннями і найчастішою причиною смерті у людей старшого віку в усьому
світі. Взаємозв’язок між раком та інсультом складний, оскільки рак призводить
до збільшення ризику інсульту внаслідок специфічних патофізіологічних механізмів. Мета роботи. Проаналізувати можливі механізми розвитку рак-асоційованого
інсульту, підходи до діагностики та лікування. інсульт, рак, рак-асоційований інсульт,
коагулопатія. DOI: https://doi.org/10.46879/ukroj.4.2022.52-61
УДК: 61.616-006.04:616-005.755 DOI: https://doi.org/10.46879/ukroj.4.2022.52-61
УДК: 61.616-006.04:616-005.755 Рак-асоційований інсульт Дубенко О.Є.1,2,ORCID: 0000-0002-4911-5613, e-mail: olgadubenko05@gmail.com 1Державна установа «Інститут медичної радіології та онкології ім. С.П. Григор’єва
Національної академії медичних наук України», Харків, Україна
2Харківська медична академія післядипломної освіти
Міністерства охорони здоров’я України, Харків, Україна DOI: https://doi.org/10.46879/ukroj.4.2022.52-61
УДК: 61.616-006.04:616-005.755 © Дубенко О.Є., 2022 Для цитування: Для цитування: Дубенко О.Є. Рак-асоційований інсульт. Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61. DOI: https://doi.org/10.46879/ukroj.4.2022.52-61 Огляд літератури
Literature review Огляд літератури
Literature review Огляд літератури
Literature review Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61
Ukrainian journal of radiology and oncology. 2022;30(4):52–61 Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61
Ukrainian journal of radiology and oncology. 2022;30(4):52–61 ISSN 2708-7166 (Print)
ISSN 2708-7174 (Online) Для кореспонденції: Дубенко Ольга Євгеніївна
Харківська медична академія після-
дипломної освіти, кафедра неврології;
вул. Амосова, буд. 58, Харків, Україна,
61176;
il
l
d b
k 05@
il Матеріали та методи. Підбір публікацій виконано за базами даних PubMed
(https://pubmed.ncbi.nlm.nih.gov/),
ClinicalKeyElsevier
(https://www.clinicalkey.com),
Cochrane
Library
(https://www.cochranelibrary.com/)
та
Google
Scholar
(https://scholar.google.com/) опублікованих у 2012–2022 рр., у яких висвітлювались
відомості про рак-асоційований інсульт. Результати та їх обговорення. Відомі декілька основних патофізіологічних
механізмів рак-асоційованого інсульту, що можуть бути безпосередньо пов’язані
з раком, або викликані ускладненнями раку. Найбільш частими є внутрішньо-
судинна ракова коагулопатія, небактеріальний тромботичний ендокардит, прямий
вплив пухлини на судинну систему або терапевтичні та діагностичні втручання
для лікування раку – ускладнення хіміотерапії, променева терапія, а також активація
звичайних механізмів інсульту, насамперед атеросклерозу. Ризик інсульту також
пов’язаний з агресивністю раку. Характерними ознаками рак-асоційованого інсульту,
пов’язаного з раковою коагулопатією, є множинні ураження в різних артеріальних
ділянках, а також лабораторні дані, що вказують на коагулопатію у вигляді підвищення
рівня D-димеру, підвищеного С-реактивного протеїну, низького гематокриту. ;
e-mail: olgadubenko05@gmail.com Результати та їх обговорення. Відомі декілька основних патофізіологічних
механізмів рак-асоційованого інсульту, що можуть бути безпосередньо пов’язані
з раком, або викликані ускладненнями раку. Найбільш частими є внутрішньо-
судинна ракова коагулопатія, небактеріальний тромботичний ендокардит, прямий
вплив пухлини на судинну систему або терапевтичні та діагностичні втручання
для лікування раку – ускладнення хіміотерапії, променева терапія, а також активація
звичайних механізмів інсульту, насамперед атеросклерозу. Ризик інсульту також
пов’язаний з агресивністю раку. Характерними ознаками рак-асоційованого інсульту,
пов’язаного з раковою коагулопатією, є множинні ураження в різних артеріальних
ділянках, а також лабораторні дані, що вказують на коагулопатію у вигляді підвищення
рівня D-димеру, підвищеного С-реактивного протеїну, низького гематокриту. Висновки. Рак призводить до збільшення ризику інсульту внаслідок специфічних
патофізіологічних механізмів. Ці механізми є множинними і складними, однак
найбільш відомим та поширеним є ракова внутрішньосудинна коагулопатія, яка
має специфічні лабораторні біомаркери. Численні дослідження продовжують
спрямовуватись на вивчення факторів, пов’язаних з коагулопатією у пацієнтів, які
перенесли інсульт, особливості раку та оцінки біологічних маркерів внутрішньо-
судинної
коагулопатії,
що
викликає
інсульт,
а
також
моніторинг
ефектів
антикоагулянтної терапії у пацієнтів із рак-асоційованим інсультом. © Дубенко О.Є., 2022 Висновки. Рак призводить до збільшення ризику інсульту внаслідок специфічних
патофізіологічних механізмів. Ці механізми є множинними і складними, однак
найбільш відомим та поширеним є ракова внутрішньосудинна коагулопатія, яка
має специфічні лабораторні біомаркери. Численні дослідження продовжують
спрямовуватись на вивчення факторів, пов’язаних з коагулопатією у пацієнтів, які
перенесли інсульт, особливості раку та оцінки біологічних маркерів внутрішньо-
судинної
коагулопатії,
що
викликає
інсульт,
а
також
моніторинг
ефектів
антикоагулянтної терапії у пацієнтів із рак-асоційованим інсультом. INTRODUCTION Найбільш значними причинами тяжкості захворю-
вань у людей старшого віку залишаються серцево-
судинні захворювання та злоякісні новоутворення [1]. Поєднання раку та інсульту призводить до погіршення
дієздатності та збільшення інвалідизації. Але, поєд-
нання раку та інсульту може бути не просто випадко-
вою комбінацією двох найбільш розповсюджених
захворювань. Рак призводить до збільшення ризику
інсульту
внаслідок
специфічних
патофізіологічних
механізмів. На теперішній час накопичено чимало
наукових знань про асоціацію між раком та інсуль-
том, і можливі механізми рак-асоційованого інсульту. Взаємозв’язок між раком та інсультом відомий в усьому
світі, і викликає значний науковий та практичний
інтерес. Цей взаємозв’язок складний та багатобічний. У низці досліджень науковці намагалися з’ясувати,
які саме види раку мають сильніший зв’язок із ви-
никненням інсульту. У хворих з діагностованим раком
легенів, підшлункової залози, колоректальним раком,
молочної залози та простати існує більш висока
частота інсульту. Ризик інсульту також пов’язаний Найбільш значними причинами тяжкості захворю-
вань у людей старшого віку залишаються серцево-
судинні захворювання та злоякісні новоутворення [1]. Поєднання раку та інсульту призводить до погіршення
дієздатності та збільшення інвалідизації. Але, поєд-
нання раку та інсульту може бути не просто випадко-
вою комбінацією двох найбільш розповсюджених
захворювань. Рак призводить до збільшення ризику
інсульту
внаслідок
специфічних
патофізіологічних
механізмів. На теперішній час накопичено чимало
наукових знань про асоціацію між раком та інсуль-
том, і можливі механізми рак-асоційованого інсульту. Взаємозв’язок між раком та інсультом відомий в усьому
світі, і викликає значний науковий та практичний
інтерес. Цей взаємозв’язок складний та багатобічний. The most substantial causes of disease severity in
older people are still cardiovascular disorders and
malignancies [1]. The combination of cancer and stroke
leads to impaired functional capacity and increased
disability. However, the combination of cancer and stroke
may not just be a random one of the two most common
diseases. Cancer causes an increased risk of stroke
due to specific pathophysiological mechanisms. At pre-
sent, there is a comprehensive scientific expertise in
the relation between cancer and stroke, and possible
mechanisms of cancer-associated stroke. The relation-
ship between cancer and stroke is known worldwide and
is of a considerable scientific and practical interest. It is complex and diverse. In a number of studies, scientists were trying to
figure out what kinds of cancer were mostly associated
with stroke. The patients diagnosed with lung, pancreas,
colon and rectum, breast and prostate cancer have
a higher incidence of stroke. The risk of stroke is also
related to cancer aggressiveness. Зв’язок роботи з науковими програмами,
планами і темами The article is a fragment of the planned research
project of State Organization «Grigoriev Institute for
Medical Radiology and Oncology of the National Academy
of Medical Sciences of Ukraine» «Development of the
individual approaches to antiblastomic therapy in patients
recovered from COVID-19», code: NAMN 04.22, state
registration number: 0121U112052, period for perfor-
mance: 2022–2024; led by Director of State Organization
«Grigoriev Institute for Medical Radiology and Oncology
of the National Academy of Medical Sciences of Ukraine»,
Doctor of Medical Sciences, Professor M.V. Krasnoselskyi;
Head of Radiation Therapy Department, Doctor of Medical
Sciences, Professor V.P. Starenkyi. Стаття є фрагментом планової науково-дослідної
роботи Державної установи «Інститут медичної радіоло-
гії та онкології ім. С.П. Григор’єва Національної акаде-
мії медичних наук України», «Розроблення індивіду-
альних підходів до проведення антибластомної терапії
у пацієнтів, які перенесли COVID-19» НАМН.04.22.,
номер державної реєстрації 0121U112052, термін
виконання 2022–2024 рр., керівники: директор Держав-
ної установи «Інститут медичної радіології та онкології
ім. С.П. Григор’єва Національної академії медичних
наук України», доктор медичних наук, професор
М.В. Красносельський, завідувач Відділу радіології,
доктор медичних наук, професор В.П. Старенький. For correspondence: Dubenko Olga Evgeniyvna
Kharkiv Medical Academy of Postgraduate
Education of the Ministry of Health of
Ukraine, department of neurology;
58, Amosova Str., Kharkiv, Ukraine, 61176;
e-mail: olgadubenko05@gmail.com Results and discussion. There are several underlying pathophysiological mechanisms
of cancer-associated stroke, which may be directly related to cancer or caused by
cancer complications. The most common ones are intravascular cancer coagulopathy,
non-bacterial thrombotic endocarditis, a direct impact of the tumor on the vascular
system or therapeutic and diagnostic interventions for the treatment of cancer that is
chemotherapy, radiation therapy complications, as well as activation of the usual
mechanisms of stroke, atherosclerosis in particular. The risk of stroke is also related
to cancer aggressiveness. The characteristic features of cancer-associated stroke which
is related to cancer coagulopathy are multiple lesions in various arterial areas, as well as
laboratory data indicating coagulopathy in the form of increased level of D-dimer,
elevated C-reactive protein, low hematocrit.i © Dubenko O.E., 2022 © Dubenko O.E., 2022 Conclusions. Cancer leads to an increased risk of stroke due to specific patho-
physiological mechanisms. These mechanisms are multiple and complex, but the most
well-known and widespread is cancer intravascular coagulopathy, which has specific
laboratory biomarkers. Numerous studies continue investigating the factors associated
with coagulopathy in stroke patients, cancer characteristics, and evaluation of biological
markers of stroke-causing intravascular coagulopathy, as well as monitoring the effects
of anticoagulation therapy in patients with cancer-associated stroke. For citation: Key words: Background. Systemic cancer and stroke are the most common diseases and the
frequent death factors in older people worldwide. The relationship between cancer and
stroke is complex, since cancer leads to an increased risk of stroke through specific
pathophysiological mechanisms. stroke, cancer, cancer-associated stroke,
coagulopathy. Purpose. Analyzing possible mechanisms of development of cancer-associated stroke,
strategies of diagnosis and treatment. Materials and methods. The contributions were selected based on PubMed
(https://pubmed.ncbi.nlm.nih.gov/), Clinical Key Elsevier (https://www.clinicalkey.com), Literature review 52 ISSN 2708-7166 (Print)
ISSN 2708-7174 (Online) Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61
Ukrainian journal of radiology and oncology. 2022;30(4):52–61 Cochrane
Library
(https://www.cochranelibrary.com/)
and
GoogleScholar
(https://scholar.google.com/) databases published from 2012 to 2022, dealing with
cancer-associated stroke. Dubenko Olga Evgeniyvna
Kharkiv Medical Academy of Postgraduate
Education of the Ministry of Health of
Ukraine, department of neurology;
58, Amosova Str., Kharkiv, Ukraine, 61176;
e-mail: olgadubenko05@gmail.com INTRODUCTION Lung, pancreas, colon
and rectum cancer, representing a highest stroke risk, У низці досліджень науковці намагалися з’ясувати,
які саме види раку мають сильніший зв’язок із ви-
никненням інсульту. У хворих з діагностованим раком
легенів, підшлункової залози, колоректальним раком,
молочної залози та простати існує більш висока
частота інсульту. Ризик інсульту також пов’язаний Literature review 53 Огляд літератури ISSN 2708-7166 (Print)
ISSN 2708-7174 (Online) Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61
Ukrainian journal of radiology and oncology. 2022;30(4):52–61 Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61
Ukrainian journal of radiology and oncology. 2022;30(4):52–61 Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61
Ukrainian journal of radiology and oncology. 2022;30(4):52–61 із агресивністю раку. Рак легенів, підшлункової залози
та колоректальний рак, які представляють найвищий
ризик інсульту, як правило, діагностується на більш
пізній стадії, ніж рак грудей і простати [2, 3]. Крім того,
хворі на рак та інсульт мають спільні фактори серцево-
судинного ризику, такі як артеріальна гіпертонія,
куріння, цукровий діабет та метаболічний синдром. is usually diagnosed at a later stage, compared to
breast and prostate cancer [2, 3]. In addition, cancer and
stroke patients have the common cardiovascular risk
factors, such as arterial hypertension, smoking, diabetes
mellitus and metabolic syndrome. The nature of stroke in cancer patients can be ischemic
or hemorrhagic, while ischemic stroke is observed more
often than hemorrhagic and the risk is higher within the
first 6 months after cancer was diagnosed [4]. The types
of cancer in hemorrhagic stroke are somewhat different. A high risk of cancer of the small intestine, liver, kidney,
nervous system, thyroid gland, endocrine system, Non-
Hodgkin lymphoma, myeloma and leukemia was reported. The mechanism of hemorrhage is multifactorial including
enhanced formation of dilated thin-walled intratumoral
vessels, rupture of these newly formed vessels, tumor
invasion into existing vessels, and tumor necrosis [5, 6]. Характер інсульту у хворих на рак може бути
ішемічним або геморагічним, при цьому ішемічний
інсульт спостерігається частіше, ніж геморагічний і ри-
зик був вищим в перші 6 місяців після встановлення
діагнозу раку [4]. Типи раку при геморагічному інсульті
дещо інші. Повідомлялося про високий ризик раку
тонкої кишки, печінки, нирок, нервової системи, щито-
подібної залози, ендокринної системи, неходжкінської
лімфоми, мієломи і лейкемії. Механізм геморагії
є багатофакторним і включає посилене утворення
розширених тонкостінних внутрішньопухлинних судин,
розрив цих новоутворених судин, інвазію пухлини
у вже існуючі судини та некроз пухлини [5, 6]. МАТЕРІАЛИ ТА МЕТОДИ ДОСЛІДЖЕННЯ The contributions were selected based on Pub
Med
(https://pubmed.ncbi.nlm.nih.gov/),
Clinical
Key
Elsevier (https://www.clinicalkey.com), Cochrane Library
(https://www.cochranelibrary.com/)
and
GoogleScholar
(https://scholar.google.com/) databases published from
2012 to 2022, dealing with cancer-associated stroke. Підбір публікацій виконано за базами даних Pub
Med
(https://pubmed.ncbi.nlm.nih.gov/),
Clinical
Key
Elsevier (https://www.clinicalkey.com), Cochrane Library
(https://www.cochranelibrary.com/)
та
GoogleScholar
(https://scholar.google.com/),
опублікованих
у
2012–
2022 рр., у яких висвітлювались відомості про
рак-асоційований інсульт. INTRODUCTION Stroke can develop in patients diagnosed with cancer,
or precede cancer [7]. In the United States, one out
of 10 patients hospitalized with stroke has been diag-
nosed with cancer [8]. Інсульт може розвинутись у пацієнтів з діагностова-
ним раком, або передувати діагнозу раку [7]. У Сполу-
чених Штатах у кожного з 10 пацієнтів, госпіталізованих
з інсультом, був виявлений рак [8]. Purpose. Analyzing possible mechanisms of deve-
lopment of cancer-associated
stroke, strategies of
diagnosis and treatment. Мета роботи. Проаналізувати можливі механізми
розвитку
рак-асоційованого
інсульту,
підходи
до
діагностики та лікування. Огляд літератури Механізми ракової коагулопатії та їх роль
у розвитку рак-асоційованого інсульту One of the main causes of stroke in cancer patients
is coagulopathy. Disrupted blood coagulation, that is
disseminated intravascular coagulopathy, is the most
common cause of cardiovascular diseases in cancer
patients. The first postmortem characteristics of cerebral
intravascular coagulation were described in 1975 in pati-
ents with breast cancer, leukemia, and lymphoma in the
setting of widespread metastases and sepsis. In cancer
patients, the most common cause of cerebrovascular
thrombosis is a hypercoagulable state that accompa-
nies cancer resulting in systemic and cerebral arterial
or venous thrombosis [12, 13]. Однією з основних причин інсульту у хворих на рак
є коагулопатія. Порушення згортання крові – дисемі-
нована внутрішньосудинна коагулопатія – це най-
поширеніша причина серцево-судинних захворювань
в онкологічних хворих. Перші посмертні характеристики
церебрального внутрішньосудинного згортання крові
були описані ще в 1975 р. у хворих на рак молочної
залози, лейкемію та лімфому, на тлі поширених
метастазів і сепсису. У хворих на рак найпоширенішою
причиною цереброваскулярного тромбозу є гіпер-
коагуляційний стан, який супроводжує рак, внаслідок
чого розвиваються системні і церебральні артеріальні
або венозні тромбози [12, 13]. Intravascular coagulopathy can be caused by the
procoagulant activity of the tumor and the proinflamma-
tory response. Tumor cells express procoagulants, tissue
factor (which binds to factor VII) and cancer procoagulant,
and release inflammatory cytokines and vascular endo-
thelial growth factor, mediators that enhance procoagulant
activity and angiogenesis. They also overexpress cytokines
that attract leukocytes, possibly causing an inflammatory
response with a prothrombotic effect. The most common
types of cancer causing coagulopathy are lung and
pancreatic cancer [14]. It has been recently shown
that the so-called neutrophil extracellular traps are
important in occurring thrombosis in cancer coagulopathy,
which are formed due to the fact that activated neutro-
phils secrete decondensed chromatin, which creates
a framework for the adhesion of platelets, erythrocytes
and platelet adhesion molecules [15].i Внутрішньосудинна коагулопатія може бути викли-
кана прокоагулянтною активністю пухлини та проза-
пальною відповіддю. Пухлинні клітини експресують
прокоагулянти, тканинний фактор (який зв’язується
із фактором VII) та прокоагулянт раку, і вивільняють
запальні цитокіни та фактор росту ендотелію судин,
медіатори, які посилюють прокоагулянтну активність
і ангіогенез. Вони також надлишково експресують цито-
кіни, які приваблюють лейкоцити, можливо, викликаючи
запальну реакцію із протромботичним ефектом. Най-
більш поширені типи раку, що викликають коагуло-
патію – це рак легенів і підшлункової залози [14]. Mechanisms of cancer coagulopathy and
their role in cancer-associated stroke development Mechanisms of cancer coagulopathy and
their role in cancer-associated stroke development RESULTS AND DISCUSSION Патофізіологія рак-асоційованого інсульту Pathophysiology of cancer-associated stroke
There
are
several
essential
pathophysiological
mechanisms of stroke in cancer patients, which may
directly be related to cancer, or caused by cancer compli-
cations like coagulopathy, infections, a direct impact of
the tumor on the vascular system, or therapeutic and diag-
nostic interventions for the treatment of cancer that is
chemotherapy complications, radiation therapy, hemato-
poietic stem cell transplantation occuring during treatment
or even years after [9].l Існують кілька основних патофізіологічних механіз-
мів інсульту у хворих на рак, які можуть бути безпосе-
редньо пов’язаними з раком, або викликані ускладнен-
нями раку, такі як коагулопатія, інфекції, прямий вплив
пухлини на судинну систему, або терапевтичні та діаг-
ностичні втручання для лікування раку – ускладнення
хіміотерапії, променева терапія, трансплантація гемо-
поетичних стовбурових клітин, які можуть відбуватися
під час лікування або навіть через роки після нього [9]. Прямий вплив пухлини, або безпосередньо пов’я-
заний з пухлиною інсульт, у клінічній практиці зустріча-
ється рідко і його важко визначити. Прямі пухлинні
ефекти значно відрізняються і включають інвазію
пухлинною масою або лептоменінгеальними інфільтра-
тами в артеріальний і венозний синус, пухлинні
емболії, здавлення кровоносної судини пухлинним
ростом, набряк ложа пухлини та внутрішньопухлин-
ний крововилив [10]. A direct influence of the tumor, or stroke directly
related to the tumor, is rare in clinical practice and it is
difficult for identifying. Direct tumor effects vary consi-
derably and include invasion by the tumor mass or
leptomeningeal infiltrates into the arterial and venous
sinuses, tumor emboli, compression of a blood vessel
by tumor growth, tumor bed edema and intratumoral
hemorrhage [10]. Non-bacterial thrombotic endocarditis, also known as
marantic endocarditis, is a recognized cause of cardio-
embolic stroke in cancer patients. In non-bacterial
thrombotic endocarditis, sterile non-infectious platelet-
thrombin vegetations develop on the heart valves (almost
exclusively in the left heart valves, mitral and aortic)
associated with widespread systemic and cerebral Небактеріальний тромботичний ендокардит, також
відомий як марантичний ендокардит, є визнаною
причиною кардіоемболічного інсульту у хворих на рак. При небактеріальному тромботичному ендокардиті
на клапанах серця розвиваються стерильні неінфек-
ційні
тромбоцитарно-тромбінові
вегетації
(майже Literature review 54 Огляд літератури ISSN 2708-7166 (Print)
ISSN 2708-7174 (Online) Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61
Ukrainian journal of radiology and oncology. 2022;30(4):52–61 Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61
Ukrainian journal of radiology and oncology. RESULTS AND DISCUSSION 2022;30(4):52–61 виключно
в
лівих
клапанах
серця,
мітрального
та аортального) в асоціації з поширеним системним
та церебральним тромбозом, і це найчастіше спричи-
нено основною злоякісною пухлиною. Зазвичай він
проявляється системним і легеневим емболізмом,
причому
найпоширенішим
неврологічним
усклад-
ненням є ішемічний інсульт. Точної поширеності не-
бактеріального тромботичного ендокардиту у хворих
на рак не встановлено, для його скринінгу використо-
вуються ехокардіографічні дослідження. Патогенез
його не досить відомий, але припускається наявність
гіперкоагуляційного стану. Рак у таких випадках частіше
поширений, а інфаркт мозку є пізнім ускладненням,
однак у рідкісних випадках небактеріальний тромбо-
тичний ендокардит при церебральному інфаркті є
ознакою раку. В одному оригінальному дослідженні
повідомляється про небактеріальний тромботичний
ендокардит у 9,3% хворих на рак, і рак у 59% пацієнтів
з
небактеріальним
тромботичним
ендокардитом
(дослідження проводилися при розтині) [11]. thrombosis, and this is most often caused by an underlying
malignanсy. It is usually manifested by systemic and
pulmonary embolism, while the most common neuro-
logical complication is ischemic stroke. The exact preva-
lence of non-bacterial thrombotic endocarditis in cancer
patients has not been established, echocardiographic
studies are used for its screening. Its pathogenesis is not
well known, but the presence of a hypercoagulable state
is assumed. Cancer is more common in such cases, and
cerebral infarction is a late complication, however, in rare
cases, non-bacterial thrombotic endocarditis in cerebral
infarction is a cancer sign. One original study reported
non-bacterial thrombotic endocarditis in 9.3% of cancer
patients, and cancer in 59% of patients with non-bacterial
thrombotic endocarditis detected at autopsy [11]. Механізми ракової коагулопатії та їх роль
у розвитку рак-асоційованого інсульту Нещодавно було показано, що у виникненні тромбозів
при раковій коагулопатії мають значення так звані
нейтрофільні екстрацелюлярні пастки, які формуються
внаслідок того, що активовані нейтрофіли виділяють
деконденсований хроматин, який створює каркас
для адгезії тромбоцитів, еритроцитів і тромбоцитарних
молекул адгезії [15]. Stroke, due to cancer-related coagulopathy, is a defi-
ned disease. Studies of different populations have repor-
ted unique characteristics of this condition. Thus, neuro-
imaging on diffusion-weighted images (DWI) shows
multiple lesions involving numerous different arterial
sites. Laboratory data also indicate coagulopathy in the
form of increased D-dimer levels, elevated C-reactive
protein, and low hematocrit. The factors associated
with coagulopathy in stroke patients, cancer characteristics
and assessment of biological markers of stroke-causing
intravascular coagulopathy, as well as monitoring the
effects of anticoagulant therapy in patients with active
cancer and stroke were studied in the OASIS-Cancer
trial [16, 17]. Cancer-associated stroke patients had Інсульт внаслідок коагулопатії, пов’язаної з раком,
є визначеною хворобою. В дослідженнях різних
популяцій повідомлялося про унікальні характерис-
тики цього стану. Так, при нейровізуалізації на дифу-
зійно-зважених зображеннях (DWI) спостерігаються
множинні ураження, що охоплюють численні різні
артеріальні ділянки. Лабораторні дані також вказують
на коагулопатію у вигляді підвищення рівня D-димеру,
підвищеного С-реактивного протеїну, низького гемато-
криту. Вивчення факторів, пов’язаних із коагулопатією
у пацієнтів, які перенесли інсульт, особливості раку Literature review Literature review 55 Огляд літератури Огляд літератури ISSN 2708-7166 (Print)
ISSN 2708-7174 (Online) Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61
Ukrainian journal of radiology and oncology. 2022;30(4):52–61 increased levels of extracellular vesicles obtained from
the
cancer
cells,
correlating
with
D-dimer
levels. The levels of circulating extracellular vesicles were
shown to be higher in patients with lung adenocarcinoma,
a well-known type of cancer with cancer coagulopathy,
than in other types of cancer (such as squamous cell). When comparing two groups of patients with ischemic
stroke, one of which had cancer and the other did not,
it was found that in the group of patients who had cancer,
there was a significantly higher level of the inflammatory
marker C-reactive protein, as well as fibrinogen, D-dimer,
deep vein and pulmonary artery thrombosis. Ischemic
stroke in cancer patients was accompanied by a signi-
ficantly higher mortality rate and greater disability,
determined by the modified Rankin scale. Therefore,
the pathophysiology and mechanism of ischemic stroke
in cancer patients were determined by different risk factors,
stroke biomarkers and subtypes compared to non-cancer
cases. Механізми ракової коагулопатії та їх роль
у розвитку рак-асоційованого інсульту It was concluded that the stroke pathogenesis
in such cases was most likely due to the state of hyper-
coagulation or side effects of cancer treatment [18]. та оцінки біологічних маркерів внутрішньосудинної
коагулопатії, що викликає інсульт, а також моніторинг
ефектів антикоагулянтної терапії у пацієнтів з активним
раком
та
інсультом
вивчались
у
дослідженні
OASIS-Cancer [16, 17]. У пацієнтів з інсультом, пов’яза-
ним з раком, були підвищені рівні екстрацелюлярних
везикул, отриманих від ракових клітин, які корелювали
з рівнями D-димеру. Показано, що рівні циркулюючих
екстрацелюлярних везикул були вищими у пацієнтів
з аденокарциномою легенів, яка є добре відомим
типом раку з розвитком ракової коагулопатії, ніж
при інших типах раку (таким як плоскоклітинний). При порівнянні двох груп пацієнтів з ішемічним інсуль-
том, в одних з яких був рак, а в інших раку не було,
виявлено, що в групі пацієнтів, які хворіли на рак,
був достовірно вищий рівень маркера запалення
С-реактивного
протеїну,
а
також
фібриногену,
D-димеру, тромбозів глибоких вен та легеневої арте-
рії. Ішемічний інсульт в онкологічних хворих супро-
воджувався значно вищим рівнем смертності та
більшою інвалідністю, визначеною за модифікованою
шкалою Ренкін. Отже, патофізіологія та механізм
ішемічного інсульту у пацієнтів з онкологічними за-
хворюваннями були зумовлені різними факторами
ризику, біомаркерами інсульту та підтипами порівняно
з нераковими випадками. Було зроблено висновок,
що патогенез інсульту в таких випадках швидше
за все зумовлений станом гіперкоагуляції або побіч-
ними ефектами лікування раку [18]. A better understanding of the molecular targets
underlying stroke-related coagulopathy will help determine
stroke risk and monitor the effectiveness of cancer-asso-
ciated stroke therapy. Patients with occult cancer may have
the characteristic symptoms and neuroimaging pattern
of cerebral infarction suggesting cancer-related stroke and
demonstrating elevated D-dimer levels. Thus, screening
for occult malignancy (using serologic or radiologic studies)
should be considered for patients who have had charac-
teristic patterns of infarction, elevated D-dimer levels,
and unobvious etiology or pathogenetic subtype of stroke. Thoracoabdominal-pelvic
computed
tomography
or
positron emission tomography should be performed
first, since lung, pancreas, genitourinary tract (prostate,
ovary, or kidney), gastrointestinal tract (colorectal and
stomach) or breast cancer are the most common types
(in order of frequency) of known as well as occult
cancers in patients with cancer-related stroke. Labora-
tory predictors of malignant neoplasm in patients with
cryptogenic stroke can also be increased levels of
fibrinogen and C-reactive protein [19, 20]. Краще
розуміння
молекулярних
мішеней,
що
лежать в основі коагулопатії, пов’язаної з інсультом,
допоможе визначити ризик інсульту і моніторувати
ефективність терапії при рак-асоційованому інсульті. Огляд літератури Механізми ракової коагулопатії та їх роль
у розвитку рак-асоційованого інсульту Пацієнти з прихованим раком можуть мати характерні
симптоми та нейровізуалізаційний паттерн інфаркту
мозку, що вказують на інсульт, пов’язаний з раком,
і демонструвати підвищені рівні D-димеру. Таким чином,
у пацієнтів, які мали характерні картини інфаркту,
підвищений рівень D-димеру та відсутність очевидної
етіології та патогенетичного підтипу інсульту, слід
розглянути можливість скринінгу на приховане зло-
якісне новоутворення (за допомогою серологічних
або радіологічних досліджень). Торакоабдомінально-
тазову
комп’ютерну
томографію
або
позитронну
емісійну томографію слід проводити у першу чергу,
оскільки рак легенів, підшлункової залози, сечостате-
вих шляхів (простати, яєчників або нирок), шлунково-
кишкового тракту (колоректальний і шлунок) або молоч-
ної залози, є найбільш частими типами (у порядку
частоти) як відомих, так і прихованих ракових захво-
рювань у пацієнтів з інсультом, пов’язаним з раком. Лабораторними предикторами злоякісного новоутво-
рення у пацієнтів з криптогенним інсультом також
можуть бути підвищений рівень фібриногену та С-реак-
тивного протеїну [19, 20]. i
g
The stroke risk can also increase due to activation
of normal stroke mechanisms, primarily atherosclerosis. Cardiovascular diseases primarily result from athero-
sclerosis complications, which is defined as a chronic
and progressive inflammatory condition caused by
uncontrolled proliferation of vascular smooth muscle cells,
endothelial cells, and macrophages in situ associated
with the immune response. The progression of the disease
at different stages usually ends with a thrombotic process
that can lead to myocardial infarction or stroke. There are
lots of similarities with the progression of neoplastic
processes in cancer and atherogenesis. Deregulation
of cell proliferation and, therefore, cell cycle progression,
changes in the synthesis of important transcription
factors as well as adhesion molecules, changes in the
control of angiogenesis, and molecular similarities that
occur after chronic inflammation are just some of the
processes that become a part of the phenomena that
atherosclerosis and cancer are closely related. Thus, de-
spite the fact that these two pathologies were previously
considered to be unrelated, a careful analysis of the
molecular manifestations of both diseases has revealed
important similarities, suggesting that they are closely
related. One of the most important peculiarities of both р
у [
]
Ризик інсульту може збільшитися також внаслідок
активації звичайних механізмів інсульту, насамперед
атеросклерозу. Серцево-судинні
захворювання
є,
передусім, результатом ускладнень атеросклерозу,
який визначається як хронічний і прогресуючий запаль-
ний стан, спричинений неконтрольованою проліфе-
рацією гладком’язових клітин судин, ендотеліальних
клітин і макрофагів insitu, пов’язаних з імунною
відповіддю. Механізми ракової коагулопатії та їх роль
у розвитку рак-асоційованого інсульту Прогресування захворювання на різних
стадіях зазвичай закінчується тромботичним проце-
сом, який може призвести до інфаркту міокарда або Literature review 56 Literature review Огляд літератури Огляд літератури ISSN 2708-7166 (Print)
ISSN 2708-7174 (Online) Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61
Ukrainian journal of radiology and oncology. 2022;30(4):52–61 Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61
Ukrainian journal of radiology and oncology. 2022;30(4):52–61 diseases is uncontrolled cell proliferation, which contribu-
tes to the appearance and severity of lesions at the
late stages of both diseases. Deregulation of cell prolife-
ration caused by a state of oxidative stress facilitates
in many cases the development of atherosclerotic plaque,
as well as various types of cancer.l інсульту. Відомо багато подібностей з прогресуванням
неопластичних процесів при раку та атерогенезі. Дерегуляція проліферації клітин і, отже, прогресування
клітинного циклу, зміни в синтезі важливих факторів
транскрипції, а також молекул адгезії, зміна в контролі
ангіогенезу та молекулярні подібності, які виникають
після хронічного запалення, – це лише деякі з процесів,
що стають частиною явищ, які тісно пов’язують
атеросклероз і рак. Таким чином, незважаючи на те,
що раніше ці дві патології вважалися непов’язаними,
завдяки ретельному аналізу молекулярних проявів
обох захворювань було виявлено важливу подібність,
що свідчить про їх тісний зв’язок. Однією з найваж-
ливіших характеристик обох захворювань є неконтро-
льована проліферація клітин, що сприяє виникненню
та тяжкості уражень на пізніх стадіях обох захво-
рювань. Дерегуляція проліферації клітин, спричинена
станом окисного стресу, у багатьох випадках сприяє
розвитку атеросклеротичної бляшки, а також різних
видів раку. Запальний процес під час атерогенезу
опосередковується міграцією моноцитів до стінки
судини, що є ключовою подією у зростанні атеро-
склеротичного
ураження. Завдяки
диференціації
моноцити утверджуються як макрофаги та, зрештою,
як збагачені ліпідами пінисті клітини. Макрофаги,
отримані з моноцитів, розпізнають та інтерналізують
окиснені
ліпопротеїни
через
рецептори-переймачі,
де збагачені ліпідами пінисті клітини сприяють роз-
витку некротичного ядра, ключового елемента враз-
ливої атеросклеротичної бляшки. На молекулярному
рівні присутність кристалів холестерину також активує
запалення, вивільняючи цитокіни IL-1β, які вважаються
важливими медіаторами запалення [21, 22]. З іншого
боку, моноцитарні макрофаги, що часто зустрічаються
як клітини-господарі в пухлинах, функціонують як ком-
поненти запальної відповіді, що створює підтримуючу
строму, яка бере участь у зростанні пухлини. Однак
багато хворих на рак і серцево-судинні захворювання
мають загальний субстрат, який пов’язує ці два захво-
рювання. Наприклад, у хворих на рак часто виявляють
метаболічні та судинні розлади, серед яких абдомі-
нальне
ожиріння,
змінений
метаболізм
глюкози,
аномалії ліпопротеїдів і гіпертонія [23, 24]. Механізми ракової коагулопатії та їх роль
у розвитку рак-асоційованого інсульту yp
The inflammatory process during atherogenesis is
mediated by monocyte migration to the vessel wall, which
is a key event in the growth of atherosclerotic lesions. Due to differentiation, monocytes become established
as macrophages and eventually as lipid-enriched foam
cells. Monocyte-derived macrophages recognize and
internalize oxidized lipoproteins via receptors that receive
signals, where lipid-enriched foam cells promote the
development of the necrotic core, a key element of
a vulnerable atherosclerotic plaque. At the molecular
level, cholesterol crystals also activate inflammation
by releasing cytokines IL-1β, which are considered to be
important inflammation mediators [21, 22]. On the other
hand, monocytic macrophages, often found as host cells
in tumors, function as inflammatory response сomponents
creating a supportive stroma that participates in tumor
growth. However, a lot of cancer and cardiovascular
disease patients share a common substrate that links
these two diseases. For example, cancer patients often
have
metabolic
and
vascular
disorders,
including
abdominal obesity, altered glucose metabolism, lipo-
protein abnormalities, and hypertension [23, 24]. Огляд літератури Cancer treatment and stroke risk Пошкод-
ження ендотелію зі змінами його проникності, призво-
дить до додаткового фіброзу та неоваскуляризації
в стінках судин у пацієнтів з наявними раніше атеро-
склеротичними бляшками, що ще більше сприяє
нестабільності бляшок. Пацієнти з наявною ішемічною
хворобою серця особливо вразливі. (наприклад, IL-6 та IL-8), міжклітинних молекул адгезії
(наприклад, ICAM-1), фактора росту фібробластів
і стимулює реакцію нейтрофілів із вторинним вивіль-
ненням цитокінів. Крім того, радіація може індукувати
апоптоз через p53 відповідь на пошкодження ДНК
або апоптоз, активований мітохондріями. Пошкод-
ження ендотелію зі змінами його проникності, призво-
дить до додаткового фіброзу та неоваскуляризації
в стінках судин у пацієнтів з наявними раніше атеро-
склеротичними бляшками, що ще більше сприяє
нестабільності бляшок. Пацієнти з наявною ішемічною
хворобою серця особливо вразливі. a neutrophil response with secondary cytokine release. Besides, radiation can induce apoptosis through the
p53 DNA damage response or mitochondria-activated
apoptosis. Damage to the endothelium with changes in
its permeability leads to additional fibrosis and neovas-
cularization in the walls of vessels in patients with
previously existing atherosclerotic plaques, which further
contributes to plaque instability. Patients with existing
coronary heart disease are particularly vulnerable. Chemotherapy-induced vascular toxicity is associated
with the onset and progression of atherosclerosis. Vascular
damage associated with chemotherapy is often mani-
fested by endothelial dysfunction with loss of vasodilation
effect and inhibition of anti-inflammatory and vascular
reparative function. Platelets are the connecting link
between atherosclerosis and cancer. Activated platelets
contribute to the severity of inflammation in atherosclerosis
by releasing inflammatory mediators. Platelets are able
to oxidize low-density lipoproteins, which are the main
factor in the formation of atherosclerotic plaques, and
also interact with neutrophils and eosinophils and contri-
bute to the formation, expansion and thrombosis of
atherosclerotic plaques. Platelets attract inflammatory cells
to tumors, which promotes tumor growth. In addition,
the procoagulant effect of cancer itself increases the
activity of platelets and reduces the production of nitric
oxide in the endothelium, which increases the formation
of atherosclerotic plaque and leads to an increased risk
of cardiovascular events [25–27]. Індукована
хіміотерапією
судинна
токсичність
пов’язана з початком і прогресуванням атеросклерозу. Пошкодження судин, пов’язані з хіміотерапією, часто
проявляються ендотеліальною дисфункцією із втратою
ефекту вазодилатації та пригніченням протизапаль-
ної
та
судиннорепаративної
функції. Тромбоцити
є зв’язуючою ланкою між атеросклерозом і раком. Активовані тромбоцити сприяють запальній тяжкості
при атеросклерозі, вивільняючи медіатори запалення. Діагностичні та терапевтичні підходи
при рак-асоційованому інсульті The time interval from the diagnosis of cancer to the
development of a stroke varies considerably and also
depends on cancer type. Usually, in cases of solid tumors,
longer periods of time pass in comparison with hemato-
logical malignant tumors. If a cancer patient has had
stroke, the clinician must determine the exact cause
by carefully examining the clinical setting, taking into
account the type of cancer and its treatment. Інтервал часу від діагностики раку до розвитку
інсульту значно відрізняється і залежить також від
типу раку. Зазвичай при солідних пухлинах проходять
більш тривалі періоди часу, ніж при гематологічних
злоякісних пухлинах. Якщо у хворого на рак стався
інсульт, клініцист повинен точно визначити причину,
уважно вивчивши клінічну обстановку, враховуючи
тип раку та його лікування. У разі криптогенного інсульту у хворих на рак
важливе значення можуть мати лабораторні показ-
ники, важливішими з яких є високий рівень D-димеру,
низький рівень тромбоцитів, порушені тести коагуля-
ційної функції, високий рівень фібриногену та збіль-
шення часткового тромбопластинового часу [28]. In the case of cryptogenic stroke in cancer patients,
laboratory indicators can be important, the most important
of which are a high level of D-dimer, a low level of platelets,
impaired coagulation function tests, a high level of fibrino-
gen, and an increase in partial thromboplastin time [28]. g
p
p
[
]
Recanalization therapy, such as thrombolysis with
recombinant tissue plasminogen activator (rtPA) and
mechanical thrombectomy, are methods with proven
efficacy in treatment of ischemic stroke, however their
efficacy and safety in cancer patients remain controversial. Still there is a growing number of research showing
that acute stroke in cancer patients can be treated
with rtPA, and active cancer should not be considered
as an absolute contraindication for rtPA use. In a number
of studies, it has been shown that intravenous thrombo-
lysis is not associated with a higher risk of bleeding in
cancer patients, and the neurological condition of such
patients improves faster [29, 30]. Current guidelines
for the treatment of acute stroke indicate that the use
of recanalization approaches, such as thrombolysis
and endovascular therapy, are not contraindicated to
cancer patients, but the response to recanalization р
у [
]
Реканалізаційна терапія, така як тромболізис реком-
бінантним тканинним активатором плазміногену (rTPA)
та механічна тромбектомія, є методами з доведеною
ефективністю в лікуванні ішемічного інсульту, але
їх ефективність та безпека хворих на рак залишаються
суперечливими. Cancer treatment and stroke risk Тромбоцити здатні окиснювати ліпопротеїни низької
щільності, які є головним фактором утворення атеро-
склеротичних бляшок, а також взаємодіють з нейтро-
філами і еозинофілами та сприяють утворенню, роз-
ширенню та тромбозу атеросклеротичних бляшок. Тромбоцити залучають запальні клітини до пухлин,
що сприяє зростанню пухлини. Крім того, прокоагулянт-
ний ефект раку сам по собі підвищує активність
тромбоцитів і знижує вироблення оксиду азоту в ендо-
телії, що посилює формування атеросклеротичної
бляшки та призводить до підвищення ризику серцево-
судинних подій [25–27]. Cancer treatment and stroke risk Cancer treatment and stroke risk
Although cancer can cause atherosclerosis through
various mechanisms, the most common are the effects
of anticancer drugs and radiation therapy. Cancer chemo-
therapy and radiation therapy are associated with
an increased risk of developing atherosclerosis and
cardiovascular disease.f Хоча рак може викликати атеросклероз через
різні механізми, найбільш поширеними є наслідки
використання протипухлинних препаратів і променевої
терапії. Хіміотерапія
і
променева
терапія
раку
асоціюються з підвищенням ризику розвитку атеро-
склерозу та серцево-судинних захворювань. Ionizing radiation affects not only cancerous, but
also non-cancerous cells, especially those that proli-
ferate rapidly, such as endothelial and bone marrow cells,
as well as local parenchymal cells within the irradiated
area. Cell cycle arrest, senescence, and apoptosis are
induced as a consequence of DNA damage. At high
doses, ionizing radiation can lead to depletion of paren-
chymal and vascular endothelial cells with both macro-
and microvascular effects. Radiotherapy has a direct
effect on cells in the pathogenesis of vascular damage
involving endothelial cells, which is an early sign of
radiation-induced vascular damage. This causes endo-
thelial damage with increased expression of interleukins
(eg, IL-6 and IL-8), intercellular adhesion molecules
(eg, ICAM-1), fibroblast growth factor, and stimulates Іонізуюче
випромінювання
впливає
не
лише
на ракові, а й на неракові клітини, особливо на ті,
що швидко проліферують, наприклад, клітини ендо-
телію та кісткового мозку, а також локальні парен-
хіматозні клітини в межах опроміненої ділянки. Зупинка
клітинного циклу, старіння та апоптоз індукуються
як наслідок пошкодження ДНК. У високих дозах
іонізуюче випромінювання може призвести до висна-
ження паренхіматозних і судинних ендотеліальних
клітин як з макро-, так і мікросудинними ефектами. Радіотерапія надає прямий вплив на клітини в пато-
генезі пошкодження судин із залученням ендотелі-
альних клітин, що є ранньою ознакою радіаційно-
індукованого ураження судин. Це викликає пошкод-
ження ендотелію з посиленням експресії інтерлейкінів Literature review 57 Огляд літератури Literature review ISSN 2708-7166 (Print)
ISSN 2708-7174 (Online) Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61
Ukrainian journal of radiology and oncology. 2022;30(4):52–61 Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61
Ukrainian journal of radiology and oncology. 2022;30(4):52–61 (наприклад, IL-6 та IL-8), міжклітинних молекул адгезії
(наприклад, ICAM-1), фактора росту фібробластів
і стимулює реакцію нейтрофілів із вторинним вивіль-
ненням цитокінів. Крім того, радіація може індукувати
апоптоз через p53 відповідь на пошкодження ДНК
або апоптоз, активований мітохондріями. Огляд літератури Діагностичні та терапевтичні підходи
при рак-асоційованому інсульті З’являється все більше досліджень,
які доводять, що гострий інсульт у хворих на рак можна
лікувати rTPA, і активний рак не слід розглядати
як абсолютне протипоказання для використання rTPA. В низці досліджень було продемонстровано, що
внутрішньовенний тромболізис не пов’язаний з вищим
ризиком кровотечі у хворих на рак, а неврологічний
стан таких пацієнтів покращується швидше [29, 30]. Сучасні рекомендації щодо терапії гострого інсульту
свідчать, що застосування реканалізаційних підходів,
таких як тромболізис та ендоваскулярна терапія, Literature review Literature review Огляд літератури Огляд літератури 58 ISSN 2708-7166 (Print)
ISSN 2708-7174 (Online) Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61
Ukrainian journal of radiology and oncology. 2022;30(4):52–61 не протипоказані для пацієнтів з раком, однак відпо-
відь на реканалізаційну терапію може відрізнятися
у хворих на рак, та у хворих без раку [31]. У систем-
ному огляді та мета-аналізі також було підтверджено,
що системний рак не є абсолютним протипоказанням
для внутрішньовенного тромболітичного лікування,
а ефективність та безпека не відрізняються порівняно
з пацієнтами без раку [32]. Ефективність механічної
тромбектомії при рак-асоційованому інсульті зали-
шається не досить відомою. Однак дослідження
продемонструвало,
що
механічна
тромбектомія
є придатним методом його лікування, однак підвищен-
ня D-димеру і грубий неврологічний дефіцит можуть
бути предикторами несприятливого виходу [33]. therapy may differ between cancer patients and non-
cancer patients [31]. A systematic review and meta-
analysis also confirmed that systemic cancer is not
an absolute contraindication to intravenous thrombolytic
treatment, and efficacy and safety are not different
compared to non-cancer patients [32]. The effectiveness
of
mechanical
thrombectomy
in
cancer-associated
stroke remains poorly known. However, the study has
demonstrated that mechanical thrombectomy is a suitable
method of its treatment, however the elevated D-dimer
and gross neurological deficit may be predictors of
adverse outcome [33]. In addition to revascularization therapy, low molecular
weight heparins are usually used to treat ischemic stroke
in cancer patients. When comparing low-molecular-
weight heparins with new oral anticoagulants, similar
results were obtained in terms of clinical outcome,
mortality, and the risk of hemorrhagic complications [34]. Крім реваскуляризаційної терапії для лікування
ішемічного інсульту у хворих на рак зазвичай застосо-
вуються низькомолекулярні гепарини. При порівнянні
низькомолекулярних гепаринів з новими перораль-
ними антикоагулянтами отримані схожі результати
відносно клінічного виходу, смертності та ризику
геморагічних ускладнень [34]. REFERENCES 1. Global, regional, and national burden of diseases and injuries for
adults 70 years and older: systematic analysis for the Global Burden
of Disease 2019 Study. British medical journal. 2022;376. DOI:
https://doi.org/10.1136/bmj-2021-068208 1. Global, regional, and national burden of diseases and injuries for
adults 70 years and older: systematic analysis for the Global Burden
of Disease 2019 Study. British medical journal. 2022;376. (In English). DOI: https://doi.org/10.1136/bmj-2021-068208 p
g
j
2. Kuan A.S., Teng C.J., Wu H.H., Su V.Y., Chen Y.T. et al. Risk of
ischemic stroke in patients with ovarian cancer: A nation wide
population based study. BMC medicine. 2014. Vol. 12. 53 р. DOI:
https://doi.org/10.1186/1741-7015-12-53 2. Kuan A.S., Teng C.J., Wu H.H., Su V.Y., Chen Y.T. et al. Risk of
ischemic stroke in patients with ovarian cancer: A nation wide
population based study. BMC medicine. 2014. Vol. 12. 53 р. DOI:
https://doi.org/10.1186/1741-7015-12-53 2. Kuan AS, Teng CJ, Wu HH, Su VY, Chen YT et al. Risk of ischemic
stroke in patients with ovarian cancer: A nationwide population-based
study. BMC medicine. 2014;12:53. (In English). DOI: https://doi.org/
10.1186/1741-7015-12-53 p
g
3. Navi B.B., Reiner A.S., Kamel H., Iadecola C., Elkind M.S., Panageas K.S.,
DeAngelis L.M. Association between incident cancer and subsequent
stroke. Annals of neurology. 2015. Vol. 77(2). Р. 291–300. DOI:
https://doi.org/10.1002/ana.24325 3. Navi BB, Reiner AS, Kamel H, Iadecola C, Elkind MS, Panageas KS,
DeAngelis LM. Association between incident cancer and subsequent
stroke. Annals of neurology. 2015;77(2):291–300. (In English). DOI:
https://doi.org/10.1002/ana.24325 4. Zöller B, Sundquist J, Sundquist K. Risk of haemorrhagic and ischaemic
stroke in patients with cancer: A nationwide follow-up study from
Sweden. European
Journal
of
Cancer. 2012;48(12):1875–83. (In English). DOI: https://doi.org/10.1016/j.ejca.2012.01.005 4. Zöller B., Sundquist J., Sundquist K. Risk of haemorrhagic and
ischaemic stroke in patients with cancer: A nationwide follow-up
study from Sweden. European Journal of Cancer. 2012. Vol. 48(12). Р. 1875–1883. DOI: https://doi.org/10.1016/j.ejca.2012.01.005 5. Donato J., Campigotto F., Uhlmann E. ., Coletti E., Neuberg D.,
Weber G.M., Zwicker J.I. Intracranial hemorrhage in patients with
brain metastases treated with therapeutic enoxaparin: A matched
cohort
study. Blood. 2015. Vol. 126(4). Р. 494–499. DOI:
https://doi.org/10.1182/blood-2015-02-626788 5. Donato J, Campigotto F, Uhlmann EJ, Coletti E, Neuberg D, Weber GM,
Zwicker JI. Intracranial hemorrhage in patients with brain metastases
treated with therapeutic enoxaparin: A matched cohort study. Blood. 2015;126(4):494–9. (In English). DOI: https://doi.org/10.1182/blood-
2015-02-626788 6. p
g
6. CONCLUSIONS Поєднання раку і мозкового інсульту є актуальною
проблемою клінічної медицини в усьому світі. Рак
призводить до збільшення ризику інсульту внаслідок
специфічних патофізіологічних механізмів. Ці меха-
нізми є множинними і складними, однак найбільш
відомим та поширеним є ракова внутрішньосудинна
коагулопатія, яка має специфічні лабораторні біо-
маркери. Численні дослідження продовжують спрямо-
вуватись на вивчення факторів, пов’язаних з коагуло-
патією у пацієнтів, які перенесли інсульт, особливості
раку та оцінки біологічних маркерів внутрішньосу-
динної коагулопатії, що викликає інсульт, а також
моніторинг ефектів антикоагулянтної терапії у пацієнтів
із рак-асоційованим інсультом. The combination of cancer and cerebral stroke is
an urgent problem of clinical medicine all over the world. Cancer leads to an increased risk of stroke due to specific
pathophysiological mechanisms. These mechanisms are
multiple and complex, but the most well-known and
widespread is cancer intravascular coagulopathy, which
has specific laboratory biomarkers. Numerous studies
continue investigating the factors associated with coagulo-
pathy in stroke patients, cancer characteristics, and
evaluation
of
biological
markers
of
stroke-causing
intravascular coagulopathy, as well as monitoring the
effects of anticoagulation therapy in patients with
cancer-associated stroke. REFERENCES Schwarzbach C.J., Schaefer A., Ebert A., Held V., Bolognese M.,
Kablau M., Hennerici M.G. Stroke and cancer: The importance
of cancer-associated hypercoagulation as a possible stroke etiology. Stroke. 2012. Vol. 43(11). Р. 3029–3034. DOI: https://doi.org/10.1161/
STROKEAHA.112.658625 12. Schwarzbach CJ, Schaefer A, Ebert A, Held V, Bolognese M, Kablau M,
Hennerici MG. Stroke and cancer: The importance of cancer-
associated hypercoagulation as a possible stroke etiology. Stroke. 2012;43(11):3029–34. (In
English). DOI:
https://doi.org/10.1161/
STROKEAHA.112.658625 13. Romeiro AC, Valadas A, Marques J. Acute ischemic stroke on
cancer patients, a distinct etiology? A case-control study. Acta médica
portuguesa. 2015;28(5):613–8. (In English). DOI: https://doi.org/
10.20344/amp.6156 13. RomeiroA.C., Valadas A., Marques J. Acute ischemic stroke on
cancer patients, a distinct etiology? A case-control study. Acta
médica portuguesa. 2015. Vol. 28(5). Р. 613–618, DOI: https://doi.org/
10.20344/amp.6156 p
14. Chung JW, Cho YH, Ahn MJ, Lee MJ et al. Association of cancer
cell type and extracellular vesicles with coagulopathy in patients
with lung cancer and stroke. Stroke. 2018;49(5):1282–5. (In English). DOI: https://doi.org/10.1161/STROKEAHA.118.020995 14. Chung J.W., Cho Y.H., Ahn M.J., Lee M.J. et al. Association of cancer
cell type and extracellular vesicles with coagulopathy in patients
with lung cancer and stroke. Stroke. 2018. Vol. 49(5). Р. 1282–1285. DOI: https://doi.org/10.1161/STROKEAHA.118.020995 15. Bang O.Y., Chung J.W., Cho Y.H., Oh M.J., Seo W.K., Kim G.M. et al. Circulating DNAs, a marker of neutrophil extracellular traposis
and
cancer-related
stroke:
the
OASIS-Cancer
Study. Stroke. 2019. Vol. 50(10). Р. 2944–2947. DOI: https://doi.org/10.1161/
STROKEAHA.119.026373 15. Bang OY, Chung JW, Cho YH, Oh MJ, Seo WK, Kim GM et al. Circulating DNAs, a marker of neutrophil extracellular traposis
and
cancer-related
stroke:
the
OASIS-Cancer
Study. Stroke. 2019;50(10):2944–7. (In
English). DOI:
https://doi.org/10.1161/
STROKEAHA.119.026373 16. Xie X., Chen L., Zeng J., Qin C., Cheng D., Wei X. Clinical features
and biological markers of lung cancer-associated stroke. The Journal
of international medical research. 2016. Vol. 44(6). Р. 1483–1491. DOI: https://doi.org/10.1177/0300060516666398 16. Xie X, Chen L, Zeng J, Qin C, Cheng D, Wei X. Clinical features
and biological markers of lung cancer-associated stroke. The Journal
of international medical research. 2016;44(6):1483–91. (In English). DOI: https://doi.org/10.1177/0300060516666398 17. Bang O.Y., Chung J.W., Lee M.J., Seo W-K., Kim G.M., Ahn M.J. OASIS-Cancer
Study
Investigators. Cancer-Related
Stroke:
An Emerging Subtype of Ischemic Stroke with Unique Patho-
mechanisms. Journal of Stroke. 2020. Vol. 22(1). Р. 1–10. DOI:
https://doi.org/10.5853/jos.2019.02278 17. Bang OY, Chung JW, Lee MJ, Seo W-K, Kim GM, Ahn MJ. OASIS-Cancer
Study
Investigators. REFERENCES Aarnio K., Joensuu H., Haapaniemi E., Melkas S., Kaste M.,
Tatlisumak T., Putaala J. Cancer in young adults with ischemic
stroke. Stroke. 2015. Vol. 46(6). Р. 1601–1606. DOI:
https://doi.org/10.1161/STROKEAHA.115.008694 6. Aarnio K, Joensuu H, Haapaniemi E, Melkas S, Kaste M,
Tatlisumak T, Putaala J. Cancer in young adults with ischemic
stroke. Stroke. 2015;46(6):1601–6. (In English). DOI: https://doi.org/
10.1161/STROKEAHA.115.008694 p
g
7. Navi B.B., Reiner A.S., Kamel H., Iadecola C., Okin P.M.,
Tagawa S.T. еt al. Arterial thromboembolic events preceding the
diagnosis of cancer in older persons. Blood. 2019. Vol. 133(8). Р. 781–789. DOI: https://doi.org/10.1182/blood-2018-06-860874 7. Navi BB, Reiner AS, Kamel H, Iadecola C, Okin PM, Tagawa ST еt al. Arterial thromboembolic events preceding the diagnosis of cancer
in older persons. Blood. 2019;133(8):781–9. (In English). DOI:
https://doi.org/10.1182/blood-2018-06-860874 Literature review 59 Огляд літератури Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61
Ukrainian journal of radiology and oncology. 2022;30(4):52–61 Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61
Ukrainian journal of radiology and oncology. 2022;30(4):52–61 ISSN 2708-7166 (Print)
ISSN 2708-7174 (Online) 8. Sanossian N, Djabiras C, Mack WJ, Ovbiagele B. Trends in cancer
diagnoses among in patients hospitalized with stroke. Journal of Stroke
and Cerebrovascular Diseases. 2013;22(7):1146–50. (In English). DOI: https://doi.org/10.1016/j.jstrokecerebrovasdis.2012.11.016 8. Sanossian N., Djabiras C., Mack W. J., Ovbiagele B. Trends in cancer
diagnoses among in patients hospitalized with stroke. Journal of
Stroke and Cerebrovasc Diseases. 2013. Vol. 22(7). Р. 1146–1150. DOI: https://doi.org/10.1016/j.jstrokecerebrovasdis.2012.11.016 p
g
j j
9. Dardiotis E., Aloizou A-M., Markoula S., Siokas V., Tsarouhas K. еt al. Cancer-associated
stroke:
Pathophysiology,
detection
and
management (Review). International journal of oncology. 2019. Vol. 54. Р. 779–796. DOI: https://doi.org/10.3892/ijo.2019.4669 9. Dardiotis E, Aloizou A-M, Markoula S, Siokas V, Tsarouhas K еt al. Cancer-associated stroke: Pathophysiology, detection and management
(Review). International
journal
of
oncology. 2019;54:779–96. (In English). DOI: https://doi.org/10.3892/ijo.2019.4669 10. Dearborn JL, Urrutia VC, Zeiler SR. Stroke and cancer: A complicated
relationship. Journal of neurology & translational neuroscience. 2014;2(1):1039. (In English). 10. Dearborn J.L., Urrutia V.C., Zeiler S.R. Stroke and cancer:
A complicated relationship. Journal of neurology & translational
neuroscience. 2014. Vol. 2(1). 1039 р. 11. Liu J., Frishman W.H. Nonbacterial Thrombotic Endocarditis: Pathogene-
sis, Diagnosis, and Management. Cardiology in review. 2016. Vol. 24(5). Р. 244–247. DOI: https://doi.org/10.1097/CRD.0000000000000106 11. Liu J, Frishman WH. Nonbacterial Thrombotic Endocarditis: Pathogenesis,
Diagnosis, and Management. Cardiology in review. 2016;24(5):244–7. (In English). DOI: https://doi.org/10.1097/CRD.0000000000000106 12. REFERENCES Cancer-Related
Stroke:
An Emerging Subtype of Ischemic Stroke with Unique Patho-
mechanisms. Journal of Stroke. 2020;22(1):1–10. (In English). DOI: https://doi.org/10.5853/jos.2019.02278 18. Abdelsalam M, Abu-Hegazy M, El-Hadaad HA, Wahba H, Egila H,
Esmael A. Pathophysiology, Mechanism, and Outcome of Ischemic
Stroke in Cancer Patients. Journal of stroke and cerebrovascular
diseases. 2020;29(11):105299. (In English). DOI: https://doi.org/
10.1016/j.jstrokecerebrovasdis.2020.105299 18. Abdelsalam M., Abu-Hegazy M., El-Hadaad H.A., Wahba H., Egila H.,
Esmael A. Pathophysiology, Mechanism, and Out come of Ischemic
Strokein Cancer Patients. Journal of stroke and cerebrovascular
diseases. 2020. Vol. 29(11). 105299 р. DOI: https://doi.org/10.1016/
j.jstrokecerebrovasdis.2020.105299 19. Cocho D., Gendre J., Boltes A., Espinosa J., Ricciardi A. C., Pons J.,
Jimenez M., Otermin P. Predictors of occult cancer in acute ischemic
stroke patients. Journal of Stroke and Cerebrovascular Diseases. 2015. Vol. 24(6). Р. 1324–1328. DOI: https://doi.org/10.1016/
j.jstrokecerebrovasdis.2015.02.006 19. Cocho D, Gendre J, Boltes A, Espinosa J, Ricciardi AC, Pons J,
Jimenez M, Otermin P. Predictors of occult cancer in acute ischemic
stroke patients. Journal of Stroke and Cerebrovascular Diseases. 2015;24(6):1324–8. (In
English). DOI:
https://doi.org/10.1016/
j.jstrokecerebrovasdis.2015.02.006 20. Selvik H.A., Bjerkreim A.T., Thomassen L., Waje-Andreassen U.,
Naess H., Kvistad C.E. When to Screen Ischaemic Stroke Patients
for Cancer. Cerebrovascular Diseases. 2018. Vol. 45(1–2). Р. 42–47. DOI: https://doi.org/10.1159/000484668 20. Selvik HA, Bjerkreim AT, Thomassen L, Waje-Andreassen U,
Naess H, Kvistad CE. When to Screen Ischaemic Stroke Patients for
Cancer. Cerebrovascular Diseases. 2018;45(1–2):42–7. (In English). DOI: https://doi.org/10.1159/000484668 g
21. Mayer F.J., Binder C.J., Wagner O.F., Schillinger M., Minar E. et al. Combine deffects of inflammatory status and carotid atherosclerosis. Stroke. 2016. Vol. 47(12). Р. 2952–2958. DOI: https://doi.org/10.1161/
STROKEAHA.116.013647 21. Mayer FJ, Binder CJ, Wagner OF, Schillinger M, Minar E et al. Combined effects of inflammatory status and carotid atherosclerosis. Stroke. 2016;47(12):2952–8. (In English). DOI: https://doi.org/10.1161/
STROKEAHA.116.013647 22. Wijeratne T., Menon R., Sales C., Karimi L., Crewther S. Carotid-
artery stenosis and inflammatory biomarkers: the role of in flammation-
induced immunological responses affecting the vascular systems. Annals of Translational Medicine. 2020. Vol. 8(19). 1276 р. DOI:
https://doi.org/10.21037/atm-20-4388 22. Wijeratne T, Menon R, Sales C, Karimi L, Crewther S. Carotid artery
stenosis and inflammatory biomarkers: the role of inflammation-induced
immunological responses affecting the vascular systems. Annals
of Translational Medicine. 2020;8(19):1276. (In English). DOI:
https://doi.org/10.21037/atm-20-4388 23. Darby S.C., Ewertz M., McGale P. et al. Risk of ischemic heart disease
in women after radiotherapy for breast cancer. New England Journal
of Medicine. 2013. Vol. 368. Р. 987–998. DOI: https://doi.org/
10.1056/NEJMoa1209825 23. 8.
Sanossian N., Djabiras C., Mack W. J., Ovbiagele B. Trends in cancer
diagnoses among in patients hospitalized with stroke. Journal of
Stroke and Cerebrovasc Diseases. 2013. Vol. 22(7). Р. 1146–1150.
DOI: https://doi.org/10.1016/j.jstrokecerebrovasdis.2012.11.016 ISSN 2708-7166 (Print)
ISSN 2708-7174 (Online) ISSN 2708-7166 (Print)
ISSN 2708-7174 (Online) 28. Nam KW, Kim CK, Kim TJ, An SJ, Demchuk AM, Kim.Y et al. D-dimer as a predictor of early neurologic deterioration in cryptogenic
stroke
with
active
cancer. European
Journal
of
Neurology. 2017;24(1):205–11. (In English). DOI: https://doi.org/10.1111/ene.13184 28. Nam K.W., Kim C.K., Kim T.J., An S.J., Demchuk A.M., Kim.Y. et al. D-dimer as a predictor of early neurologic deterioration in cryptogenic
stroke with active cancer. European Journal of Neurology. 2017. Vol. 24(1). Р. 205–211. DOI: https://doi.org/10.1111/ene.13184 ( )
g
29. Sobolewski P., Brola W., Szczuchniak W., Fudala Mand Sobota A. Safety of intravenous thrombolysis for acute ischaemic stroke including
concomitant neoplastic diseasesufferers – experience from Poland. International journal of clinical practice. 2015. Vol. 69(6). Р. 666–673. DOI: https://doi.org/10.1111/ijcp.12586 29. Sobolewski P, Brola W, Szczuchniak W, Fudala Mand Sobota A. Safety of intravenous thrombolysis for acute ischaemic stroke including
concomitant neoplastic disease sufferers – experience from Poland. International journal of clinical practice. 2015;69(6):666–73. (In English). DOI: https://doi.org/10.1111/ijcp.12586 g
j
30. Murthy S.B., Moradiya Y., Shah S., Shastri A., Bershad E.M.,
Suarez J.I. In-hospital outcomes of thrombolysis for acute ischemic
stroke in patients with primary brain tumors. Journal of clinical
neuroscience. 2015. Vol.22(3). Р. 474–478. DOI: https://doi.org/
10.1016/j.jocn.2014.09.016 30. Murthy SB, Moradiya Y, Shah S, Shastri A, Bershad EM, Suarez JI. In-hospital outcomes of thrombolysis for acute ischemic stroke
in patients with primary brain tumors. Journal of clinical neuro-
science. 2015;22(3):474–8. (In English). DOI: https://doi.org/10.1016/
j.jocn.2014.09.016 j j
31. Power W.J., Rabinstein A.A., Ackerson T. et al. Guidelines for the
Early Management of Patients with Acute Ischemic Stroke. Guideline
for Healthcare Professionals from the American Heart Association/Ame-
rican Stroke Association. Stroke. 2018. Vol. 49(3). Р. e46–e234. DOI: https://doi.org/10.1161/STR.0000000000000158fi j j
31. Power WJ, Rabinstein AA, Ackerson T et al. Guidelines for the Early
Management of Patients with Acute Ischemic Stroke. Guideline
for Healthcare Professionals from the American Heart Association/
American Stroke Association. Stroke. 2018;49(3):e46–234. (In English). DOI: https://doi.org/10.1161/STR.0000000000000158fi 32. Huang S., Lu X., Tang L. V., Hu Y. Efficacy and safety of intravenous
thrombolysis for acute ischemic stroke in cancer patients: a systemic
review and meta-analysis. American Journal of Translational Research. 2020. Vol. 12(8). Р. 4795–4806. 32. Huang S, Lu X, Tang LV, Hu Y. Efficacy and safety of intravenous
thrombolysis for acute ischemic stroke in cancer patients: a systemic
review and meta-analysis. American Journal of Translational Research. 2020;12(8):4795–806. (In English).f 33. ISSN 2708-7166 (Print)
ISSN 2708-7174 (Online) Lee E-J, Bae J, Jeong H-B, Lee EJ, Jeong HY, Yoon B-W. Effectiveness
of mechanical thrombectomy in cancer-related stroke and associated
factors with unfavorable outcome. BMC Neurology. 2021;21:57. (In English). DOI: https://doi.org/10.1186/s12883-021-02086-y 33. Lee E-J., Bae J., Jeong H-B., Lee E.J., Jeong H.Y., Yoon B-W. Effectiveness of mechanical thrombectomy in cancer-related stroke
and associated factors with unfavorable outcome. BMC Neurology. 2021. Vol. 21. 57 р. DOI: https://doi.org/10.1186/s12883-021-02086-y 34. Nam KE, Kim CK, Kim T-J, An SJ, Oh K, Ko SB, Yoon BW. Treatment
of Cryptogenic Stroke with Active Cancer with a New Oral
Anticoagulant. Journal of Stroke and Cerebrovascular Diseases. 2017;26(12):2976–80. (In English). DOI: https://doi.org/10.1016/
j.jstrokecerebrovasdis.2017.07.029 34. Nam K.E., Kim C.K., Kim T-J., An S.J., Oh K., Ko S.B., Yoon B.W. Treatment of Cryptogenic Stroke with Active Cancer with a New
Oral Anticoagulant. Journal of Stroke and Cerebrovascular Diseases. 2017. Vol. 26(12). Р. 2976–2980. DOI: https://doi.org/10.1016/
j.jstrokecerebrovasdis.2017.07.029 Конфлікт інтересів Автор заявляє про відсутність конфлікту інтересів та власної
фінансової зацікавленості в підготуванні даної статті. The author states no conflict of interest or own financial incentive
associated with publishing the paper. Перспективи подальших досліджень Further studying the multiple mechanisms that associate
cancer and stroke will make it possible to intentionally develop
the strategies of the treatment and prevention of cancer-
associated stroke. Подальше вивчення множинних механізмів, які поєднують
рак та мозковой інсульт дозволить цілеспрямовано розроб-
ляти підходи до лікування та профілактики рак-асоційова-
ного інсульту. Conflict of interest REFERENCES Darby SC, Ewertz M, McGale P et al. Risk of ischemic heart disease
in women after radiotherapy for breast cancer. New England Journal of
Medicine. 2013;368:987–98. (In English). DOI: https://doi.org/10.1056/
NEJMoa1209825 24. Li Q, Liu F, Tang Y, Lee S, Lang C, Bai L et al. The distribution of
cardiovascular-related comorbidities in different adult-onset cancers
and related risk factors: analysis of 10 year retrospective data. Frontier of Cardiovascular Medicine. 2021;8:695454. (In English). DOI:
https://doi.org/10.3389/fcvm.2021.695454 24. Li Q., Liu F., Tang Y., Lee S., Lang C., Bai L. et al. The distribution
of
cardiovascular-related
comorbidities
in
different
adult-onset
cancers and related risk factors: analysis of 10 year retrospective data. Frontier of Cardiovascular Medicine. 2021. Vol. 8. 695454 р. DOI:
https://doi.org/10.3389/fcvm.2021.695454 25. Min S.S., Wierzbicki A.S. Radiotherapy, chemotherapy and athero-
sclerosis. Current opinion in cardiology. 2017. Vol. 32(4). Р. 441–447. DOI: https://doi.org/10.1097/HCO.0000000000000404 f 25. Min SS, Wierzbicki AS. Radiotherapy, chemotherapy and athero-
sclerosis. Current opinion in cardiology. 2017;32(4):441–7. (In English). DOI: https://doi.org/10.1097/HCO.0000000000000404f 26. Lysov Z, Dwivedi DJ, Gould TJ, Liaw PC. Procoagulant effects of
lung cancer chemotherapy: impact on microparticles and cell-free
DNA. Blood Coagul Fibrinolysis. 2017;28(1):72–82. (In English). DOI: https://doi.org/10.1097/MBC.0000000000000546 26. Lysov Z., Dwivedi D.J., Gould T.J., Liaw P.C. Procoagulant effects
of lung cancer chemotherapy: impact on microparticles and cell-free
DNA. Blood Coagul Fibrinolysis. 2017. Vol. 28(1). Р. 72–82. DOI:
https://doi.org/10.1097/MBC.0000000000000546 27. Nardi E., Prastaro M., Santoro C. Vascular Damage Induced by
Chemotherapy and Radio therapy in Oncological Patients. American
Journal of Biomedical Science & Research. 2022. Vol. 15(5). DOI:
https://doi.org/10.34297/AJBSR.2022.15.002146 27. Nardi E, Prastaro M, Santoro C. Vascular Damage Induced by
Chemotherapy and Radiotherapy in Oncological Patients. American
Journal of Biomedical Science & Research. 2022;15(5). (In English). DOI: https://doi.org/10.34297/AJBSR.2022.15.002146 Literature review 60 Огляд літератури Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61
Ukrainian journal of radiology and oncology. 2022;30(4):52–61 Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61
Ukrainian journal of radiology and oncology. 2022;30(4):52–61 INFORMATION ABOUT AUTHORS Dubenko Olha Yevheniivna – Doctor of Medical Sciences,
Professor, Senior Scientist of Radiation Pathology and Palliative
Care Department of State Organization «Grigoriev Institute
for Medical Radiology and Oncology of the National Academy
of Medical Sciences of Ukraine»; Professor of Neurology
Department of Kharkiv Medical Academy of Postgraduate
Education; 58, Amosov Str., Kharkiv, Ukraine, 61176; Дубенко Ольга Євгеніївна – доктор медичних наук,
професор, старший науковий співробітник відділення проме-
невої патології та паліативної медицини Державної установи
«Інститут медичної радіології та онкології ім. С.П. Григор’єва
Національної академії медичних наук України»; професор
кафедри неврології Харківської медичної академії після-
дипломної освіти, вул. Амосова, буд. 58, Харків , Україна, 61176
e-mail: olgadubenko05@gmail.com e-mail: olgadubenko05@gmail.com g
@g
Phone: +38 (050) 660-14-42 Внесок автора: інформаційний пошук та аналіз
наукових публікацій за темою статті, написання
тексту статті. Author contributions: information search and analysis
of scientific publications on the subject of the article,
writing the article. Прийнято до друку
Accepted for printing
08.12.2022
Опубліковано
Published
10.12.2022 Прийнято до друку
Accepted for printing
08.12.2022
Опубліковано
Published
10.12.2022 Отримано після рецензування
Received after review
27.10.2022 Отримано після рецензування
Received after review
27.10.2022 61 Literature review Огляд літератури
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https://www.frontiersin.org/articles/10.3389/fnut.2023.1103303/pdf
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Investigating the causal association between branched-chain amino acids and Alzheimer's disease: A bidirectional Mendelian randomized study
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Frontiers in nutrition
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TYPE Original Research
PUBLISHED 31 March 2023
DOI 10.3389/fnut.2023.1103303 TYPE Original Research
PUBLISHED 31 March 2023
DOI 10.3389/fnut.2023.1103303 TYPE Original Research
PUBLISHED 31 March 2023
DOI 10.3389/fnut.2023.1103303 OPEN ACCESS EDITED BY
Lais Bhering Martins,
University of Texas Health Science Center at
Houston, United States
REVIEWED BY
Wen Liu,
Capital Medical University, China
Baruh Polis,
Yale University, United States
Andrew C. Shin,
Texas Tech University, United States
*CORRESPONDENCE
Gang-yu Ding
jonathandin@126.com
Hui-dong Tang
thd10495@rjh.com.cn
† Xiao-hang Qian1,2,3†, Xiao-li Liu4†, Bin Zhang4, Yuan Lin5,
Jian-hua Xu6, Gang-yu Ding6* and Hui-dong Tang1,2,3* 1Department of Geriatrics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai,
China, 2Medical Center on Aging of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine,
Shanghai, China, 3Department of Neurology and Institute of Neurology, Ruijin Hospital, Shanghai Jiao
Tong University School of Medicine, Shanghai, China, 4Department of Neurology, Shanghai University of
Medicine and Health Sciences Afliated Sixth People’s Hospital South Campus, Shanghai, China,
5Department of Gastroenterology, Jiading District Central Hospital Afliated Shanghai University of
Medicine and Health Sciences, Shanghai, China, 6Department of Neurology, Jiading District Central
Hospital Afliated Shanghai University of Medicine and Health Sciences, Shanghai, China †These authors have contributed equally to this
work SPECIALTY SECTION
This article was submitted to
Nutrition, Psychology and Brain Health,
a section of the journal
Frontiers in Nutrition
RECEIVED 20 November 2022
ACCEPTED 06 March 2023
PUBLISHED 31 March 2023
CITATION
Qian X-h, Liu X-l, Zhang B, Lin Y, Xu J-h,
Ding G-y and Tang H-d (2023) Investigating the
causal association between branched-chain
amino acids and Alzheimer’s disease: A
bidirectional Mendelian randomized study. Front. Nutr. 10:1103303. doi: 10.3389/fnut.2023.1103303 Background: There are many metabolic pathway abnormalities in Alzheimer’s
disease (AD). Several studies have linked branched-chain amino acid (BCAA)
metabolism disorders with AD but have not obtained consistent results. The
purpose of this study is to explore the causal association between BCAA
concentration and the risk of AD. Methods: A bidirectional Mendelian randomized (MR) study was applied to explore
the causal efect between BCAA level and the risk of AD. Genetic instrumental
variables from the genome-wide association study (GWAS) of serum BCAA levels
[total BCAAs (115,047 participants), valine (115,048 participants), leucine (115,074
participants), and isoleucine (115,075 participants)] from the UK Biobank and
AD (21,982 AD cases and 41,944 controls) from the International Genomics of
Alzheimer’s Project were applied to explore the causal efect through the inverse
variance-weighted (IVW) method, MR-Egger, and weighted median, accompanied
by multiple pluripotency and heterogeneity tests. © 2023 Qian, Liu, Zhang, Lin, Xu, Ding and
Tang. Alzheimer’s disease, branched-chain amino acids, valine, leucine, isoleucine, Mendelian
randomized study Frontiers in Nutrition KEYWORDS Data sources and selection of genetic
instruments Branched-chain amino acids (BCAAs), including leucine (Leu),
isoleucine (Ile), and valine (Val), are three types of essential amino
acids in the human diet (9). They are not only involved in
protein synthesis but also possess various metabolic pathways (10,
11). Altered BCAA metabolism has been shown to be associated
with AD in an increasing number of studies (12). However,
different studies showed controversial and mixed results. For
instance, a previous study of the cerebrospinal fluid (CSF) and
plasma amino acid composition has demonstrated a significant
reduction of valine in AD patients compared to healthy controls
(HC) (13). In a study of sporadic AD patients without receiving
any medication, alterations of 23 metabolites were detected,
including significantly decreased valine levels (14). In addition, a
longitudinal study in APP/PS1 transgenic mice involving profiling
of the brain and the plasma metabolome has found seriously
disturbed polyamines and BCAA metabolism (15). Notably, the
plasma levels of valine were shown to be significantly reduced
in AD mice. In a more recent study, lower plasma valine level
was shown to correlate with the rate of cognitive decline (16). Nonetheless, there were elevated BCAA concentrations in the
serum of AD patients in a small sample study (17). Similarly,
elevated isoleucine levels have been observed in patients with mild
cognitive impairment (MCI) (18). These heterogeneous results
may be related to the susceptibility of metabolites influenced by
multiple factors, such as lifestyle and diet, immune response,
genetic variations, and gut microbiota (19). These uncontrollable
confounding factors make it difficult to distinguish symptoms from
causes. Therefore, the use of more reliable research methods will
help to elucidate the causal association between BCAA levels and
the risk of AD. The genetic summary statistics of AD (21,982 AD cases
and 41,944 controls) were accessed from the International
Genomics of Alzheimer’s Project (IGAP), which was composed
of four consortia: Cohorts for Heart and Aging Research in
Genomic Epidemiology Consortium (CHARGE), the European
Alzheimer’s
Disease
Initiative
(EADI),
Alzheimer’s
Disease
Genetics Consortium (ADGC), and Genetic and Environmental
Risk in AD/Defining Genetic, Polygenic and Environmental Risk
for Alzheimer’s Disease Consortium (GERAD/PERADES) (22). Study design overview Alzheimer’s disease (AD), the main type of dementia, becomes
one of the most serious public health threats in the world (1). It is estimated that there are currently more than 50 million
people worldwide with dementia, which is expected to triple in
2050 (2). Among them, AD accounts for 60–80% of dementia
(3). Typical pathological features of AD include extracellular
amyloid-β (Aβ) plaques accumulated by Aβ and intracellular
neurofibrillary tangles formed by phosphorylated tau (4). However,
there is still a lack of accurate explanation of the pathogenesis
and effective disease-modifying treatment for AD (5). Increased
evidence suggests that the disruption of various metabolic pathways
is another important feature of AD (6, 7). Metabolites are the
end biochemical products of various biological pathways, such as
amino acids, peptides, lipids, and nucleic acids (8). They can reflect
the alteration in the complex biological pathways involved in AD
caused by the interaction of genetic, environmental, and behavioral
factors (6). We performed a bidirectional Mendelian randomized study
to assess the causal effect between BCAA levels and the risk
of AD (Figure 1). The bidirectional Mendelian randomization
study was built on the following three assumptions: First of
all, the instrumental variables (IVs) were not related to the
confounders. Second, there was a strong correlation between IVs
and exposure. Third, IVs can affect outcomes (AD) only through
exposure and not through other pathways. The analysis of this
study was based on genome-wide association study (GWAS)
data of BCAAs and Alzheimer’s disease in the public database. Therefore, ethics approval and consent are not required for
this study. Frontiers in Nutrition OPEN ACCESS 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. Results: The forward MR analysis showed that there was no causal efect of total
BCAAs (OR: 1.067, 95% CI: 0.838–1.358; p = 0.838), valine (OR: 1.106, 95% CI:
0.917–1.333; p = 0.292), leucine (OR: 1.096, 95% CI: 0.861–1.396; p = 0.659), and
isoleucine (OR: 1.457, 95% CI: 1.024–2.742; p = 0.037) levels on the risk of AD. The
reverse analysis showed that AD was related to reduced levels of total BCAAs (OR:
0.979, 95% CI: 0.989–0.990; p < 0.001), valine (OR: 0.977, 95% CI: 0.963–0.991;
p = 0.001), leucine (OR: 0.983, 95% CI: 0.973–0.994; p = 0.002), and isoleucine
(OR: 0.982, 95% CI: 0.971–0.992; p = 0.001). Conclusion: We provide robust evidence that AD was associated with a decreased
level of BCAAs, which can serve as a marker for early diagnosis of AD. Alzheimer’s disease, branched-chain amino acids, valine, leucine, isoleucine, Mendelian
randomized study 01 Frontiers in Nutrition frontiersin.org Qian et al. 10.3389/fnut.2023.1103303 frontiersin.org Mendelian randomized analysis method. In addition, MR-Egger and weighted median were applied
to verify the results of the IVW method. After removing SNPs, the
results showed that there were no casual effects on the levels of
total BCAAs (IVW, OR: 1.067, 95% CI: 0.838–1.358; p = 0.838),
valine (IVW, OR: 1.106, 95% CI: 0.917–1.333; p = 0.292), leucine
(IVW, OR: 1.096, 95% CI: 0.861–1.396; p = 0.659), and isoleucine
(IVW, OR: 1.457, 95% CI: 1.024–2.742; p = 0.037) on the risk of
AD (Figures 2A–D, Table 1). Similarly, the results for MR-Egger
and weighted median do not suggest an effect of BCAA levels on
the risk of AD. Based on these results, we found no causal effect of
BCAA levels on the risk of AD. Two-sample MR was applied to explore the causal effect
between BCAA levels and the risk of AD. The inverse variance-
weighted (IVW) method was applied to the standard MR
analysis. In addition, the MR-Egger and weighted median were
also performed to verify the causal effect (21, 26). Mendelian
randomization pleiotropy residual sum and outlier (MR-PRESSO)
global test was conducted to identify the horizontal pleiotropic
of IVs (26). Cochran’s Q statistic was applied to assess the
heterogeneity of IVW and MR-Egger (27). The MR-Egger intercept
test was also used to estimate the potential horizontal pleiotropy
of the MR results. In addition, the leave-one-out analysis was
also used to eliminate potential pleiotropy by assessing the effects
of a single IV on causal effect by removing each IV from the
IVW method (28). All analyses in this study were performed
through the “two-sample MR” and the “MR-PRESSO” packages in
R software (v3.60). As four types of exposures were analyzed, the
statistically significant threshold was set as P < 0.0125 after the
Bonferroni correction. Data sources and selection of genetic
instruments For BCAAs (total BCAAs, valine, leucine, and isoleucine),
the
GWAS
statistics
were
obtained
from
the
MRC
IEU
OpenGWAS
project
(https://gwas.mrcieu.ac.uk/),
including
115,047 participants for total BCAAs (GWAS ID “met-d-
Total_BCAA”),
115,048
participants
for
valine
(GWAS
ID
“met-d-Val”), 115,074 participants for leucine (GWAS ID “met-
d-Leu”), and 115,075 participants for isoleucine (GWAS ID
“met-d-Ile”) from the UK Biobank. All participants were of
European ancestry. Instrumental variables (IVs) for AD and BCAAs were extracted
under the same criteria. To be specific, the GWAS statistical
difference threshold of all relevant SNPs from each GWAS was set
to P < 5 × 10−8. The PLINK clumping algorithm was applied
to prune for the independence of SNPs in linkage disequilibrium
under the threshold of r2 < 0.001 in a 10,000 kb window (23). The palindromic SNPs with a minor allele frequency (MAF) of
<0.01 were excluded from the aforementioned instrument SNPs. The PhenoScanner GWAS database (http://phenoscanner.medschl. cam.ac.uk) was applied to remove possible confounding of the
exposure–outcome associations (24). In this study, gender, age,
diabetes, and cardiovascular disease were selected as confounders. The SNPs associated with them were excluded. Furthermore, the
F statistic was calculated to assess the strength of the selected
genetic variants (25). The SNPs used as IVs in this study are
presented in Supplementary Table S1. The F statistic is presented
in Supplementary Table S2. Mendelian randomization (MR) study is a methodology that
can be applied to explore the causal relationships between
exposures (risk factors) and outcomes (diseases) by using genetic
variants (20). The genetic variants are only related to the risk
factors but are not affected by any confounders (21). In this
study, a bidirectional Mendelian randomized approach was applied
to explore the causal effects between BCAA levels and the risk
of AD. 02 frontiersin.org Qian et al. 10.3389/fnut.2023.1103303 FIGURE 1
Summary of this bidirectional MR study between the level of BCAAs and the risk of AD in this study. SNP, single-nucleotide polymorphism; BCAAs,
branched-chain amino acids; AD, Alzheimer’s disease. FIGURE 1
Summary of this bidirectional MR study between the level of BCAAs and the risk of AD in this study. SNP, single-nucleotide polymorphism; BCAAs,
branched-chain amino acids; AD, Alzheimer’s disease. FIGURE 1
Summary of this bidirectional MR study between the level of BCAAs and the risk of AD in this study. SNP, single-nucleotide polymorphism; BCAAs,
branched-chain amino acids; AD, Alzheimer’s disease. Genetically predicted AD on the BCAA
levels In the reverse MR analysis, we explored the causal effect of
AD on the BCAA levels through IVW, MR-Egger, and weighted
median. Our investigation revealed that AD was related to a
decreased level of total BCAAs (IVW, OR: 0.979, 95% CI: 0.989–
0.990, p < 0.001), valine (IVW, OR: 0.977, 95% CI: 0.963–
0.991; p = 0.001), leucine (IVW, OR: 0.983, 95% CI: 0.973-
−0.994, p = 0.002), and isoleucine (IVW, OR: 0.982, 95%
CI: 0.971–0.992, p = 0.001) (Figures 2E–H, Table 2). Similarly,
the results of MR-Egger and weighted median were consistent
with the results of the IVW method. Taken together, these
results revealed that AD was obviously associated with decreased
BCAA levels. Frontiers in Nutrition frontiersin.org Sensitivity analyses to the risk of AD. Conversely, the reverse MR analysis suggests
that AD is significantly associated with decreased BCAA levels,
suggesting that AD is the cause but not the result of changed
BCAA levels. There were no obvious heterogeneities in the causal effect
between AD and BCAAs (Tables 1, 2). The intercept of the
MR-Egger
regression
and
MR-PRESSO
test
revealed
no
obvious horizontal pleiotropy (Tables 1, 2). In addition, the
included
instrumental
variables
show
apparent
symmetry
in
the
funnel
plot
to
exclude
the
directional
pleiotropy
(Supplementary Figure S1). Meanwhile, the leave-one-out analysis
showed all SNPs contributing to consistent causal estimates
(Supplementary Figure S2). The aforementioned analysis proves
the reliability of the results of the study. Several previous observational cohort studies have linked
BCAAs to AD. An earlier study found that there was reduced
levels of leucine and valine in the CSF of patients with AD, but
no statistically significant differences in plasma (13). In Alzheimer’s
Disease Neuroimaging Initiative-1 (ADNI-1) cohort, there was a
decreased serum valine level in AD patients compared to HC and
stable mild cognitive impairment (sMCI) patients (32). Similarly,
serum valine levels were lower in patients with progressive MCI
(pMCI) than in HC (32). More importantly, serum valine levels
in pMCI patients were negatively correlated with Tau and pTau
levels in CSF and could be used to predict the risk of progression
from MCI to AD (32). In a prospective study of eight cohorts,
BCAAs were found to be negatively associated with dementia and
the risk of AD (31). However, in another observational study,
serum isoleucine levels were elevated in dementia, compared with
HC (33). Similarly, elevated blood levels of BCAAs in patients
with AD were reported in a small sample study cohort (17). In APP/PS1 mice model, there was an increased plasma BCAA
level compared with wide-type mice (34). After high-fat feeding,
the plasma BCAA level was significantly upregulated in the WT
group, which was not observed in the APP/PS1 group (34). A high
saturated fat/glycemic index diet significantly increased CSF BCAA
levels in MCI patients but had no significant effect on the HC
group (35). Combined with the results of this study, we speculate Genetically predicted BCAA levels on the
risk of AD The causal effect of BCAA levels on the risk of AD was
conducted through the inverse variance-weighted (IVW) MR 03 frontiersin.org Qian et al. 10.3389/fnut.2023.1103303 FIGURE 2
Scatterplot of the genetic causal efect sbetween BCAAs level the risk of AD. (A) Total BCAAs on AD, (B) valine on AD, (C) leucine on AD, (D) isoleucine
on AD, (E) AD on total BCAAs, (F) AD on valine, (G) AD on leucine, (H) AD on isoleucine. BCAAs, branched-chain amino acids; AD, Alzheimer’s disease. Scatterplot of the genetic causal efect sbetween BCAAs level the risk of AD. (A) Total BCAAs on AD, (B) valine on AD, (C) leucin
on AD, (E) AD on total BCAAs, (F) AD on valine, (G) AD on leucine, (H) AD on isoleucine. BCAAs, branched-chain amino acids; A enetic causal efect sbetween BCAAs level the risk of AD. (A) Total BCAAs on AD, (B) valine on AD, (C) leucine on AD, (D) isoleucine
otal BCAAs, (F) AD on valine, (G) AD on leucine, (H) AD on isoleucine. BCAAs, branched-chain amino acids; AD, Alzheimer’s disease. Scatterplot of the genetic causal efect sbetween BCAAs level the risk of AD. (A) Total BCAAs on AD, (B) valine on AD, (C) leucine on AD, (D) isoleucine
on AD, (E) AD on total BCAAs, (F) AD on valine, (G) AD on leucine, (H) AD on isoleucine. BCAAs, branched-chain amino acids; AD, Alzheimer’s disease. Frontiers in Nutrition frontiersin.org P
’s dise
al ef
P M
−
−
M
−
− −
−
M
−
− Discussion Alzheimer’s disease, the most common type of dementia,
currently lacks disease-modifying treatment strategies. To make
matters worse, the current understanding of the pathogenesis
of AD is limited. As a type of essential amino acid, BCAAs
have been proven to be closely associated with AD (3, 29). The
alteration of BCAA concentration in AD patients is correlated with
disease progression and AD-related pathological features (30, 31). However, a causal association between AD and BCAA has not
been established. In this study, we attempt to explore the causal
effect between AD and BCAAs (total BCAAs, valine, leucine,
and isoleucine) using a bidirectional two-sample MR study. The
results do not show evidence that BCAA levels are causally related 04 frontiersin.org MR-egger
intercept
(p value)
MR-PRESSO
global test
(p value)
Cochran’s Q
(p value)
−0.011 (0.474)
0.135
10.312 (0.244)
0.001 (0.900)
0.451
8.131 (0.701)
−0.011 (0.482)
0.164
10.311 (0.325)
0.031 (0.435)
0.199
8.888 (0.113)
MR-egger
intercept
(p value)
MR-PRESSO
global test
(p value)
Cochran’s Q
(p value)
0.001 (0.632)
0.495
18.464 (0.425)
0.002 (0.385)
0.022
31.089 (0.028)
−0.0004 (0.816)
0.905
10.835 (0.901)
−0.0001 (0.910)
0.707
12.588 (0.815) ue
ue
1 MR analysis
β value
OR
95% CI
P value
0.065
1.067
0.838–1.358
0.838
0.177
1.193
0.918–1.550
0.918
0.225
1.252
0.773–2.028
0.772
0.100
1.106
0.917–1.333
0.292
0.119
1.126
0.894–1.419
0.315
0.081
1.085
0.767–1.535
0.656
0.092
1.096
0.861–1.396
0.659
0.216
1.241
0.944–1.634
0.869
0.275
1.316
0.763–2.269
0.352
0.376
1.457
1.024–2.742
0.037
0.361
1.434
1.014–2.029
0.414
−0.150
0.860
0.248–2.986
0.824
sease; OR, odd ratio; CI, confidence interval. efects of AD on the level of BCAAs. MR analysis
β value
OR
95% CI
P value
−0.021
0.979
0.989–0.990
<0.001
−0.020
0.980
0.964–0.994
0.008
−0.024
0.976
0.961–0.991
0.007
−0.023
0.977
0.963–0.991
0.001
−0.020
0.980
0.964–0.995
0.011
−0.029
0.971
0.952–0.990
0.009
−0.017
0.983
0.973–0.994
0.002
−0.019
0.982
0.968–0.995
0.010
−0.016
0.984
0.970–0.999
0.050
−0.019
0.982
0.971–0.992
0.001
−0.019
0.981
0.966–0.996
0.013
−0.018
0.982
0.967–0.997
0.031
sease; OR, odd ratio; CI, confidence interval. Qian et al. 10.3389/fnut.2023.1103303 diagnosis of AD. Future studies need to further clarify how AD
leads to lower levels of BCAAs. diagnosis of AD. Future studies need to further clarify how AD
leads to lower levels of BCAAs. that these inconsistent results occurred in ordinary observational
studies due to the limited sample size and the susceptibility of
BCAAs to various confounding factors such as diet. 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 We sincerely appreciated the data provided by the MRC IEU
OpenGWAS project. Funding Branched-chain amino acids (BCAAs) are essential amino
acids and account for one-third of the total amino acid content
of the human body (30). After being absorbed by the gut,
BCAAs are widely distributed in many organs for metabolism,
mainly including the muscle, brain, liver, and adipose tissue
(37). Among them, the muscle is the main metabolic place of
BCAAs, accounting for approximately 50% (30, 38). In addition,
the brain also absorbs a proportion of BCAAs in the blood
(30). In the mammalian brain, BCAAs are involved in multiple
functions, including neurotransmitter synthesis, protein synthesis,
and energy production (39). In this study, AD patients are found
to have reduced BCAA levels, which may be related to BCAA
intake and metabolic processes. Circulating BCAAs are derived
from diet, proteolysis, and gut microbiota (40). First, changes in
gastrointestinal function have been observed in the AD mice model
(41, 42). This may lead to reduced absorption of BCAAs from
the diet in AD. Second, AD or MCI patients usually experience
subclinical malnutrition (43). This resulted in reduced protein
reserve and proteolysis levels in AD. Third, a series of studies in
recent years have demonstrated the imbalance of gut microbiota
in AD (12, 41). This may lead to changes in the abundance
of gut microbiota related to BCAAs metabolism and ultimately
reduce the level of circulating BCAAs. However, these are just
some possible reasons why AD has reduced BCAA levels. Further
studies are needed to clarify the specific mechanism. In addition,
supplementing seven amino acids (leucine, phenylalanine, lysine
hydrochloride, isoleucine, histidine hydrochloride, valine, and
tryptophan) can improve cognitive, psychological, and social
functions in middle-aged and older adults (44). Furthermore,
supplementing L-norvaline (an isoform of valine) for 2 months
can reverse cognitive decline and synaptic loss in triple-transgenic
(3 × Tg) mice at the age of 4 months (45). Therefore, the
supplementation of BCAAs may be one of the strategies to delay
cognitive decline in AD patients. This study was supported by the National Natural Science
Foundation of China [81971014], the Hundred Faculty Talent Pool
Program at Shanghai University of Medicine & Health Sciences
in China, and the Shanghai Municipal Commission of Health and
Family Planning [20184Y0056]. Discussion The results of
our study explain the causal relationship between AD and BCAAs
from the root. Supplementary material The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fnut.2023. 1103303/full#supplementary-material 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 H-dT and G-yD designed the study and prepared the
manuscript. X-hQ and X-lL developed the methodology and
analyzed the data. All authors discussed the results and approved
the manuscript. Data availability statement In 2017, Larsson and Markus also reported the results of a study
using Mendelian randomization to explore the causal association
of BCAAs with the risk of AD (36). The results showed that only
higher isoleucine levels were associated with the risk of AD but
not leucine and valine (36). Our study differs from other studies in
several ways. In this study, the GWAS datasets related to BCAAs are
selected with a larger sample size, containing 115,047 participants. From the perspective of methodology, Larsson and Markus (36)
only used a single methodology to illustrate the effect of BCAAs
on the risk of AD. In our study, IVW, MR-Egger, and weighted
median are adopted in the analysis for Mendelian randomization,
which provides an important guarantee for the reliability of our
research results. The original contributions presented in the study are included
in the article/Supplementary material, further inquiries can be
directed to the corresponding authors. Frontiers in Nutrition SUPPLEMENTARY FIGURE S1
Funnel plot of the causal efect between BCAAs level and the risk of AD. (A)
Total BCAAs on AD, (B) valine on AD, (C) leucine on AD, (D) isoleucine on
AD, (E) AD on total BCAAs, (F) AD on valine, (G) AD on leucine, (H) AD on frontiersin.org References 19. Horgusluoglu E, NeffR, Song WM, Wang M, Wang Q, Arnold M,
et
al. Integrative
metabolomics-genomics
approach
reveals
key
metabolic
pathways
and
regulators
of
Alzheimer’s
disease. Alzheimer’s
Demen. (2022)
18:1260–78. doi: 10.1002/alz.12468 1. Jia L, Quan M, Fu Y, Zhao T, Li Y, Wei C, et al. Dementia in China:
epidemiology, clinical management, and research advances. Lancet Neurol. (2020)
19:81–92. doi: 10.1016/S1474-4422(19)30290-X 2. Irwin
MR,
Vitiello
MV. Implications
of
sleep
disturbance
and
inflammation
for
Alzheimer’s
disease
dementia. Lancet
Neurol. (2019)
18:296–306. doi: 10.1016/S1474-4422(18)30450-2 20. Skrivankova VW, Richmond RC, Woolf BAR, Yarmolinsky J, Davies NM,
Swanson SA. Strengthening the reporting of observational studies in epidemiology
using mendelian randomization: The STROBE-MR statement. Jama. (2021) 326:1614–
21. doi: 10.1001/jama.2021.18236 3. Jasbi P, Shi X, Chu P, Elliott N, Hudson H, Jones D, et al. Metabolic profiling of
neocortical tissue discriminates Alzheimer’s disease from mild cognitive impairment,
high pathology controls, and normal controls. J Proteome Res. (2021) 20:4303–
17. doi: 10.1021/acs.jproteome.1c00290 21. Bowden J, Davey Smith G, Burgess S. Mendelian randomization with invalid
instruments: effect estimation and bias detection through Egger regression. Int J
Epidemiol. (2015) 44:512–25. doi: 10.1093/ije/dyv080 4. Fani L, Ahmad S, Ikram MK, Ghanbari M, Ikram MA. Immunity and amyloid
beta, total tau and neurofilament light chain: Findings from a community-based cohort
study. Alzheimer’s Demen. (2021) 17:446–56. doi: 10.1002/alz.12212 22. Kunkle BW, Grenier-Boley B, Sims R, Bis JC, Damotte V, Naj AC, et al. Genetic meta-analysis of diagnosed Alzheimer’s disease identifies new risk loci and
implicates Aβ, tau, immunity and lipid processing. Nat Genet. (2019) 51:414–30. doi: 10.1038/s41588-019-0358-2 5. Arvanitakis Z, Shah RC, Bennett DA. Diagnosis and management of dementia:
review. Jama. (2019) 322:1589–99. doi: 10.1001/jama.2019.4782 23. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet. (2007) 81:559–75. doi: 10.1086/519795 6. Varma VR, Oommen AM, Varma S, Casanova R, An Y, Andrews RM,
et al. Brain and blood metabolite signatures of pathology and progression
in
Alzheimer
disease:
A
targeted
metabolomics
study. PLoS
Med. (2018)
15:e1002482. doi: 10.1371/journal.pmed.1002482 24. Staley JR, Blackshaw J, Kamat MA, Ellis S, Surendran P, Sun BB, et al. PhenoScanner: a database of human genotype-phenotype associations. Bioinformatics. (2016) 32:3207–9. doi: 10.1093/bioinformatics/btw373 7. An Y, Varma VR, Varma S, Casanova R, Dammer E, Pletnikova O, et al. Evidence
for brain glucose dysregulation in Alzheimer’s disease. Alzheimer’s Demen. (2018)
14:318–29. References doi: 10.1016/j.jalz.2017.09.011 25. Lawlor DA, Harbord RM, Sterne JA, Timpson N, Davey Smith G. Mendelian
randomization: using genes as instruments for making causal inferences in
epidemiology. Stat Med. (2008) 27:1133–63. doi: 10.1002/sim.3034 8. Lord J, Jermy B, Green R, Wong A, Xu J, Legido-Quigley C, et al. Mendelian
randomization identifies blood metabolites previously linked to midlife cognition
as causal candidates in Alzheimer’s disease. Proc Natl Acad Sci U S A. (2021)
118:e2009808118. doi: 10.1073/pnas.2009808118 26. Verbanck
M,
Chen
CY,
Neale
B,
Do
R. Detection
of
widespread
horizontal
pleiotropy
in
causal
relationships
inferred
from
Mendelian
randomization
between
complex
traits
and
diseases. Nat
Genet. (2018)
50:693–8. doi: 10.1038/s41588-018-0099-7 9. Biswas D, Duffley L, Pulinilkunnil T. Role of branched-chain amino acid-
catabolizing enzymes in intertissue signaling, metabolic remodeling, and energy
homeostasis. FASEB J. (2019) 33:8711–31. doi: 10.1096/fj.201802842RR 27. Hemani G, Bowden J, Davey Smith G. Evaluating the potential role of
pleiotropy in Mendelian randomization studies. Hum Mol Genet. (2018) 27:R195–
r208. doi: 10.1093/hmg/ddy163 10. Dullius A, Fassina P, Giroldi M, Goettert MI, Volken CF, de Souza. A
biotechnological approach for the production of branched chain amino acid containing
bioactive peptides to improve human health: A review. Food Res Int. (2020)
131:109002. doi: 10.1016/j.foodres.2020.109002 28. Hemani G, Zheng J, Elsworth B, Wade KH, Haberland V, Baird D, et al. The MR-
Base platform supports systematic causal inference across the human phenome. Elife. (2018) 7:e34408. doi: 10.7554/eLife.34408 11. Sivanand S, Vander Heiden MG. Emerging roles for branched-chain amino acid
metabolism in cancer. Cancer Cell. (2020) 37:147–56. doi: 10.1016/j.ccell.2019.12.011 29. Jia L, Yang J, Zhu M, Pang Y, Wang Q, Wei Q, et al. A metabolite panel
that differentiates Alzheimer’s disease from other dementia types. Alzheimer’s Demen. (2022) 18:1345–56. doi: 10.1002/alz.12484 12. Qian XH, Song XX, Liu XL, Chen SD, Tang HD. Inflammatory pathways
in Alzheimer’s disease mediated by gut microbiota. Ageing Res Rev. (2021)
68:101317. doi: 10.1016/j.arr.2021.101317 30. Polis B, Samson AO. Role of the metabolism of branched-chain amino acids in
the development of Alzheimer’s disease and other metabolic disorders. Neural Regen
Res. (2020) 15:1460–70. doi: 10.4103/1673-5374.274328 13. Basun H, Forssell LG, Almkvist O, Cowburn RF, Eklöf R, Winblad B, et al. Amino acid concentrations in cerebrospinal fluid and plasma in Alzheimer’s disease
and healthy control subjects. J Neural Transm Parkinson’s Dis Dement Section. (1990)
2:295–304. doi: 10.1007/BF02252924 31. Tynkkynen J, Chouraki V, van der Lee SJ, Hernesniemi J, Yang Q, Li S, et al. SUPPLEMENTARY TABLE S2 F statistic in this study. References Association of branched-chain amino acids and other circulating metabolites with risk
of incident dementia and Alzheimer’s disease: A prospective study in eight cohorts. Alzheimer’s Demen. (2018) 14:723–33. doi: 10.1016/j.jalz.2018.01.003 14. González-Domínguez R, García-Barrera T, Gómez-Ariza JL. Metabolite profiling
for the identification of altered metabolic pathways in Alzheimer’s disease. J Pharm
Biomed Anal. (2015) 107:75–81. doi: 10.1016/j.jpba.2014.10.010 32. Xiong YL, Therriault J, Ren SJ, Jing XJ, Zhang H. The associations of serum valine
with mild cognitive impairment and Alzheimer’s disease. Aging Clin Exp Res. (2022)
34:1807–17. doi: 10.1007/s40520-022-02120-0 15. Pan
X,
Nasaruddin
MB,
Elliott
CT,
McGuinness
B,
Passmore
AP,
Kehoe
PG,
et
al. Alzheimer’s
disease-like
pathology
has
transient
effects
on
the
brain
and
blood
metabolome. Neurobiol
Aging. (2016)
38:151–63. doi: 10.1016/j.neurobiolaging.2015.11.014 33. Socha E, Ko´sli´nski P, Koba M, Madra-Gackowska K, Kedziora-Kornatowska K,
Gackowski M, et al. Amino acid levels as potential biomarker of elderly patients with
dementia. Brain Sci. (2020) 10:917. doi: 10.3390/brainsci10120914 16. Toledo JB, Arnold M, Kastenmüller G, Chang R, Baillie RA, Han X, et al. Metabolic network failures in Alzheimer’s disease: A biochemical road map. Alzheimer’s
Demen. (2017) 13:965–84. doi: 10.1016/j.jalz.2017.01.020 16. Toledo JB, Arnold M, Kastenmüller G, Chang R, Baillie RA, Han X, et al. Metabolic network failures in Alzheimer’s disease: A biochemical road map. Alzheimer’s
Demen. (2017) 13:965–84. doi: 10.1016/j.jalz.2017.01.020 34. Ruiz HH, Chi T, Shin AC, Lindtner C, Hsieh W, Ehrlich M, et al. Increased
susceptibility to metabolic dysregulation in a mouse model of Alzheimer’s disease is
associated with impaired hypothalamic insulin signaling and elevated BCAA levels. Alzheimer’s Demen. (2016) 12:851–61. doi: 10.1016/j.jalz.2016.01.008 17. Li H, Ye D, Xie W, Hua F, Yang Y, Wu J, et al. Defect of branched-chain
amino acid metabolism promotes the development of Alzheimer’s disease by targeting
the mTOR signaling. Biosci Rep. (2018) 38:BSR20180127. doi: 10.1042/BSR201
80127 17. Li H, Ye D, Xie W, Hua F, Yang Y, Wu J, et al. Defect of branched-chain
amino acid metabolism promotes the development of Alzheimer’s disease by targeting
the mTOR signaling. Biosci Rep. (2018) 38:BSR20180127. doi: 10.1042/BSR201
80127 35. Russin KJ, Nair KS, Montine TJ, Baker LD, Craft S. Diet effects on cerebrospinal
fluid amino acids levels in adults with normal cognition and mild cognitive
impairment. J Alzheimer’s Dis. (2021) 84:843–53. doi: 10.3233/JAD-210471 18. Wang X, Sun G, Feng T, Zhang J, Huang X, Wang T, et al. SUPPLEMENTARY FIGURE S1 This bidirectional MR study indicates that AD is associated
with decreased BCAA levels, which can serve as a marker for early 06 frontiersin.org 10.3389/fnut.2023.1103303 Qian et al. isoleucine. Each instrumental SNP was represented by a black dot. SNPs,
single-nucleotide polymorphisms; BCAAs, branched-chain amino acids;
AD, Alzheimer’s disease. (H) AD on isoleucine. BCAAs, branched-chain amino acids; AD,
Alzheimer’s disease. SUPPLEMENTARY TABLE S1
Characteristics of selected SNPs for analysis of the causal efect between
BCAA level and the risk of AD from IGAP. Frontiers in Nutrition SUPPLEMENTARY FIGURE S2 Leave-one-out analysis of the causal efect between BCAAs level and the
risk of AD. (A) Total BCAAs on AD, (B) valine on AD, (C) leucine on AD, (D)
isoleucine on AD, (E) AD on total BCAAs, (F) AD on valine, (G) AD on leucine, 45. Polis B, Srikanth KD, Elliott E, Gil-Henn H, Samson AO. L-Norvaline reverses
cognitive decline and synaptic loss in a murine model of Alzheimer’s disease.
Neurotherapeutics. (2018) 15:1036–54. doi: 10.1007/s13311-018-0669-5 41. Qian XH, Liu XL, Chen G, Chen SD, Tang HD. Injection of amyloid-β to
lateral ventricle induces gut microbiota dysbiosis in association with inhibition of
cholinergic anti-inflammatory pathways in Alzheimer’s disease. J Neuroinflam. (2022)
19:236. doi: 10.1186/s12974-022-02599-4 42. Pellegrini C, Fornai M, D’Antongiovanni V, Antonioli L, Bernardini N,
Derkinderen P. The intestinal barrier in disorders of the central nervous system. Lancet
Gastroenterol Hepatol. (2023) 8:66–80. doi: 10.1016/S2468-1253(22)00241-2 37. Brosnan JT, Brosnan ME. Branched-chain amino acids: enzyme and substrate
regulation. J Nutr. (2006) 136:207s–11s. doi: 10.1093/jn/136.1.207S 38. Trautman ME, Richardson NE, Lamming DW. Protein restriction and
branched-chain amino acid restriction promote geroprotective shifts in metabolism.
Aging Cell. (2022) 21:e13626. doi: 10.1111/acel.13626 37. Brosnan JT, Brosnan ME. Branched-chain amino acids: enzyme and substrate
regulation. J Nutr. (2006) 136:207s–11s. doi: 10.1093/jn/136.1.207S
38. Trautman ME, Richardson NE, Lamming DW. Protein restriction and
branched-chain amino acid restriction promote geroprotective shifts in metabolism.
Aging Cell. (2022) 21:e13626. doi: 10.1111/acel.13626
39. J.D. Fernstrom. Branched-chain amino acids and brain function. J Nutr. (2005)
135:1539s–46s. doi: 10.1093/jn/135.6.1539S
40. McGarrah RW, White PJ. Branched-chain amino acids in cardiovascular disease.
Nat Rev Cardiol. (2023) 20:77–89. doi: 10.1038/s41569-022-00760-3
41. Qian XH, Liu XL, Chen G, Chen SD, Tang HD. Injection of amyloid-β to
lateral ventricle induces gut microbiota dysbiosis in association with inhibition of
cholinergic anti-inflammatory pathways in Alzheimer’s disease. J Neuroinflam. (2022)
19:236. doi: 10.1186/s12974-022-02599-4 43. S. Ogawa. Nutritional management of older adults with cognitive decline and
dementia. Geriatr Gerontol Int. (2014) 14:17–22. doi: 10.1111/ggi.12252 40. McGarrah RW, White PJ. Branched-chain amino acids in cardiovascular disease.
Nat Rev Cardiol. (2023) 20:77–89. doi: 10.1038/s41569-022-00760-3 39. J.D. Fernstrom. Branched-chain amino acids and brain function. J Nutr. (2005)
135:1539s–46s. doi: 10.1093/jn/135.6.1539S Frontiers in Nutrition 42. Pellegrini C, Fornai M, D’Antongiovanni V, Antonioli L, Bernardini N,
Derkinderen P. The intestinal barrier in disorders of the central nervous system. Lancet
Gastroenterol Hepatol. (2023) 8:66–80. doi: 10.1016/S2468-1253(22)00241-2
43. S. Ogawa. Nutritional management of older adults with cognitive decline and
dementia. Geriatr Gerontol Int. (2014) 14:17–22. doi: 10.1111/ggi.12252
44. Suzuki H, Yamashiro D, Ogawa S, Kobayashi M, Cho D, Iizuka A, et al.
Intake of seven essential amino acids improves cognitive function and psychological
and social function in middle-aged and older adults: A double-blind, randomized,
placebo-controlled trial. Front Nutr. (2020) 7:586166. doi: 10.3389/fnut.2020.
586166
45. Polis B, Srikanth KD, Elliott E, Gil-Henn H, Samson AO. L-Norvaline reverses
cognitive decline and synaptic loss in a murine model of Alzheimer’s disease.
Neurotherapeutics. (2018) 15:1036–54. doi: 10.1007/s13311-018-0669-5 References Sodium oligomannate
therapeutically remodels gut microbiota and suppresses gut bacterial amino acids-
shaped neuroinflammation to inhibit Alzheimer’s disease progression. Cell Res. (2019)
29:787–803. doi: 10.1038/s41422-019-0216-x 36. Larsson
SC,
Markus
HS. Branched-chain
amino
acids
and
Alzheimer’s
disease:
a
Mendelian
randomization
analysis. Sci
Rep. (2017)
7:13604. doi: 10.1038/s41598-017-12931-1 07 frontiersin.org Qian et al. 10.3389/fnut.2023.1103303 10.3389/fnut.2023.1103303 42. Pellegrini C, Fornai M, D’Antongiovanni V, Antonioli L, Bernardini N,
Derkinderen P. The intestinal barrier in disorders of the central nervous system. Lancet
Gastroenterol Hepatol. (2023) 8:66–80. doi: 10.1016/S2468-1253(22)00241-2 38. Trautman ME, Richardson NE, Lamming DW. Protein restriction and
branched-chain amino acid restriction promote geroprotective shifts in metabolism. Aging Cell. (2022) 21:e13626. doi: 10.1111/acel.13626 43. S. Ogawa. Nutritional management of older adults with cognitive decline and
dementia. Geriatr Gerontol Int. (2014) 14:17–22. doi: 10.1111/ggi.12252 39. J.D. Fernstrom. Branched-chain amino acids and brain function. J Nutr. (2005)
135:1539s–46s. doi: 10.1093/jn/135.6.1539S 44. Suzuki H, Yamashiro D, Ogawa S, Kobayashi M, Cho D, Iizuka A, et al. Intake of seven essential amino acids improves cognitive function and psychological
and social function in middle-aged and older adults: A double-blind, randomized,
placebo-controlled trial. Front Nutr. (2020) 7:586166. doi: 10.3389/fnut.2020. 586166 40. McGarrah RW, White PJ. Branched-chain amino acids in cardiovascular disease. Nat Rev Cardiol. (2023) 20:77–89. doi: 10.1038/s41569-022-00760-3 08 Frontiers in Nutrition frontiersin.org
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https://cadmus.eui.eu/bitstream/1814/60669/2/Beverelli_et_al-2019-The_World_Economy.pdf
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English
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Domestic value chains as stepping stones to global value chain integration
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World economy
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public-domain
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Domestic value chains as stepping stones to
global value chain integration∗ Cosimo Beverelli†
Victor Stolzenburg†‡ Robert B. Koopman†‡ Cosimo Beverelli† Simon Neumueller§ January 12, 2019 Abstract We study the role of Domestic Value Chains (DVCs) for Global Value Chain (GVC) inte-
gration. In the presence of industry specific fixed costs of fragmenting production and of
switching across input suppliers, DVCs can either be stepping stones or stumbling blocks
for GVCs. Focusing on backward linkages, that is the sourcing of intermediates, we provide
robust empirical evidence in favour of the stepping stone hypothesis. In our benchmark
specification a one standard deviation increase in DVC integration raises subsequent GVC
integration by about 0.4%. To identify the mechanisms at work, we exploit two dimensions
of industry level heterogeneity: product differentiation (a proxy of fragmentation costs) and
relationship specificity (a proxy of the costs of switching between suppliers). We find that
DVC integration is less conducive to GVC integration in industries that are characterized
by relatively high switching costs and relatively low fragmentation costs. ted
K A
1 In the remainder, a distinction will be drawn between ‘industries’, ‘broad industries’ and ‘sectors’. Industries
will refer to the ISIC Rev. 3 two digit codes (or aggregation thereof) listed in the first column of Table A-2. Broad
industries will refer to the following six aggregates of industries, listed in the last column of Table A-2: Primary,
Raw material processing, Light manufacturing, Heavy manufacturing, Electronics and Services. Sectors will be
the three macro aggregates of economic activity: primary (ISIC codes 01-14), manufacturing (ISIC codes 15-37),
and services (ISIC codes 40-95). Keywords: Domestic value chains; global value chains; input-output linkages. p
JEL Classification: F14; F15; F63. p
JEL Classification: F14; F15; F63. ce
∗The opinions expressed in this paper should be attributed to its authors. They are not meant to represent the
positions or opinions of the WTO and its Members and are without prejudice to Members’ rights and obligations
under the WTO. Any errors are attributable to the authors. This paper builds upon background research for the
Global Value Chain Development Report 2017, a joint project by IDE-JETRO, OECD, RCGVC, World Bank,
and WTO. Without implicating them, we thank seminar participants at the GVC Development Report 2016
Background Paper Conference, the Geneva Trade and Development Workshop, the University of Trento, the 16th
RIEF meetings, and the Graduate Institute, Geneva for useful comments. c
g ,
,
†Economic Research Division, World Trade Organization. Rue de Lausanne 154, 1202 Geneva, Switzerland. E-mail: cosimo.beverelli@wto.org; robert.koopman@wto.org; victor.stolzenburg@wto.org (corresponding author). ‡Graduate Institute of International and Development Studies, Chemin Eug`ene-Rigot 2, 1202 Geneva, Switzer- c
g ,
,
†Economic Research Division, World Trade Organization. Rue de Lausanne 154, 1202 Geneva, Switzerland. E-mail: cosimo.beverelli@wto.org; robert.koopman@wto.org; victor.stolzenburg@wto.org (corresponding author). c
E-mail: cosimo.beverelli@wto.org; robert.koopman@wto.org; victor.stolzenburg@wto.org (corresponding author). ‡Graduate Institute of International and Development Studies, Chemin Eug`ene-Rigot 2, 1202 Geneva, Switzer-
land. c
‡Graduate Institute of International and Development Studies, Chemin Eug`ene-Rigot 2, 1202 Geneva, Switzer-
land. §Office for West Africa, United Nations Economic Commission for Africa, P.O. BOX 744 Niamey, Niger. E-mail: NSimon@uneca.org. §Office for West Africa, United Nations Economic Commission for Africa, P.O. BOX 744 Niamey, Niger. E-mail: NSimon@uneca.org. A
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/twec.12779 This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. 1
Introduction Article
Global value chains (GVCs) are an important phenomenon of 21st century trade. Not only
final goods or services get traded, but intermediate products and stages get outsourced and
production becomes more and more fragmented. GVCs are often developed by large firms that
coordinate input sourcing and assembly decisions, establishing industry linkages across borders.1
Seminal work by Hummels et al. (2001) unveiled the growing importance of this international
production sharing. In more recent work, Johnson and Noguera (2012a, 2012b) characterize
the difference between value added trade and gross trade, showing that the GVC revolution, as
measured by trade in value added, is ongoing. In a similar fashion, Timmer et al. (2014) show
that global fragmentation, proxied by the foreign value added content of production, has rapidly
increased since the early 1990s. pted
This development is of considerable interest for policy makers since both theory and em-
pirics suggest that integrating in GVCs can lead to higher productivity and GDP. For instance,
Baldwin and Robert-Nicoud (2014) show how GVCs cause productivity improvements akin to
technological change by embedding the prominent features of the Grossman and Rossi-Hansberg
(2008) model into a general equilibrium setting. Empirically, Kummritz (2016) finds that at the
industry level higher GVC integration results in higher labor productivity and value added. ccep
A central question that arises in this context is what determines GVC integration. Hummels
et al. (2001), Johnson and Noguera (2012a), and Baldwin and Lopez Gonzalez (2015) find that
structural factors such as country size, industrial structure, and location play a key role. In
particular, small economies that are located close to GVC hubs such as Germany, Japan, and
the United States exhibit strong linkages into GVCs. Kowalski et al. (2015) show that in addition
policy matters. Countries with open trade and investment policies and sound institutions tend to integrate into GVCs more easily. to integrate into GVCs more easily. Article
A substantial part of the variation in GVC integration, however, remains unexplained. To
provide an illustration, Figure 1 plots the levels of GVC integration (measured as foreign value
added in exports as a share of total exports) of three country pairs: Sweden and Belgium;
Switzerland and Singapore; Romania and Bulgaria. Within each pair, countries are similar in
terms of the structural and policy factors used in the literature to explain GVC participation. This article is protected by copyright. All rights reserved. 1
Introduction One can still see significant differences in levels of GVC integration within pairs of matched
countries, especially in the last two pairs. Such differences cannot be explained by the drivers
of GVC integration identified in the literature. @ Figure 1 about here A y
y (
)
4 See, for example, Antr`as and Helpman (2004) and Grossman and Helpman (2005). @ Figure 1 about here A Accepted
A recent and growing strand of research, revisiting earlier work by Hirschman (1958), dis-
cusses the positive implications of domestic linkages within and across industries.2 Building on
this literature, in this paper we investigate whether such domestic linkages are stepping stones
or stumbling blocks for GVCs, and the mechanisms behind the relationship between GVCs and
domestic value chains (DVCs). Various papers have modeled firms’ sourcing choices as either one
between global offshoring and in-house production,3 or a simultaneous decision between global
offshoring, domestic outsourcing and in-house production.4 Our interest is to empirically assess
a sequential development, in which the amount of globally sourced intermediates is dependent
on the pre-existing level of domestic outsourcing because fragmenting further and replacing do-
mestic for foreign suppliers may be costly. To identify the sequential relationship, we exploit
the fact that the international fragmentation of production only started to expand at a global
scale in the 1990s, as a result of a dramatic fall in cross-border trade and communication costs. Domestic fragmentation, in turn, has been available to firms much longer. Therefore, initial
2 See, among others, Jones (2011), Bartelme and Gorodnichenko (2015), Bernard et al. (2015), and Dhyne
and Rub´ınov´a (2016). 3 See, for example, Jones and Kierzkowski (1990, 2001), Antr`as (2003), Grossman and Helpman (2003), and
Fally and Hillberry (2015). 4 See, for example, Antr`as and Helpman (2004) and Grossman and Helpman (2005). This article is protected by copyright. All rights reserved. levels of DVC integration are unlikely to be affected by current levels of GVC integration. d Article
The mechanism linking GVC and DVC integration that we focus on relates to the frag-
mentation costs associated with slicing up production, and to the costs of switching suppliers. We refer to fragmentation costs as all the costs paid by a firm deciding to source inputs ex-
ternally, rather than producing them in-house.5 Here, we focus on that part of fragmentation
costs that can be considered as one-offfixed costs. Such fixed costs entail, among other things,
the necessary codifying of tacit knowledge, the downsizing of plants and workforce, the pro-
tection of intellectual property, the adaptation of the firm’s structure, and learning about the
fragmentation process.6 Once these costs have been paid for domestic fragmentation, they are
not incurred again. A
6 This is analogous to the literature on learning to export where firms start their internationalization by
exporting small amounts to easily accessible markets to learn about the export process and their export potential
before expanding their activities (see for instance Schmeiser, 2012). @ Figure 1 about here A This implies that DVC integration should serve as a stepping stone to subse-
quent GVC integration, particularly in industries characterized by relatively large fragmentation
costs. Other things being equal, this would be reflected in a positive relationship between DVC
and GVC integration. epted
Conversely, we refer to switching costs as the costs paid by a firm when it decides to stop
sourcing an input from a supplier and starts sourcing the same input from another supplier, either
located in the same country or abroad. Examples are contract termination fees and supplier
searching and training costs. For a given level of domestic fragmentation, high switching costs
should reduce subsequent international fragmentation making DVCs a stumbling block to GVC
integration. Other things being equal, this would be reflected in a negative relationship between
DVC and GVC integration. Acc
Thus, in the presence of both fragmentation costs and switching costs, the sign of the rela-
tionship between DVCs and GVCs is dependent on the relative importance of switching vis-`a-vis
fragmentation costs and needs to be determined empirically. To that effect, we propose a novel
measure of DVCs, equal to the share of domestically sourced inputs in domestic output, where This article is protected by copyright. All rights reserved. rticle
the latter is computed excluding foreign sourced inputs. We are able to compute this measure
for up to 61 countries and 34 industries.7 We show that higher levels of domestic fragmentation
at the beginning of our sample (mid 1990s) positively affect GVC integration, as measured by
foreign value added in exports, at the end of the sample (late 2000s). In the benchmark specifi-
cation, a one unit increase in DVC integration raises subsequent GVC integration by 2.4%. This
suggests that DVC integration is a stepping stone for GVC integration, and that, overall, the
fragmentation cost channel dominates the switching cost channel. Moreover, by taking DVCs
into account, we are able to reduce the unexplained variation in GVC integration by about 30%. Accepted Ar
To investigate the hypothesized role of fragmentation and switching costs for the relation-
ship between DVCs and GVCs, we exploit manufacturing industry-level heterogeneity along
two dimensions: the degree of product differentiation and contract intensity (i.e. relationship
specificity). p
g
g
9 The case of high fragmentation and low switching costs is not observed in the data. A complete list of countries and industries is available in Appendix A, respectively in Tables A-1 and A- A
p
pp
,
p
y
8 Since Nunn (2007)’s contract intensity measure employs Rauch (1999)’s classification, we show in the
robustness section that our results hold when alternative proxies for switching and fragmentation costs are used.
9 This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. 9 The case of high fragmentation and low switching costs is not observed in the data. @ Figure 1 about here A Fixed fragmentation costs are likely to be higher in industries producing a high
share of differentiated goods since these industries tend to be more innovation- and skill- in-
tensive, therefore they have higher costs of codifying tacit knowledge or downsizing as shown
by Voigtl¨ander (2014). Switching suppliers, in turn, is costlier in industries characterized by a
high degree of contract intensity because it involves the payment of contract termination fees
and similar costs that tend to increase with the relationship-specificity of inputs. Accordingly,
we use Rauch (1999)’s classification for differentiated goods to proxy for fragmentation costs,
and Nunn (2007)’s contract intensity measure to proxy for switching costs.8 The 26 manufac-
turing industries are then grouped into three categories: industries with high fragmentation and
high switching costs, industries with low fragmentation and low switching costs, and industries
with low fragmentation and high switching costs.9 In line with the suggested mechanism, we
find that the positive role of DVCs for subsequent GVC integration is driven by the first two
groups of industries, suggesting that fragmentation costs tend to be more relevant than switch- This article is protected by copyright. All rights reserved. ing costs. For the third group of industries, which is characterized by high switching costs and
low fragmentation costs, the positive effect of DVCs disappears entirely. icle
The remainder of the paper is organized as follows. Section 2 provides details on the
measures employed to proxy for DVC and GVC, describes the data used and the identification
strategy. Section 3 presents the empirical results. Section 4 concludes and suggests some policy
implications. This article is protected by copyright. All rights reserved. ti
2.1
Measuring value chain integration and industry level characteristics cepted Ar
The literature has developed several measures for GVC participation that can be split up into
backward and forward linkages. The former are related to intermediates sourced from abroad. The latter are related to domestic intermediates exported abroad. Since our channel between
DVCs and GVCs relates to the costs of sourcing intermediate goods, we focus on backward
linkages measures. These measures are typically based on Inter Country Input Output (ICIO)
tables, which are jl ik matrices that represent supply and demand relationships within and
across industries (i,j) and countries (k,l). A generic element mji
lk of an ICIO matrix gives the
value of intermediate goods supplied by industry j of country l to industry i of country k. ICIOs
indicate not only cross country linkages but also within country linkages. This implies that the
same data source can be used to build both our DVC and GVC integration indicators, limiting
the scope for measurement error. In the next subsections, we first discuss the standard measures
of GVC integration proposed in the literature and also used in this study. Next, we move to our
novel measure of DVC integration. rt
2
Empirical methodology Ar
In this section we discuss the methodology to examine the role of DVCs for subsequently linking
into GVCs. To do so, we postulate the following reduced form model at the country-industry
level: gvcikt βdvciks λι γxikt εikt,
(2.1) (2.1) pted
where i indexes industries; k indexes countries; t and s @ t index time; the scalar β is the
coefficient of interest, to be estimated; γ is a column vector of coefficients to be estimated; x
is a column vector of controls; λ is a column vector of fixed effects; ι is a column vector of 1’s;
and ε is a random error term. The variables in x, and the elements in and dimension of λ, are
specified in Section 2.3, where the identification strategy is discussed. Accep
While equation (2.1) can give the net effect of DVCs for GVC integration, it does not
provide information on the mechanism driving the relationship. Therefore, in a second step we
proceed estimating a variant of equation (2.1) that exploits the industry-level variation in the
data to analyze the suggested role of fragmentation and switching costs. For this purpose, we
build proxies for industries’ fragmentation and switching costs and then classify industries based
on the relative magnitude of the costs into three groups: industries with high fragmentation and
high switching costs (HFHS industries), industries with low fragmentation and low switching
costs (LFLS industries), and industries with low fragmentation and high switching costs (LFHS This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. industries). We postulate the following reduced form model with interactions between DVCs
and these industry-level characteristics: industries). We postulate the following reduced form model with interactions between DVCs
and these industry-level characteristics: gvcikt δdvciks c
i λι γxikt εikt,
(2.2) (2.2) c
where δ is a column vector of coefficients to be estimated and ci HFHS i LFLS i LFHS i).10 c
where δ is a column vector of coefficients to be estimated and ci HFHS i LFLS i LF ic
where δ is a column vector of coefficients to be estimated and ci HFHS i LFLS i LFHS i).10 ic
where δ is a column vector of coefficients to be estimated and ci HFHS i LFLS i LFHS i).10 c
2.1.1
GVC integration Ac
The recent literature on GVCs has used various indicators to capture the rise of global production
networks. Recently, Hummels et al. (2001)’s Vertical Specialization measure and its refinements
by Wang et al. (2013) and Koopman et al. (2014) have emerged as standard indicators, also
10 A
10 The c vector does not contain a HFLS dummy since, as mentioned in footnote 9, the case of high fragmen-
tation and low switching costs is not observed. A
10 The c vector does not contain a HFLS dummy since, as mentioned in footnote 9, the case of high fragmen-
tation and low switching costs is not observed. This article is protected by copyright. All rights reserved. adopted in the present analysis. adopted in the present analysis. icle
The baseline GVC indicator is fvax, the foreign value added content in the production
of exports. To construct this indicator, value added needs to cross at least two borders to
be counted towards GVC trade. To obtain the required value added flows, it is necessary to
decompose gross exports using information from ICIOs.11 In the simplest case with two countries
and two industries, the decomposition can be illustrated as follows: V I A1E
vaeii
kk
vaeij
kk
vaeii
kl
vaeij
kl
vaeji
kk
vaejj
kk
vaeji
kl
vaejj
kl
vaeii
lk
vaeij
lk
vaeii
ll
vaeij
ll
vaeji
lk
vaejj
lk
vaeji
ll
vaejj
ll
, V I A1E
vaeii
kk
vaeij
kk
vaeii
kl
vaeij
kl
vaeji
kk
vaejj
kk
vaeji
kl
vaejj
kl
vaeii
lk
vaeij
lk
vaeii
ll
vaeij
ll
vaeji
lk
vaejj
lk
vaeji
ll
vaejj
ll
, cepted
where E is a 4 4 matrix in which the diagonal elements give each industry’s gross exports for
N industries and G countries, V is a 4 4 matrix in which the diagonal elements give each
industry’s value added to output ratio, and A is the 4 4 Input Output coefficient matrix, i.e. each element of A gives the intermediates that each industry supplies for $1 of output in every
other industry.12 The intuition behind the decomposition becomes clear by recalling that these
intermediate flows are the output of other industries, which means that they consist of value
added and other intermediates themselves. 13A more exhaustive explanation of the approach can be found in Wang et al. (2013). 11 This is technically implemented using the R package decompr by Quast and Kummritz (2015).
12 In the general case of G countries and N industries, the dimension of the E, V , A (and, conseque 11 This is technically implemented using the R package decompr by Quast and Kummritz (2015).
12 In the general case of G countries and N industries, the dimension of the E, V , A (and, conseque
matrices is GN GN. 11 This is technically implemented using the R package decompr by Quast and Kummritz (2015).
12 This article is protected by copyright. All rights reserved. c
2.1.1
GVC integration I A1 approximates these indirect links between
industries and when combined with the values in the V matrix gives the actual value added flows
so that the elements of the vae matrix are estimates of the industry-level value added origins of
each industry’s exports.13 Our benchmark GVC indicator fvax for industry i in country k is then given by: Our benchmark GVC indicator fvax for industry i in country k is then given by: Our benchmark GVC indicator fvax for industry i in country k is then given by: fvax ik lnFVAX ik lnQ
l
Q
j
vaeji
lk
(2.3) (2.3) 11 This is technically implemented using the R package decompr by Quast and Kummritz (2015). 12 In the general case of G countries and N industries, the dimension of the E, V , A (and, consequently, vae)
matrices is GN GN 13A more exhaustive explanation of the approach can be found in Wang et al. (2013). This article is protected by copyright. All rights reserved. where l x k. Thus, fvax ik is equal to the sum of value added from all industries j of all foreign
countries l in the exports of industry i in country k.14 where l x k. Thus, fvax ik is equal to the sum of value added from all industries j of all foreign
countries l in the exports of industry i in country k.14 e cle
As a simple alternative indicator, used in robustness exercises, we additionally calculate
the log of imported inputs, i2p, following Baldwin and Lopez Gonzalez (2015). This measure
implies a broader definition of GVC and can easily be derived from ICIOs as follows: i2pik lnI2Pik lnQ
l
Q
j
mji
lk,
(2.4) (2.4) (2.4) r
where l x k.15 A
15 In this case, too, sourcing from ISIC Rev. 3 group C (mining industry) is excluded.
16 Note that, as for GVC measures, we exclude intermediates sourced from the mining industry (ISIC Rev. 3
group C). This article is protected by copyright. All rights reserved. 14 Sourcing from ISIC Rev. 3 group C (mining industry) is excluded to avoid effects stemming fr
variations in commodities. In Section 3.3 we use further strategies to deal with the mining industry.
15 g
g
y
In this case, too, sourcing from ISIC Rev. 3 group C (mining industry) is excluded. 15 In this case, too, sourcing from ISIC Rev. 3 group C (mining industry) is excluded.
16 Note that, as for GVC measures, we exclude intermediates sourced from the mining indu cc
18 A number of recent papers that measure gains from trade in the presence of intermediates consider the
relationship between foreign input use and domestic input use. Blaum et al. (2015) combine domestic and foreign
inputs in a constant elasticity of substitution (CES) function, with elasticity of substitution ε ranging between 1.4
and 2.4. Similarly, in Tintelnot et al. (2017), firms use a CES input bundle of workers and domestic and foreign
inputs, with elasticity of substitution ρ A 1. The authors use values of ρ ranging from 1.5 to 2. Antr`as et al.
(2017) assume that, from the perspective of firms, inputs are differentiated by country of origin, with an elasticity
of substitution across inputs from any two locations equal to 1 θ.
Their benchmark estimate of θ – which
corresponds to the shape parameter of the Fr´echet distribution of firm-specific intermediate input efficiencies – is
equal to 1.789. A
19 A complete list by country and industry is available in the Appendix. See Tables A-3 and A-4.
20 The Rauch classification treats goods as differentiated if they are neither reference priced nor traded on
organized exchanges. A
2.1.2
DVC integration pted A
For DVC integration (the explanatory variable of interest in equation (2.1)) we suggest a novel
indicator, applying the following approach. First, we decompose an industry’s output into
three parts based on the different inputs required to produce it: i) its own value added; ii)
domestically sourced intermediate goods; and iii) foreign sourced intermediate goods. We refer
to the combination of i) and ii) as ‘virtual domestic output’, since it is the virtual part of an
industry’s output that is produced with domestic content only. With this distinction at hand,
we proceed to define our DVC indicator as the share of domestically sourced intermediate goods
in virtual domestic output: dvcik
Pj mji
kk
Pj mji
kk vaik
. (2.5) (2.5) c
In other words, dvc is the share of domestic inputs in total output excluding foreign inputs. c
In other words, dvc is the share of domestic inputs in total output excluding foreign inputs. c
In other words, dvc is the share of domestic inputs in total output excluding foreign inputs. cc
That is, it captures how much of an establishment’s domestically produced output is supplied
by other establishments instead of being produced in-house.16 cc
That is, it captures how much of an establishment’s domestically produced output is supplied
by other establishments instead of being produced in-house.16 by other establishments instead of being produced in-house.16 The reason for calculating the DVC indicator in this particular way is as follows. W A
The reason for calculating the DVC indicator in this particular way is as follows. We only
14 S
i
f
ISIC R
3
C (
i i
i d
t
) i
l d d t
id
ff
t
t
i
f
i A
,
,
g
g
p
(
g
y)
16 Note that, as for GVC measures, we exclude intermediates sourced from the mining industry (ISIC Rev. 3
group C). ed Article
look at virtual domestic output instead of total output to avoid a mechanical correlation with
the dependent variable which, as discussed in Section 2.1.1 above, is a subset of foreign sourced
intermediate goods. These, in turn, are a subset of total output (recall that we decompose total
output into three parts: own value added, domestically sourced intermediate goods, and foreign
sourced intermediate goods). Hence, using total output as denominator of our dvc measure
would correlate dependent and independent variable. This article is protected by copyright. All rights reserved. omplete list by country and industry is available in the Appendix. See Tables A-3 and A-4. Rauch classification treats goods as differentiated if they are neither reference priced nor traded on
exchanges. The difference between total output and virtual domestic output in 1995 was in any case minimal an
ore, as shown in Section 3.3, the results are not sensitive to the choice of denominator of the dvc variable A
2.1.2
DVC integration By considering only virtual domestic
content, we break this mechanical link.17 An alternative solution to the problem of mechanical
correlation between the main explanatory variable (dvc) and the dependent variable (gvc) would
be the use of levels, rather than shares, of the dvc variable. This approach, however, would also
create a positive (negative) mechanical correlation between the two variables in the presence
of any complementarity (substitutability) between foreign and domestic inputs in production.18
To summarize, we avoid a mechanical correlation between our GVC and DVC indicators by
constructing the former in levels and the latter in shares, using in its denominator only virtual
domestic content. Table 1 shows the top and bottom ten countries in terms of the dvc variable
in 1995 (averaged across all industries for each country).19 ed Article
look at virtual domestic output instead of total output to avoid a mechanical correlation with
the dependent variable which, as discussed in Section 2.1.1 above, is a subset of foreign sourced
intermediate goods. These, in turn, are a subset of total output (recall that we decompose total
output into three parts: own value added, domestically sourced intermediate goods, and foreign
sourced intermediate goods). Hence, using total output as denominator of our dvc measure
would correlate dependent and independent variable. By considering only virtual domestic
content, we break this mechanical link.17 An alternative solution to the problem of mechanical
correlation between the main explanatory variable (dvc) and the dependent variable (gvc) would
be the use of levels, rather than shares, of the dvc variable. This approach, however, would also
create a positive (negative) mechanical correlation between the two variables in the presence
of any complementarity (substitutability) between foreign and domestic inputs in production.18
To summarize, we avoid a mechanical correlation between our GVC and DVC indicators by
constructing the former in levels and the latter in shares, using in its denominator only virtual
domestic content. Table 1 shows the top and bottom ten countries in terms of the dvc variable
in 1995 (averaged across all industries for each country).19 @ Table 1 about here A q
19 A complete list by country and industry is available in the Appendix. See Tables A-3 and A-4.
20 The Rauch classification treats goods as differentiated if they are neither reference priced nor
i
d
h A
(
)
22 A detailed explanation of the classifications of industries is available in Appendix B. Note that Nunn uses
the Rauch classification to determine if an intermediate is sourced on a contractual basis. Thus, our switching and
fragmentation cost proxies depend indirectly and directly on the Rauch classification. However, since the Nunn
classification looks at an industry’s intermediates and the Rauch classification at an industry’s output, there is no
strong mechanical correlation between the two. We show in Section 3.3 that our results are robust to employing
alternative proxies for switching and fragmentation costs such as Costinot (2009)’s complexity classification. p
2.1.3
Industry fragmentation costs and switching costs e
As benchmark proxy for fragmentation costs, we use Rauch (1999)’s classification for differenti-
ated goods concorded to our industry-level data.20 The reason for this choice is that industries ated goods concorded to our industry-level data.20 The reason for this choice is that in e
with a high share of differentiated goods tend to be more innovation and skill intensive.21 As
a result, we can assume that codifying tacit knowledge, downsizing, and other fragmentation
related activities are more costly for these industries. Article
Regarding switching costs, Nunn (2007)’s contract intensity (or relationship specificity)
measure is a straightforward proxy. Different industries require different amounts of relation-
ship specific inputs. When these inputs are supplied at arm’s length, complex contracts are
needed to address the resulting hold up problem. Nunn (2007) shows that, as a result, in-
dustries differ in their contract intensity. A substantive share of fixed switching costs, such
as contract cancellation fees, only occur when intermediates cannot be sourced from organized
markets, but are sourced using contract-based relations with suppliers. Nunn (2007)’s indicator
measures the share of intermediates that are sourced through such contract-based relations in
total intermediates, and is thus a natural proxy for our exercise.22 epted
Table 2 lists the industries separated into the three categories and shows that the selected
proxies create a sensible allocation. LFLS industries like ‘Food and beverages’ typically source
inputs from organized markets and, thus, should find it easy to switch between suppliers. At
the same time, their production tends to be neither skill nor innovation intensive and so frag-
mentation should be equally simple. In contrast, the identified HFHS industries such as ‘Motor
vehicles’ or ‘Electronics’ are dependent on a highly skilled workforce and complex intermediates. Lastly, LFHS industries such as ‘Electrical machinery’ (e.g. cables) require complex intermedi-
ates but production is homogeneous and non complex. p
Lastly, LFHS industries such as ‘Electrical machinery’ (e.g. cables) require complex intermedi-
ates but production is homogeneous and non complex. 21 See, for example, Voigtl¨ander (2014) for evidence on this relationship. This article is protected by copyright. All rights reserved. for example, Voigtl¨ander (2014) for evidence on this @ Figure 2 about here A ted Ar
Similarly, when we look at our DVC integration indicator by country (Table 1), we see
that countries that are well integrated into GVCs also had strong domestic linkages in 1995. Interestingly, however, while the top ten is dominated by countries traditionally highly involved
into GVCs, such as China, Slovakia, or Korea, it also contains countries like Croatia and New
Zealand, which do not exhibit strong linkages into GVCs. The same pattern holds when exam-
ining the bottom ten. While Cambodia and Greece lag behind in GVC participation, Mexico is
highly integrated. This suggests heterogeneity in the effects of DVCs, which will be taken into
account in the empirical analysis. @ Table 2 about here A This article is protected by copyright. All rights reserved. 2.2
Descriptive evidence ticle
When taking our GVC and DVC indicators to the data, we find preliminary evidence that DVC
integration is a stepping stone for GVC integration. Figure 2 unconditionally correlates GVCs
in 2008 (in logs) with the measure of DVCs in 1995. The slope coefficient is equal to 0.47. The stepping-stone effect of DVC integration for GVC integrations also constitutes suggestive
evidence that the fragmentation cost channel dominates the switching cost channel. @ Figure 2 about here A p
2.3
Identification Accep
Estimation results of the reduced form equations (2.1) and (2.2) could in particular be affected
by omitted variable bias. For instance, high levels of GVC integration could be driven by a coun-
try’s institutions, which might also affect domestic production fragmentation. To deal with this
concern, we use a combination of fixed effects and control variables that account for structural
and policy determinants of GVC participation identified in the literature. More specifically,
we use industry fixed effects to control for structural differences across industries that might
correlate at once with GVC and DVC integration. Heavy manufactures and downstream indus-
tries, for instance, are more likely to develop backward linkages than agriculture. In addition, This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. le
we include country-broad industry fixed effects to take care of (non time-varying) policy and
structural differences across countries – in terms of country size, economic development, remote-
ness, institutions, endowments as well as the industrial structure of the country – that may vary
across broadly defined industries in each country.23 le
we include country-broad industry fixed effects to take care of (non time-varying) policy and
structural differences across countries – in terms of country size, economic development, remote-
ness, institutions, endowments as well as the industrial structure of the country – that may vary
across broadly defined industries in each country.23 pted Artic
Industry and country-broad industry fixed effects fail to control for any potentially omitted
variable that varies across countries-industries and might be correlated with DVC integration,
even within broad industries. We include two such variables: i) a dummy equal to one for
comparative advantage industries, ca; and ii) industry level ‘composite’ import tariffs, ctau. The
comparative advantage dummy synthetically accounts for all those determinants of patterns of
specialization of a given country in a given industry (e.g., country labour abundance in a labour-
intensive industry). It is calculated using Balassa (1965)’s revealed comparative advantage index,
but on the basis of value added exports.24
The ‘composite’ import tariffs variable captures
differences in the trade policy environment at the country-industry level. For each industry in a
given country, it is calculated as the weighted average of tariffs that the inputs into the industry
face, with weights being given by the input output coefficient. Ac
23 As the dependent variable is at the country-industry level, we cannot use country-industry fixed effects to
control for a country’s industrial structure. To be nevertheless as conservative as possible, we form country-broad
industry fixed effects by interacting country fixed effects with fixed effects for the six ‘broad industries’ defined in
the last column of Table A-2. These country-broad industry fixed effects control for the fact that larger industries
within a country have more scope for fragmentation. A
y
p
g
24 The Balassa index is given by
eik~ek
Pk eik~ Pk ek . It compares an industry’s share in a country’s exports, ek, to
that industry’s share in global exports. An index value larger than 1 indicates comparative advantage industries. This article is protected by copyright. All rights reserved. y
g
p
25 See, for instance, Hsieh and Klenow (2009). A
y
g
24 The Balassa index is given by
eik~ek
Pk eik~ Pk ek . It compares an industry’s share in a country’s exports, ek, to
that industry’s share in global exports. An index value larger than 1 indicates comparative advantage industries.
25 See, for instance, Hsieh and Klenow (2009). that industry’s share in global exports. An index value larger than 1 indicates comparative advantage industries.
25 See, for instance, Hsieh and Klenow (2009). 25 See, for instance, Hsieh and Klenow (2009). p
2.3
Identification We follow the literature and
use United States’ input output coefficients under the assumption that they are determined by
technological factors as opposed to market distortions.25 ccep
Considering that the aim is to examine how initial patterns in DVCs have affected subse-
quent GVC integration, we use dvc values for 1995 and fvax values for 2008. This also addresses
potential concerns about a reverse causality bias since the rapid expansion of GVCs only started
around 1995 (Wang et al., 2017 show that in 1995 GVCs only accounted for 3% of global pro-
duction). This limits any potential reverse effect on DVC integration values in 1995. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. e
The benchmark reduced form model for the effect of DVCs on GVC integration (equation
(2.1)) is then estimated as: gvcik2008 βdvcik1995 γ1caik2008 γ2ctauik2008 λ λi λbk εik2008,
(2.6) gvcik2008 βdvcik1995 γ1caik2008 γ2ctauik2008 λ λi λbk εik2008,
(2.6) (2.6) rtic
where β is the coefficient of interest; λ is a constant; and λi and λbk are respectively industry
and broad industry-country fixed effects. Since the mechanism determining the role of DVCs
for GVC integration is ambiguous as outlined in the introduction, we do not have a prior on the
sign of β. A
The benchmark reduced form model for testing the mechanism behind the DVC-GVC link
(equation (2.2)), in turn, is estimated as: The benchmark reduced form model for testing the mechanism behind the DVC-GVC link
(equation (2.2)), in turn, is estimated as: gvcik2008 δ1dvcik1995 LFLSi δ2dvcik1995 HFHSi δ3dvcik1995 LFHSi γ1caik2008
γ2ctauik2008 λ λi λbk εik2008. (2.7) gvcik2008 δ1dvcik1995 LFLSi δ2dvcik1995 HFHSi δ3dvcik1995 LFHSi γ1caik2008
γ2ctauik2008 λ λi λbk εik2008. (2.7) (2.7) γ2ctauik2008 λ λi λbk εik2008. γ2ctauik2008 λ λi λbk εik2008. te
In equation (2.7), δ1 gives the effect of DVCs on subsequent GVC integration in LFLS industries;
δ2 gives the effect for HFHS industries; and δ3 gives the effect for LFHS industries. Accept
A positive δ1 would suggest that fragmentation costs play a larger role than switching costs,
while a negative δ1 would imply the opposite. p
2.3
Identification We include the HFHS dummy as falsification
test, since we expect δ2 not to be significantly different from δ1 since in both industry categories
the relative level of fragmentation vis-`a-vis switching costs is identical. In other words, if the
channel we propose is at work, there is no reason to believe that the effect of DVCs on GVCs
should be different in HFHS industries as compared to LFLS industries. The coefficient of
interest in equation (2.7) is δ3, which is expected to be significantly smaller than δ1 and δ2. This
is because in LFHS industries the relative level of switching costs, as compared to fragmentation
costs, is higher than in the benchmark LFLS industries. Accordingly, DVC integration in these
industries is less likely to lead to GVC integration. This article is protected by copyright. All rights reserved. Article
The proposed strategy of adding industry and broad-industry country fixed effects, as well
as the ca and ctau controls, does not guarantee that the empirical correlation between dvc
(alone or interacted with industry fragmentation/switching costs dummies) and gvc is free of
any omitted variable bias. Therefore, as described in Section 3.3, panel estimations using data
for the years 2000 and 2005 in addition to 1995 and 2008 are presented. These estimations allow
to include a rich structure of fixed effects, namely country-year, industry-year, and industry-
country, controlling for any unobserved factor that might be correlated with GVCs and DVCs
and varies across two of the three dimensions (countries, industries and time) of the data. This
should minimize concerns related to omitted variable bias. pted A
Moreover, the approach of using 1995 values for the dvc variable and 2008 values for the gvc
variable does not automatically exclude any reverse causality. As also detailed in Section 3.3,
we thus estimate regressions with an interaction term between gvc and a high income dummy
as a way of addressing these concerns. The reason is that if there is reverse causality flowing
from GVCs to DVCs, we would like our dvc values to be from a point in time where the extent
of GVCs was negligible, so that the estimates would be unbiased even in the presence of reverse
causality. In 1995 the extent of GVC integration might not have been negligible in developed
countries, but was certainly so in many developing countries. A
26 The instrument for dvcikt is, therefore, the variable dvcIV
ikt Plxk dvcilt gdplt, where gdplt is the share of
country l in total GDP of all countries in the sample at time t. The weights sum up to one. This article is protected by copyright. All rights reserved. p
2.3
Identification Accep
Killing two birds (omitted variables and reverse causality) with one stone, we further propose
an instrumental variable (IV) strategy that exploits exogenous variation in the DVC variable. To obtain such exogenous variation, we regress dvc in the first stage on the weighted average of
other countries’ dvc values, with the weights given by country size (as proxied by GDP).26 The
intuition is the following. Think of DVC values as having three components: a country-specific,
an industry-specific and a country-industry component. The first two components are absorbed
by fixed effects, and are not a source of concern. The third component itself can further be
decomposed in two parts: i) an idiosyncratic part specific to each country-industry (e.g. man- This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. ted Article
agerial ability, reputation); and ii) a part correlated across countries due to the interaction of
structural industry characteristics and country characteristics (e.g. market size matters more
for domestic fragmentation in complex industries than in non-complex industries). The idiosyn-
cratic part might introduce an omitted variable bias – for instance if high managerial ability
specific to an industry in a country might lead to both domestic and foreign fragmentation. In
contrast, the correlated part should be free of such bias, because the individual contribution of
the industry- and country-specific components is controlled for by fixed effects, so that identi-
fication is obtained only through the exogenous interaction of these variables. By using other
countries’ DVC variables as an instrument, the estimates are only identified by this exogenous
part, which is by definition uncorrelated with the endogenous idiosyncratic part. Therefore, the
average value of dvc in other countries is a legitimate instrument for dvc, since it both meets the
relevance and the exclusion requirements. We additionally weight the instrument by country
size since we assume that the DVC values of larger countries in our sample are closest to a true
optimal undistorted DVC value because large countries have a larger set of available suppliers,
which means that their DVC values are not artificially restricted by the absence of adequate
domestic outsourcing opportunities. This article is protected by copyright. All rights reserved. p
2.4
Data p
2
F Accep
For the calculation of the DVC and GVC indicators, as well as the revealed comparative ad-
vantage measure, we employ the most recent and most advanced release of OECD ICIO tables,
covering 61 countries and 34 2 digit ISIC Rev. 3 industries for the years 1995, 2000, 2005, and
2008 to 2011. To create ICIOs, the OECD combines national IO tables with international trade
data. As OECD countries have a harmonized construction methodology, potential discrepancies
between national IO tables should be minor. Furthermore, the advanced harmonization across
countries reduces to a minimum the use of proportionality assumptions to derive the ratio of
imported intermediates in an industry’s demand. The OECD has used elaborate techniques This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. to deal with processing trade. Due to the outstanding role of processing trade in GVCs, this
implies a significant improvement for the reliability of the database.27 Article
For the tariffmeasure, we additionally take HS 2 digit MFN tariffs from the UNCTAD
TRAINS database and concord them to the ICIOs’ ISIC Rev. 3 classification employing a
concordance table provided by WITS. The Rauch classification is available at the 4 digit SITC
Rev. 2 level. To concord it to ISIC Rev. 3, we need to apply a crosswalk via SITC Rev. 3. The necessary concordance tables are provided by Eurostat. In cases where ISIC industries are
concorded to both homogeneous and differentiated SITC commodities, we use trade weights
from COMTRADE to determine if an ISIC industry is differentiated or homogeneous.28 Accepted A
For additional specifications we employ as supplementary controls the distance of country k
to the closest GVC hub (China, Germany, Japan, and the United States) from CEPII and GDP
data from the World Development Indicators. We also perform several robustness checks, some
of which necessitate additional data. Firstly, in one robustness exercise we replace the Rauch
classification for fragmentation costs with Costinot (2009)’s measure of industrial complexity. It assesses how many days of training an average worker in a given industry needs to complete
a job, and is hence a direct measure of an industry’s skill content. The data is available for
a subset of 3 digit SIC codes at the 1972 revision. To match these with ISIC codes, we first
concord them to the 1987 SIC revision using concordance tables by the NBER. A
pp
29 Details are provided in Appendix B. All concordances and classifications are available from the authors upon
request. This article is protected by copyright. All rights reserved. 27 See Koopman et al. (2012) for an analysis of China’s processing trade.
28 This article is protected by copyright. All rights reserved. 27 See Koopman et al. (2012) for an analysis of China’s processing trade.
28 28 See Appendix B for details. Koopman et al. (2012) for an analysis of China’s processing trade.
A
di
B f
d t il S
K
t
l (2012) f
l
i
f Chi
’
i
t
d 28 See Appendix B for details.
29 te
3
Results cept
OThis section is structured in three parts. We start by discussing the outcome of equation (2.6),
which estimates the net effect that higher values of DVC integration have for subsequent GVC
integration. In the second part, we assess the mechanism behind this effect by disentangling the
role of fragmentation and switching costs as identified by equation (2.7). In the last part of the
section we present a series of robustness checks. p
2.4
Data We then use a
concordance table between SIC87 and ISIC Rev. 3 built by Statistics Canada.29 Secondly, in a
separate exercise we replace the Nunn’s proxy for contract intensity with US capital intensity
data from the OECD. In capital intensive industries, suppliers are often required to make large
sunk investments. Consequently, they are likely to demand contracts with high cancellation fees. This makes capital intensity a likely proxy for switching costs. This data is readily available at
the ISIC level. d Article
The country coverage comprises developing and developed economies, allowing to examine
whether the level of development plays any role in shaping the relationship between domestic
and global value chains. In addition, the industry coverage includes primary, manufacturing,
and services sectors. The latter play an increasingly dominant role in value chains. However,
there are no tariffs nor Rauch measures available for services. Therefore, we use estimates
based only on the manufacturing and primary sectors as benchmark but perform robustness
checks that include services without the tariffcontrol, assuming that all services industries
are differentiated and excluding non-tradeable services industries such as education, health, or
construction. Concerning the time period examined, we use 1995 and 2008 because these are the
earliest and latest available data points before the start of the prolonged global financial crisis. Finally, we exclude in the benchmark estimations the oil exporting countries Saudi Arabia
and Brunei; Chinese Taipei, for which no tariffdata is available; and the United States, to
avoid endogeneity stemming from the construction of the weighted tariffand capital intensity
variables.30 3.1
The net effect of DVCs on GVCs A
Table 3 reports the results for the first part of this exercise. The coefficient on dvc is positive
and statistically significant in all specifications, indicating that domestic value chains facilitate
30 Exclusion of the United States from the sample is standard practice in the literature that uses US IO weights
– see for instance Beverelli et al. (2017). A
Table 3 reports the results for the first part of this exercise. The coefficient on dvc is positive
and statistically significant in all specifications, indicating that domestic value chains facilitate A
30 Exclusion of the United States from the sample is standard practice in the literature that uses US IO weights
– see for instance Beverelli et al. (2017). A
30 Exclusion of the United States from the sample is standard practice in the literature that uses US IO weights
– see for instance Beverelli et al. (2017). This article is protected by copyright. All rights reserved. subsequent GVC integration. @ Table 3 about here A @ Table 3 about here A This article is protected by copyright. All rights reserved. @ Table 3 about here A rticle
Columns (1) and (2) give the results for primary and manufacturing sectors only, while
columns (3) and (4) include industries in the services sector, but at the cost of not controlling
for tariffs. While all estimates are similar in magnitude and significance, we take the point
estimate in column (2) as benchmark because it includes the full set of fixed effects described in
equation (2.6) and it is comparable to the analysis in Section 3.2. d Ar
The benchmark specification suggests that a one unit increase in initial DVC integration
leads to 2.4% higher subsequent GVC integration. This implies that a one standard deviation
increase in DVC integration raises GVC integration by approximately 0.2 standard deviations
or 0.4%. In addition, back of the envelope calculations comparing R squared of the benchmark
regression with and without the dvc measure suggest that DVCs can reduce the unexplained
variation in contemporaneous GVC integration by about 30%. Accepte
Since our benchmark estimates only allow for within-broad-industry-country variation, they
might hide gains that stem from variation in DVC integration across countries which are empha-
sized by some studies such as Bartelme and Gorodnichenko (2015). Therefore, we additionally
report in columns (1) and (3) results that only control for industry fixed effects, including in the
vector of controls the following three variables: log of per capita GDP at constant 2005 prices;
log of GDP at constant 2005 prices; and log distance to the closest GVC hub (CHN, DEU,
JPN, or USA). These variables capture basic country characteristics such as size, development
status, and remoteness. Since the estimates in columns (1) and (3) are potentially subject to an
omitted variable bias due to the exclusion of country fixed effects, we interpret them as upper
bounds of the DVC effect. Table 3 shows that the coefficients are indeed larger in magnitude
than the benchmark estimates, although the difference is not large. This article is protected by copyright. All rights reserved. icle
In summary, these findings highlight a further advantage of comprehensive domestic linkages
through their contribution to GVC participation. In the light of the literature that has unveiled
productivity enhancing effects of GVC integration (Kummritz, 2016), our results can help explain
why efficient DVCs facilitate economic development. Our findings also speak to the literature
on the determinants of GVC integration. We show that differences in pre-existing domestic
linkages can account for differences in observed GVC linkages. @ Table 3 about here A Arti
The descriptive evidence discussed in Section 2.1 has however illustrated that there are
countries with strong DVC linkages which are not well integrated into GVCs. To unveil the
heterogeneity in the GVC-DVC relationship, we now empirically assess the relevance of the
mechanism related to the industry-specific costs discussed above. A
3.2
The role of fragmentation and switching costs ed
In this section, we estimate equation (2.7), which separates the effects of DVCs on GVCs in LFHS
(low fragmentation, high switching costs) industries as opposed to HFHS (high fragmentation,
high switching costs) industries, and LFLS (low fragmentation, low switching costs) industries. cepte
As a reminder, we expect the coefficient on the LFHS dummy to be significantly smaller
than the coefficients on the HFHS and the LFLS dummies. This is due to the fact that the
former dummy captures industries in which switching costs tend to be high, which makes a
change from domestic to foreign suppliers costly, but fragmentation costs low, which shuts down
the stepping stone channel of DVCs for GVCs. In addition, we expect the coefficients on the
HFHS and the LFLS dummies to be not significantly different, since the relative incidence of
fragmentation and switching costs should be identical in the two cases. Ac
Table 4 reports the corresponding results. As in Table 3, columns (1) and (2) exclude
industries in the services sector, while columns (3) and (4) include them. We replicate in
columns (1) and (3) the basic results presented in Table 3 for comparison, while columns (2)
and (4) report the new estimates from equation (2.7). This article is protected by copyright. All rights reserved. @ Table 4 about here A icle
case of Singapore, ‘Textiles, leather and footwear – TEX’ in the case of Bulgaria).31 This means
that in these three under-performing countries the structure of the economy causes domestic
linkages to prevent GVC integration, despite the fact that other factors, such as location, would
facilitate GVC integration. For policy makers this implies that they need to take into account
both the strength of domestic linkages and the structure of their economies when they develop
GVC strategies. @ Table 4 about here A t c e
The results are in line with our expectations. The coefficients on the interaction of the
dvc variable with the HFHS and the LFLS dummies (δ1 and δ2 in equation (2.7)) are positive
and statistically significant in both samples, while the coefficients on the interaction with the
LFHS dummy (δ3 in equation (2.7)) is smaller and, in fact, not statistically different from zero. This is strong evidence in favour of the proposed channel which links initial DVC patterns to
subsequent GVC integration by taking into account the varying importance of fragmentation
and switching costs across industries. Ar
Our preferred specification in column (2) suggests that in HFHS and LFLS industries, a one
percentage point increase in initial DVC linkages increases contemporaneous GVC integration
by 2.3 to 3.1%. However, this positive effect of DVCs is not observed in LFHS industries. pted
In terms of our proposed channel, these findings are further evidence that fragmentation
costs are more important than switching costs for the DVC-GVC link. This is because in
industries where both costs are high (low), the positive effect of DVCs prevails. Similarly, even
when switching costs are high but fragmentation costs are low, the net effect is around zero
instead of turning negative. Thus, the net effect that we found in Section 3.1 is confirmed and
can be explained by the fact that fragmentation costs are more relevant than switching costs for
moving from DVCs to GVCs. Acce
These findings can also explain the observed heterogeneity in the DVC-GVC link as pre-
sented in the introduction. In Figure 3 we revisit the matched country pairs from Figure 1. There, we grouped countries based on the known structural and policy determinants of GVC
integration and showed that there remains substantial unexplained variation in the data. Here,
we add leading export industries to the chart. It is striking that all the leading export industries
of the under-performers (respectively, ‘Paper – PAP’ for Sweden, and ‘Chemicals – CHM’ for
Switzerland and for Romania) fall into the LFHS category whereas the comparison group is
more specialized in HFHS industries (‘Computer, electronic and optical products – CEQ’ in the This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. A
31 An exception is Belgium, which is also specialized in ‘Chemicals’. However, its DVC integration levels in
1995 were fairly low in this industry while Switzerland, Sweden and Romania had high values of DVC integration
in this industry.
32 Column (1) of Table 5 reproduces the estimates of the preferred specification of column (2) of Table 3 for
convenience. A
in this industry.
32 Column (1) of Table 5 reproduces the estimates of the preferred specification of column (2) of Table 3 for
convenience. This article is protected by copyright. All rights reserved. Ar
3.3
Robustness tests and IV regressions ccepted A
We expose our results to a battery of robustness checks starting with the identification assump-
tions. While we have argued that in 1995 reverse causality from GVCs to DVCs should have
been minor, since only 3% of global production was related to GVCs, there is evidence that
in high income countries GVCs did already play a role. We augment equation (2.6) with an
interaction term between the DVC measure and a high income dummy, equal to one if a country
was classified as high income in the World Bank classification in 1995. This allows us to see
if the effect is also present in developing economies, for which the assumption of the absence
of reverse causality is not questionable. This estimation can also provide insights on a possi-
ble development dimension to our findings. The results are in column (2) of Table 5.32 The
high income interaction term is not significant. We interpret this result (which is robust to
several alternative constructions of country groups) as an indication that the effect of DVC on
GVC integration is not driven by reverse causality, since the effect is also present in developing
economies, where GVCs linkages only developed in relatively recent years. @ Table 5 about here A A
31 An exception is Belgium, which is also specialized in ‘Chemicals’. However, its DVC integration levels in
1995 were fairly low in this industry while Switzerland, Sweden and Romania had high values of DVC integration
in this industry. 32 Column (1) of Table 5 reproduces the estimates of the preferred specification of column (2) of Table 3 for
convenience. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Article
Next, we further address concerns about omitted variable bias by fully exploiting the time
variation in our data. More specifically, we additionally use data for the years 2000 and 2005
and estimate a panel version of equation (2.6). The specification includes a one period lagged
DVC variable (following Bartelme and Gorodnichenko, 2015) and country-year, industry-year,
and industry-country fixed effects. The specification without the time dimension remains our
preferred model since the objective is to assess the effect of initial DVC conditions unaffected
by the rise of GVCs. However, the advantage of this robustness check is that we can add a
rich structure of fixed effects, controlling for any unobserved factor that might be correlated
with GVC and DVC and varies across two of the three dimensions (countries, industries and
time) of the data. Column (3) of Table 5 shows that the coefficient on dvc remains positive and
statistically significant. Its magnitude decreases by 75%. This can be explained by a potential
linear accumulation of the effect over time. cepted
The results of the instrumental variable regression using the variable dvcIV described in
Section 2.3 as an instrument for dvc are in column (4) of Table 5. All coefficients are in line, in
terms of sign, magnitude and statistical significance, with the benchmark coefficients reported
in column (1). The Cragg-Donald Wald F statistic of 176.6 is well above the corresponding
critical values tabulated in Stock and Yogo (2005), suggesting that the instrument is relevant. Note further that the first-stage correlation between the instrument and dvc is statistically
significant. Taken together, the results of columns (1)-(3) of Table 5 allow to conclude that
domestic linkages causally affect GVC participation, in other words they are stepping stones for
integration in global value chains. Acc
The stepping-stone result is subject to five further robustness checks addressing measure-
ment and sample selection issues. The results of these tests are displayed in columns (5)-(9)
of Table 5. @ Table 5 about here A First, we test if our results are dependent on the selected GVC measure. For this
purpose, we replace fvax with the i2p indicator of Baldwin and Lopez Gonzalez (2015), which
is simply the value of intermediate imports (see equation 2.4 and Section 2.1.1). We find in This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. column (5) of Table 5 that this variation has no relevant impact on our estimates. column (5) of Table 5 that this variation has no relevant impact on our estimates. icle
We then proceed to vary the sample composition by including the previously excluded
countries Saudi Arabia, Brunei, and the United States and, in a separate exercise, non tradeable
services industries to examine if the results are sensitive to specific sample changes (see Table
A-2 for the list of non-tradeable industries). Columns (6) and (7) of Table 5 show that the
results are not affected in any meaningful way. pted Arti
In a next step, we replace the GVC value of 2008 with the values of 2005 (in column (8)) or
2011 (in column (9)). This allows assessing whether the results are dependent on a specific time
period, in particular whether the global financial crisis is relevant for the DVC-GVC relationship. The estimates in columns (8) and (9) of Table 5 suggest that this is not the case. In addition, the
latter result that uses GVC values for 2011, combined with the estimates from the robustness
exercise that uses a panel setup (column (3)) reveals suggestive evidence on the role of DVCs in
the current slowdown in GVC expansion.33 The panel estimates suggest that the effect of DVCs
accumulates over time until 2008. However, the coefficient for 2008 is not statistically different
from the coefficient for 2011, implying that the accumulation has ended. This would be in line
with the fact that the boost that initial DVC integration provided to GVC expansion has been
fully exploited which, in turn, would contribute to explaining the observed slowdown in GVC
expansion. Accep
We now turn to a set of robustness checks concerning the mechanism behind the DVC-
GVC link of Table 4, starting with the results of IV regressions. A
(
)
34 The benchmark coefficients of column (1) of Table 4 are the ones of column (2) of Table 4, and are reported
for convenience. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. A
33 See, for instance, Degain et al. (2017) for recent evidence that the GVC expansion has stalled since 2008. @ Table 5 about here A The IV results, presented in
column (2) of Table 6, are similar, in terms of sign, magnitude and statistical significance, to
the benchmark coefficients of column (1).34 Namely, the coefficients on the interaction of the
dvc variable with the HFHS and the LFLS dummies are positive and statistically significant,
and the coefficient on the interaction with the LFHS dummy is smaller and not statistically ticle
different from zero. The value of the Cragg-Donald Wald F statistic of 54.65 again suggests that
the instrument is relevant, and the first-stage correlation between the instruments (interaction
terms between dvcIV and industry dummies) and the variables to be instrumented (interaction
terms between dvc and industry dummies) – presented in columns (2.a, 2.b and 2.c) – are
statistically significant. We therefore confirm that the mechanisms behind the causal effect of
initial DVC patterns on subsequent GVC integration are related to the varying importance of
fragmentation and switching costs across industries, as discussed in Section 3. @ Table 6 about here A @ Table 6 about here A cepted A
The next three columns of Table 6 present the results of three robustness checks that
concern the measuring of industry dummies that proxy for fragmentation and switching costs. Firstly, we vary the cutoffcondition which classifies industries as differentiated and, indirectly,
as contract intensive. For this, it is necessary to recall that the Rauch classification is at the 4
digit SITC level, while our industry classification used to construct the GVC and DVC variables
is the 2 digit ISIC. As a results, several SITC industries are assigned to one ISIC industry. As explained in detail in Appendix B, in the benchmark estimation, we define a 2 digit ISIC
industry as differentiated if the trade share of differentiated 4 digit SITC industries matched
with the 2 digit ISIC industry exceeds the mean value across all ISIC industries. We now use
the median as cutoffinstead. This reduces our LFHS industries to ‘Pulp and paper products’,
‘Chemicals’, and ‘Mining and quarrying’, with the other two in the last column of Table 2 being
now classified as HFHS industries. Acc
Column (3) of Table 4 shows the estimates for this slightly stricter cutoff. Qualitatively,
the results hold while the magnitude of the LFHS coefficient increases at the expense of the
coefficient on the HFHS interaction. @ Table 5 about here A Since we increased the cutoff, the ratio of switching to frag-
mentation costs in LFHS industries has increased causing a reclassification of LFHS industries
as HFHS industries. Therefore, the change in magnitudes is sensible and confirms our channel. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. rticle
In the two next robustness exercises, we change the measures used to classify the industries
into their respective categories. First, we use Costinot (2009)’s industrial complexity indicator to
proxy for fragmentation costs. As discussed in Section 2.4, it is a proxy for an industry’s average
skill intensity and can accordingly capture the costs of downsizing and protecting intellectual
property well. The results in column (4) of Table 6 show that this change has no relevant impact
on our estimates. As a second variation, we use capital intensity as an alternative proxy for
switching costs and equally find no meaningful impact on our results, as can be seen in column
(5) of Table 6. d Ar
Overall, the two main results concerning the net effect of DVCs for GVC integration, and
the mechanisms behind this link, are robust to variations in the identification assumptions, the
sample composition, and to the employed indicators and proxies. Therefore, we conclude that
DVCs and their interaction with a country’s industrial structure are an important driving factor
for integration in GVCs. e
4
Conclusions cept
GVCs have become a dominant factor in international trade. Recent theoretical and empirical
research has shown that integration in GVCs can lead to gains in productivity and welfare. This
begs the question of what drives GVC participation. While previous research has revealed a set
of important structural factors and policies, there remains substantial unexplained variation in
GVC integration patterns across countries. Acc
This paper has shed light on a new determinant of these patterns by providing evidence
that initial levels of DVC integration affect contemporaneous GVC integration. In other words,
levels of domestic fragmentation established before the rise of GVCs can explain a relevant
share of current variation in GVC integration. The results hold for countries at varying stages
of development, over varying time periods, and when changing the sample composition and
identifying assumptions. In our preferred specification, a one standard deviation increase in This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. DVC integration raises subsequent GVC integration by 0.4%. Besides, we are able to decrease
the unexplained variation in GVC integration by about 30%. Article
The mechanisms behind the DVC-GVC link investigated in this paper are related to industry-
specific fixed costs of fragmentation and of switching suppliers. On the one hand, high fixed
fragmentation costs allow, due their sunk nature, DVCs to act as stepping stones for GVCs. On the other hand, high fixed costs of switching suppliers should lead to a negative association
between DVCs and GVCs. Our findings on the net effect suggest that the fragmentation cost
channel is more important than the switching cost channel. Exploiting the industry variation in
fragmentation and switching costs in the data, we have confirmed that this is indeed the case. The facilitating effect of DVCs is absent in industries characterized by low fragmentation costs,
but high switching costs. ed
The policy implications are relevant and straightforward. Key barriers to GVC integration
might stem from the interplay of domestic linkages and industrial structure. These linkages
should be taken into account when designing and evaluating GVC integration strategies. This article is protected by copyright. All rights reserved. References e
Antr`as, P., 2003, “Firms, Contracts, and Trade Structure,” Quarterly Journal of Economics,
118, 1375–1418. c
Antr`as, P., T. C. Fort, and F. Tintelnot, 2017, “The Margins of Global Sourcing: Theory and
Evidence from U.S. Firms,” American Economic Review, 107, 2514–64. rti
Antr`as, P., and E. Helpman, 2004, “Global Sourcing,” Journal of Political Economy, 112, 552–
580. This article is protected by copyright. All rights reserved. 580. Ar
Balassa, B., 1965, “Trade Liberalisation and “Revealed” Comparative Advantage,” The Manch-
ester School, 33, 99–123. A
Baldwin, R., and J. Lopez Gonzalez, 2015, “Supply-chain Trade: A Portrait of Global Patterns d
Baldwin, R., and J. Lopez Gonzalez, 2015, “Supply-chain Trade: A Portrait of Global Patterns
and Several Testable Hypotheses,” The World Economy, 38, 1682–1721. ed
Baldwin, R., and F. Robert-Nicoud, 2014, “Trade-in-goods and trade-in-tasks: An integrating
framework,” Journal of International Economics, 92, 51–62. pt
Bartelme, D., and Y. Gorodnichenko, 2015, “Linkages and Economic Development,” NBER
Working Paper No. 21251, National Bureau of Economic Research. t
Bartelme, D., and Y. Gorodnichenko, 2015, “Linkages and Economic Development,” NBER pt
Working Paper No. 21251, National Bureau of Economic Research. p
Working Paper No. 21251, National Bureau of Economic Research. ep
Bernard, A. B., A. Moxnes, and Y. U. Saito, 2015, “Production networks, geography and firm
performance,” NBER Working Paper No. 21082, National Bureau of Economic Research. Acc
Beverelli, C., M. Fiorini, and B. Hoekman, 2017, “Services trade policy and manufacturing
productivity: The role of institutions,” Journal of International Economics, 104, 166–182. Blaum, J., C. Lelarge, and M. Peters, 2015, “The Gains From Trade in Firm-Based Models of
Importing,” unpublished manuscript, available at https://www.brown.edu/Departments/
Economics/Faculty/Joaquin_Blaum/GainsFromInputTrade.pdf. cc
Beverelli, C., M. Fiorini, and B. Hoekman, 2017, “Services trade policy and manufacturing
productivity: The role of institutions,” Journal of International Economics, 104, 166–182. Acc
Beverelli, C., M. Fiorini, and B. Hoekman, 2017, “Services trade policy and manufacturing
productivity: The role of institutions,” Journal of International Economics, 104, 166–182. Blaum, J., C. Lelarge, and M. Peters, 2015, “The Gains From Trade in Firm-Based Models of
I
ti
”
bli h d
i t
il bl
t htt
//
b
d /D
t
t / Ac
Blaum, J., C. Lelarge, and M. Peters, 2015, “The Gains From Trade in Firm-Based Models of
Importing,” unpublished manuscript, available at https://www.brown.edu/Departments/
Economics/Faculty/Joaquin_Blaum/GainsFromInputTrade.pdf. This article is protected by copyright. All rights reserved. Costinot, A., 2009, “On the origins of comparative advantage,” Journal of International Eco-
nomics, 77, 255–264. cle
Degain, C., B. Meng, and Z. Wang, 2017, “Recent trends in global trade and global value
chains,” in Global Value Chain Development Report 2017, World Bank, IDE-JETRO, OECD,
RCGVC-UIBE, WTO. RCGVC-UIBE, WTO. RCGVC-UIBE, WTO. rti
Dhyne, E., and S. Rub´ınov´a, 2016, “The supplier network of exporters: Connecting the dots,”
Working Paper No. 296, National Bank of Belgium. Fally, T., and R. Hillberry, 2015, “A Coasian Model of International Production Chains,” NBER
Working Paper No. 21520, National Bureau of Economic Research. d
Grossman, G. M., and E. Helpman, 2003, “Outsourcing Versus FDI in Industry Equilibrium,”
Journal of the European Economic Association, 1, 317–327. ed
Grossman, G. M., and E. Helpman, 2005, “Outsourcing in a Global Economy,” Review of
Economic Studies, 72, 135–159. pte
Grossman, G. M., and E. Rossi-Hansberg, 2008, “Trading Tasks: A Simple Theory of Off-
shoring,” American Economic Review, 98, 1978–97. ep
Hirschman, A. O., 1958, The strategy of economic development, New Haven: Yale University
Press. ce
Hsieh, C.-T., and P. J. Klenow, 2009, “Misallocation and Manufacturing TFP in China and
India,” Quarterly Journal of Economics, 124, 1403–1448. Ac
Hummels, D., J. Ishii, and K.-M. Yi, 2001, “The nature and growth of vertical specialization in
world trade,” Journal of International Economics, 54, 75–96. A
Johnson, R. C., and G. Noguera, 2012a, “Accounting for intermediates: Production sharing and
trade in value added,” Journal of International Economics, 86, 224–236. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Johnson, R. C., and G. Noguera, 2012b, “Fragmentation and Trade in Value Added over Four
Decades,” NBER Working Paper No. 18186, National Bureau of Economic Research. cle
Jones, C. I., 2011, “Intermediate Goods and Weak Links in the Theory of Economic Develop-
ment,” American Economic Journal: Macroeconomics, 3, 1–28. rtic
Jones, R. W., and H. Kierzkowski, 1990, “The role of services in production and international
trade: a theoretical framework,” in R. W. Jones, and A.O. Krueger (ed.), The Political Econ-
omy of International Trade: Essays in Honour of Robert A. Mundell, MIT Press, Cambridge,
MA. A
Jones, R. W., and H. Kierzkowski, 2001, “A framework for fragmentation,” in S.W. Arndt,
and H. Kierzkowski (ed.), Fragmentation: New Production Patterns in the World Economy,
Oxford University Press, New York. ed
Koopman, R. B., Z. Wang, and S.-J. Wei, 2012, “Estimating domestic content in exports when
processing trade is pervasive,” Journal of Development Economics, 99, 178–189. te
Koopman, R. B., Z. Wang, and S.-J. Wei, 2014, “Tracing Value-Added and Double Counting in
Gross Exports,” American Economic Review, 104, 459–94. ep
Kowalski, P., J. Lopez Gonzalez, A. Ragoussis, and C. RCGVC-UIBE, WTO. Ugarte, 2015, “Participation of Developing
Countries in Global Value Chains,” OECD Trade Policy Paper No. 179, Organisation for
Economic Co-operation and Development. c
Kummritz, V., 2016, “Do Global Value Chains Cause Industrial Development?,” CTEI Working
Paper No. 2016-01, Centre for Trade and Economic Integration. c
Nunn, N., 2007, “Relationship-Specificity, Incomplete Contracts, and the Pattern of Trade,”
Quarterly Journal of Economics, 122, 569–600. A
Quast, B. A., and V. Kummritz, 2015, “decompr: Global Value Chain decomposition in R,”
CTEI Working Paper No. 2015-01, Centre for Trade and Economic Integration. This article is protected by copyright. All rights reserved. Rauch, J. E., 1999, “Networks versus markets in international trade,” Journal of International
Economics, 48, 7–35. cle
Schmeiser, K. N., 2012, “Learning to export: Export growth and the destination decision of
firms,” Journal of International Economics, 87, 89–97. Stock, J., and M. Yogo, 2005, “Testing for Weak Instruments in Linear IV Regression,” in
Donald W. K. Andrews (ed.), Identification and Inference for Econometric Models, New York:
Cambridge University Press. Ar
Timmer, M. P., A. A. Erumban, B. Los, R. Stehrer, and G. J. de Vries, 2014, “Slicing Up Global
Value Chains,” Journal of Economic Perspectives, 28, 99–118. Tintelnot, F., K. Kikkawa, M. Mogstad, and E. Dhyne, 2017, “Trade and Domestic Production
Networks,” unpublished manuscript, available at http://felix-tintelnot.wdfiles.com/
local-files/research/TKMD_draft.pdf. Tintelnot, F., K. Kikkawa, M. Mogstad, and E. Dhyne, 2017, “Trade and Domestic Production Tintelnot, F., K. Kikkawa, M. Mogstad, and E. Dhyne, 2017, “Trade and Domestic Production
Networks,” unpublished manuscript, available at http://felix-tintelnot.wdfiles.com/ local-files/research/TKMD_draft.pdf. Voigtl¨ander, N., 2014, “Skill bias magnified: Intersectoral linkages and white-collar labor demand
in US manufacturing,” Review of Economics and Statistics, 96, 495–513. p
Wang, Z., S.-J. Wei, X. Yu, and K. Zhu, 2017, “Characterizing Global Value Chains,” NBER
Working Paper No. 23261, National Bureau of Economic Research. p
Working Paper No. 23261, National Bureau of Economic Research. ce
Wang, Z., S.-J. Wei, and K. Zhu, 2013, “Quantifying International Production Sharing at the
Bilateral and Sector Levels,” NBER Working Paper No. 19677, National Bureau of Economic
Research. Revised March 2014. Research. Revised March 2014. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Article
Figures
Figure 1: GVC integration of matched countries
0! 5! 10! 15! 20! 25! 30! 35! 40! 45! 50! SWE! BEL! CHE! SGP! ROU! BGR! Notes: OECD data for 2008. Data are averaged across industries. GVC in-
tegration is measured as foreign value added in exports (normalized by total
exports). Country names are displayed in Table A-1. Figures Figure 1: GVC integration of matched countries 0! 5! 10! 15! 20! 25! 30! 35! 40! 45! 50! SWE! BEL! CHE! SGP! ROU! BGR! Notes: OECD data for 2008. Data are averaged across industries. GVC in-
tegration is measured as foreign value added in exports (normalized by total
exports). Country names are displayed in Table A-1. 0! 5! 10! 15! 20! 25! 30! 35! 40! 45! 50! SWE! BEL! CHE! SGP! ROU! BGR! Notes: OECD data for 2008. Data are averaged across industries. GVC in-
tegration is measured as foreign value added in exports (normalized by total
exports). Country names are displayed in Table A-1. Notes: OECD data for 2008. Data are averaged across industries. GVC in-
tegration is measured as foreign value added in exports (normalized by total
exports). Country names are displayed in Table A-1. Figure 2: Correlation scatterplot between GVC in 2008 and DVC in 1995 Notes: OECD data. Each point is a country-industry combination. The
GVC measure is from equation (2.3). The DVC measure is from equation
(2.5). Notes: OECD data. Each point is a country-industry combination. The
GVC measure is from equation (2.3). The DVC measure is from equation
(2.5). Notes: OECD data. Each point is a country-industry combination. The
GVC measure is from equation (2.3). The DVC measure is from equation
(2.5). This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Figure 3: Differences in GVC integration of matched countries – revisited 0! 5! 10! 15! 20! 25! 30! 35! 40! 45! 50! SWE! BEL! CHE! SGP! ROU! BGR! CHM! CHM! PAP! CHM! CEQ! TEX! Notes: OECD data for 2008. Data are averaged across industries. GVC in-
tegration is measured as foreign value added in exports (normalized by total
exports). Country names are displayed in Table A-1. Notes: OECD data for 2008. Data are averaged across industries. GVC in-
tegration is measured as foreign value added in exports (normalized by total
exports). Country names are displayed in Table A-1. This article is protected by copyright. This article is protected by copyright. All rights reserved. All rights reserved. es
Table 1: DVC integration by country in 1995
Top 10
Bottom 10
Country
DVC
Country
DVC
China
57.73%
Austria
38.68%
Singapore
56.03%
Israel
38.11%
Czech Republic
53.59%
Canada
37.92%
Slovakia
53.14%
Greece
37.56%
Bulgaria
51.37%
Hong Kong
36.85%
Korea
50.14%
Malta
35.92%
New Zealand
49.91%
Mexico
34.69%
Estonia
48.52%
Luxembourg
29.76%
Hungary
48.07%
Cyprus
28.84%
Croatia
47.95%
Cambodia
23.50%
Notes: OECD data for 1995. Data are averaged across industries. The DVC
measure is from equation (2.5). Tables Tables Table 1: DVC integration by country in 1995 Table 1: DVC integration by country in 1995 Top 10
Bottom 10
Country
DVC
Country
DVC
China
57.73%
Austria
38.68%
Singapore
56.03%
Israel
38.11%
Czech Republic
53.59%
Canada
37.92%
Slovakia
53.14%
Greece
37.56%
Bulgaria
51.37%
Hong Kong
36.85%
Korea
50.14%
Malta
35.92%
New Zealand
49.91%
Mexico
34.69%
Estonia
48.52%
Luxembourg
29.76%
Hungary
48.07%
Cyprus
28.84%
Croatia
47.95%
Cambodia
23.50%
Notes: OECD data for 1995. Data are averaged across industries. The DVC
measure is from equation (2.5). Top 10
Bottom 10 Table 2: Industries by category Table 2: Industries by category
LFLS
HFHS
LFHS
Agriculture
Textile and leather products
Mining and quarrying
Food and beverages
Wood products
Pulp and paper products
Coke and refined petroleum
Non metallic mineral products
Chemicals
Basic metals
Fabricated metals
Rubber and plastics
Machinery n.e.c. Electrical machinery n.e.c. Electronic and optical products
Motor vehicles
Other transport equipment
Manufacturing n.e.c. Notes: Based on the Rauch and Nunn classifications. L - low, H - high, F - fragmentation costs, S -
switching costs. Industries are defined using the ISIC Rev. 3, 2 digit classification (see Table A-2). Notes: Based on the Rauch and Nunn classifications. L - low, H - high, F - fragmentation costs, S -
switching costs. Industries are defined using the ISIC Rev. 3, 2 digit classification (see Table A-2). This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Table 3: The net effect of DVC integration on GVC integration
(1)
(2)
(3)
(4)
Primary & Manufacturing
All sectors
dvc
2.566***
2.275***
2.900***
2.422***
[0.451]
[0.484]
[0.379]
[0.373]
ca
1.519***
1.099***
1.472***
1.025***
[0.074]
[0.073]
[0.065]
[0.062]
ctau
-0.002
-0.014
[0.013]
[0.019]
Observations
1,025
1,025
1,449
1,449
R-squared
0.705
0.877
0.704
0.872
Notes: *** p@0.01, ** p@0.05, * p@0.1. Robust standard errors in parentheses. Dependent
variable: fvax (see equation (2.3)). This article is protected by copyright. All rights reserved. Columns (1) and (3) include industry fixed effects and
log of GDP, log of per capita GDP, and log of hub distance as controls. Columns (2) and
(4) include industry and broad industry-country fixed effects. All sectors include primary,
manufacturing and services. Table 3: The net effect of DVC integration on GVC integration Table 4: Industrial heterogeneity in the effect of DVC integration on GVC integration ble 4: Industrial heterogeneity in the effect of DVC integration on GVC integration ble 4: Industrial heterogeneity in the effect of DVC integration on GVC integrat
(1)
(2)
(3)
(4)
Primary & Manufacturing
All sectors
dvc
2.275***
2.422***
[0.484]
[0.373]
dvc LFLS
3.068***
3.081***
[0.706]
[0.690]
dvc HFHS
2.336***
2.552***
[0.788]
[0.484]
dvc LFHS
0.196
0.123
[0.845]
[0.818]
ca
1.099***
1.085***
1.025***
1.014***
[0.073]
[0.074]
[0.062]
[0.062]
ctau
-0.014
-0.010
[0.019]
[0.018]
Observations
1,025
1,025
1,449
1,449
R-squared
0.877
0.879
0.872
0.873
Notes: *** p@0.01, ** p@0.05, * p@0.1. Robust standard errors in parentheses. Dependent
variable: fvax (see equation (2.3)). Columns (1) and (3) report the benchmark estimations
of columns (2) and (4) of Table 3. All columns include industry and broad industry-country
fixed effects.All sectors include primary, manufacturing and services. Notes: *** p@0.01, ** p@0.05, * p@0.1. Robust standard errors in parentheses. Dependent
variable: fvax (see equation (2.3)). Columns (1) and (3) report the benchmark estimations
of columns (2) and (4) of Table 3. All columns include industry and broad industry-country
fixed effects.All sectors include primary, manufacturing and services. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Table 5: The net effect of DVC integration on GVC integration – Robustness
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
Benchmark
High income
Panel
IV
GVC measure
All countries
All sectors
2005
2011
Dep. Variable:
fvax
fvax
fvax
i2p
fvax
fvax
fvax
fvax
fvax
dvc
2.275***
1.892***
0.568**
2.744***
2.008***
1.717***
2.598***
2.127***
2.448***
[0.484]
[0.716]
[0.222]
[0.630]
[0.449]
[0.506]
[0.346]
[0.462]
[0.530]
dvc high income
0.770
[0.867]
ca
1.099***
1.098***
0.240***
1.103***
0.749***
1.167***
1.090***
1.210***
1.157***
[0.073]
[0.072]
[0.032]
[0.063]
[0.060]
[0.081]
[0.063]
[0.071]
[0.085]
ctau
-0.014
-0.014
0.002
-0.015
0.020
0.009
0.001
-0.014
[0.019]
[0.019]
[0.006]
[0.016]
[0.016]
[0.024]
[0.016]
[0.019]
Cragg-Donald Wald F statistic
176.6
First stage (dep. This article is protected by copyright. All rights reserved. variable: dvc)
dvcIV
-19.173***
[3.761]
Observations
1,025
1,025
2,896
1,025
1,026
1,078
1,883
1,025
989
R-squared
0.877
0.877
0.748
0.877
0.884
0.881
0.858
0.881
0.873
Notes: *** p@0.01, ** p@0.05, * p@0.1. Robust standard errors in parentheses. Dependent variable: fvax (see equation (2.3)) in columns (1)-(3) and (5)-(9); i2p (see equation
(2.4)) in column (4). All columns excluding column (3) include industry and broad industry-country fixed effects. Column (3) includes country-year, industry-year, and
industry-country fixed effects. All columns excluding column (7) exclude services industries. Number of observations in first stage regression: 1,025. dvcIV defined in footnote
26. protected by copyright. All rights reserved. e net effect of DVC integration on GVC integration – Robustness This article is protected by copyright. All rights reserved. Table 6: Industrial heterogeneity in the effect of DVC integration on GVC integration – Robustness Table 6: Industrial heterogeneity in the effect of DVC integration on GVC integration – Robustness
(1)
(2)
(3)
(4)
(5)
Benchmark
IV
Cutoff
Complexity
Capital intensity
dvc LFLS
3.068***
2.271***
3.106***
3.050***
3.093***
[0.706]
[0.637]
[0.766]
[0.841]
[0.776]
dvc HFHS
2.336***
4.603***
1.962***
2.472***
1.929***
[0.788]
[1.025]
[0.694]
[0.684]
[0.694]
dvc LFHS
0.196
1.169
0.701
1.236
1.621
[0.845]
[0.863]
[0.930]
[0.976]
[1.082]
ca
1.085***
1.074***
1.093***
1.100***
1.100***
[0.074]
[0.064]
[0.075]
[0.075]
[0.074]
ctau
-0.010
-0.012
-0.012
-0.020
-0.014
[0.018]
[0.016]
[0.018]
[0.020]
[0.019]
(2.a)
(2.b)
(2.c)
Cragg-Donald Wald F statistic
54.65
First stage dep. variable:
dvc LFLS
dvc HFHS
dvc LFHS
dvcIV LFLS
-18.462***
0.026
1.065
[4.864]
[0.710]
[0.655]
dvcIV HFHS
1.533*
-25.414***
0.186
[0.917]
[4.819]
[0.769]
dvcIV LFHS
3.607***
-0.146
-22.595***
[1.308]
[1.366]
[3.089]
Observations
1,025
1,025
1,025
911
1,025
R-squared
0.879
0.878
0.878
0.879
0.878
Notes: *** p@0.01, ** p@0.05, * p@0.1. Robust standard errors in parentheses. Dependent variable: fvax (see equation (2.3)). All columns
include industry and broad industry-country fixed effects. All columns exclude services industries. Number of observations in first stage
regressions: 1,025. dvcIV defined in footnote 26. Table 6: Industrial heterogeneity in the effect of DVC integration o This article is protected by copyright. All rights reserved. Note: Countries in bold excluded in benchmark estimations (Tables 3 and 4). This article is protected by copyright. All rights reserved. pted Article
Appendices
A
Data
Table A-1: OECD ICIO country coverage
ISO3
Country
ISO3
Country
AUS
Australia
ITA
Italy
ARG
Argentina
JPN
Japan
AUT
Austria
KHM
Cambodia
BEL
Belgium
KOR
Korea
BGR
Bulgaria
LTU
Lithuania
BRA
Brazil
LUX
Luxembourg
BRN
Brunei
LVA
Latvia
CAN
Canada
MEX
Mexico
CHE
Switzerland
MLT
Malta
CHL
Chile
MYS
Malaysia
CHN
China
NLD
Netherlands
COL
Colombia
NOR
Norway
CRI
Costa Rica
NZL
New Zealand
CYP
Cyprus
PHL
Philippines
CZE
Czech Republic
POL
Poland
DEU
Germany
PRT
Portugal
DNK
Denmark
ROU
Romania
ESP
Spain
RUS
Russia
EST
Estonia
SAU
Saudi Arabia
FIN
Finland
SGP
Singapore
FRA
France
SVK
Slovak Republic
GBR
United Kingdom
SVN
Slovenia
GRC
Greece
SWE
Sweden
HKG
Hong Kong, China
THA
Thailand
HRV
Croatia
TUN
Tunisia
HUN
Hungary
TUR
Turkey
IDN
Indonesia
TWN
Chinese Taipei
IND
India
USA
United States
IRL
Ireland
VNM
Viet Nam
ISL
Iceland
ZAF
South Africa
ISR
Israel
Note: Countries in bold excluded in benchmark estimations (Tables 3 and 4). Table A-1: OECD ICIO country coverage ISO3
Country
ISO3
Country
AUS
Australia
ITA
Italy
ARG
Argentina
JPN
Japan
AUT
Austria
KHM
Cambodia
BEL
Belgium
KOR
Korea
BGR
Bulgaria
LTU
Lithuania
BRA
Brazil
LUX
Luxembourg
BRN
Brunei
LVA
Latvia
CAN
Canada
MEX
Mexico
CHE
Switzerland
MLT
Malta
CHL
Chile
MYS
Malaysia
CHN
China
NLD
Netherlands
COL
Colombia
NOR
Norway
CRI
Costa Rica
NZL
New Zealand
CYP
Cyprus
PHL
Philippines
CZE
Czech Republic
POL
Poland
DEU
Germany
PRT
Portugal
DNK
Denmark
ROU
Romania
ESP
Spain
RUS
Russia
EST
Estonia
SAU
Saudi Arabia
FIN
Finland
SGP
Singapore
FRA
France
SVK
Slovak Republic
GBR
United Kingdom
SVN
Slovenia
GRC
Greece
SWE
Sweden
HKG
Hong Kong, China
THA
Thailand
HRV
Croatia
TUN
Tunisia
HUN
Hungary
TUR
Turkey
IDN
Indonesia
TWN
Chinese Taipei
IND
India
USA
United States
IRL
Ireland
VNM
Viet Nam
ISL
Iceland
ZAF
South Africa
ISR
Israel
Note: Countries in bold excluded in benchmark estimations (Tables 3 and 4). This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Table A-2: OECD ICIO industry coverage pted Article
ISIC
Industry
Industry
Broad
Rev. pt
Notes: Industries in bold are classified as non-tradeable. Benchmark estimations (Tables 3 and 4) only include
industries up to ISIC 37. This article is protected by copyright. All rights reserved. 3
code
description
industry
01T05
AGR
Agriculture
Primary
10T14
MIN
Mining and quarrying
Primary
15T16
FOD
Food products, beverages, and tobacco
Light manufacturing
17T19
TEX
Textiles, leather and footwear
Light manufacturing
20
WOD
Wood and products of wood and cork
Light manufacturing
21T22
PAP
Pulp, paper, paper products, printing and publishing
Light manufacturing
23
PET
Coke, refined petroleum products and nuclear fuel
Raw material processing
24
CHM
Chemicals and chemical products
Raw material processing
25
RBP
Rubber and plastics products
Raw material processing
26
NMM
Other non-metallic mineral products
Raw material processing
27
MET
Basic metals
Heavy manufacturing
28
FBM
Fabricated metal products
Heavy manufacturing
29
MEQ
Machinery and equipment n.e.c
Heavy manufacturing
30,32,33
CEQ
Computer, electronic and optical products
Electronics
31
ELQ
Electrical machinery and apparatus n.e.c
Electronics
34
MTR
Motor vehicles, trailers and semi-trailers
Heavy manufacturing
35
TRQ
Other transport equipment
Heavy manufacturing
36T37
OTM
Manufacturing n.e.c; recycling
Light manufacturing
40T41
EGW
Electricity, gas and water supply
Services
45
CON
Construction
Services
50T52
WRT
Wholesale and retail trade
Services
55
HTR
Hotels and restaurants
Services
60T63
TRN
Transport and storage
Services
64
PTL
Post and telecommunications
Services
65T67
FIN
Finance and insurance
Services
70
REA
Real estate activities
Services
71
RMQ
Renting of machinery and equipment
Services
72
ITS
Computer and related activities
Services
73T74
BZS
Research and development and other business services
Services
75
GOV
Public administration and defence
Services
80
EDU
Education
Services
85
HTH
Health and social work
Services
90T93
OTS
Other community, social and personal services
Services
95
PVH
Private households with employed persons
Services
Notes: Industries in bold are classified as non-tradeable. Benchmark estimations (Tables 3 and 4) only include
industries up to ISIC 37. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Notes: Industries are ranked by decreasing DVC integration. Only industries
included in benchmark estimations (Tables 3 and 4) are reported. The DVC
measure is defined in equation (2.5).
Industry descriptions are displayed in
Table A-2. ti
B
Construction of cost proxies rt
B.1
Fragmentation costs Table A-5: Summary statistics for main variables Table A-5: Summary statistics for main variables Variable
Observations
Mean
Std. Dev. Min
Max
dvc
1025
0.54
0.13
0.06
0.94
fvax
1025
6.28
2.13
-2.65
12.23
ca
1025
0.36
0.48
0.00
1.00
ctau
1025
4.92
4.48
0.00
30.54
Notes: In-sample statistics based on benchmark estimations (column (2) of Table 3). The variable dvc (see equation (2.5)) is for the year 1995. The variable fvax (see
equation (2.3)) is for the year 2008. This article is protected by copyright. All rights reserved. Table A-3: DVC integration, country average across industries Table A-3: DVC integration, country average across industries ISO3
DVC
ISO3
DVC
CHN
0.58
CHL
0.44
SGP
0.56
TUR
0.43
CZE
0.54
SVN
0.43
SVK
0.53
RUS
0.43
BGR
0.51
IDN
0.43
KOR
0.50
USA
0.43
NZL
0.50
FRA
0.42
EST
0.49
PHL
0.42
TWN
0.48
DEU
0.42
HUN
0.48
CHE
0.41
HRV
0.48
ISL
0.41
ROU
0.48
MYS
0.41
POL
0.48
CRI
0.40
AUS
0.47
TUN
0.39
VNM
0.47
DNK
0.39
BEL
0.47
COL
0.39
IRL
0.47
NOR
0.39
ESP
0.47
ARG
0.39
PRT
0.46
AUT
0.39
ITA
0.46
ISR
0.38
LVA
0.46
CAN
0.38
FIN
0.45
GRC
0.38
IND
0.45
HKG
0.37
LTU
0.45
MLT
0.36
BRA
0.44
MEX
0.35
JPN
0.44
LUX
0.30
ZAF
0.44
CYP
0.29
GBR
0.44
SAU
0.28
NLD
0.44
BRN
0.27
THA
0.44
KHM
0.24
SWE
0.44
Notes: Countries are ranked by decreasing DVC integration. Only countries
included in benchmark estimations (Tables 3 and 4) are reported. The DVC
measure is defined in equation (2.5). Country names are displayed in Table A-1. Notes: Countries are ranked by decreasing DVC integration. Only countries
included in benchmark estimations (Tables 3 and 4) are reported. The DVC
measure is defined in equation (2.5). Country names are displayed in Table A-1. Notes: Countries are ranked by decreasing DVC integration. Only countries
included in benchmark estimations (Tables 3 and 4) are reported. The DVC
measure is defined in equation (2.5). Country names are displayed in Table A-1. This article is protected by copyright. All rights reserved. Table A-4: DVC integration, industry average across countries Table A-4: DVC integration, industry average across countries ISIC Rev. 3
Industry code
DVC
34
MTR
0.70
15T16
FOD
0.69
27
MET
0.66
17T19
TEX
0.64
20
WOD
0.63
25
RBP
0.62
35
TRQ
0.61
24
CHM
0.60
36T37
OTM
0.60
30,32,33
CEQ
0.59
29
MEQ
0.59
31
ELQ
0.58
21T22
PAP
0.58
28
FBM
0.54
26
NMM
0.52
23
PET
0.49
60T63
TRN
0.44
01T05
AGR
0.41
65T67
FIN
0.38
73T74
BZS
0.36
50T52
WRT
0.35
72
ITS
0.35
10T14
MIN
0.32
71
RMQ
0.31
64
PTL
0.31
Notes: Industries are ranked by decreasing DVC integration. Only industries
included in benchmark estimations (Tables 3 and 4) are reported. The DVC
measure is defined in equation (2.5). Industry descriptions are displayed in
Table A-2. This article is protected by copyright. All rights reserved. Ar
Rauch (1999)’s product differentiation d A
As discussed in Section 2, we use the Rauch classification as benchmark measure for fragmen-
tation costs. The classification is available in two variants (liberal and conservative version) at
the 4 digit SITC Rev. 2 level. We choose the liberal version, which we concord to ISIC Rev. 3
using a crosswalk via SITC Rev. 3 based on concordance tables provided by Eurostat. pte
For the analysis, this creates two problems. First, the classification is only available for
the primary and manufacturing sectors. Second, due to the higher disaggregation several SITC
categories are matched with each individual ISIC industry. We take the first issue into account
by excluding industries in the services sector from the benchmark estimates and, in addition,
by including services in robustness checks in which we label them as differentiated. Acce
The second point is only problematic when the SITC categories concorded to ISIC industries
differ in their categorization. To determine the ISIC industry’s category in this case, we use gobal
trade data from WITS at the SITC 4 digit level. We classify ISIC industries as differentiated
if the trade share of differentiated SITC industries matched with them exceeds the mean value
across all ISIC industries. Alternatively, we use the median value for robustness checks. A
Costinot (2009)’s complexity A
Costinot (2009)’s complexity In cases where there are several complexity observations
matched with one ISIC industry, we use the simple mean. Table B-1 presents the resulting
industry level values of the complexity variable. ed A
categories. This means several SIC industries have identical complexity values. We match these
with our ISIC industries using a crosswalk via the 1987 SIC revision using concordance tables
by the NBER and Statistics Canada. In cases where there are several complexity observations
matched with one ISIC industry, we use the simple mean. Table B-1 presents the resulting
industry level values of the complexity variable. pt
B.2
Switching costs p
Nunn (2007)’s contract intensity p
Nunn (2007)’s contract intensity A
Costinot (2009)’s complexity A
Costinot’s measure is available for 41 unique observations covering 85 3 digit manufacturing SIC A
Costinot’s measure is available for 41 unique observations covering 85 3 digit manufacturing SIC This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Table B-1: Industry complexity Table B-1: Industry complexity ISIC Rev. 3
Industry code
Complexity
35
TRQ
28.23
24
CHM
26.32
30,32,33
CEQ
25.36
29
MEQ
21.98
34
MTR
21.77
28
FBM
19.42
27
MET
18.29
21T22
PAP
17.29
31
ELQ
15.84
26
NMM
15.59
20
WOD
12.47
15T16
FOD
12.38
36T37
OTM
11.41
25
RBP
10.10
17T19
TEX
5.04
Notes: Industries are ranked by decreasing value of complexity. Complexity is the
average number of training days a new workers requires to be prepared for a job in a
given industry. The data are taken from Costinot (2009) and concorded to ISIC Rev. 3. industry descriptions displayed in Table A-2. Notes: Industries are ranked by decreasing value of complexity. Complexity is the
average number of training days a new workers requires to be prepared for a job in a
given industry. The data are taken from Costinot (2009) and concorded to ISIC Rev. 3. industry descriptions displayed in Table A-2. ed A
categories. This means several SIC industries have identical complexity values. We match these
with our ISIC industries using a crosswalk via the 1987 SIC revision using concordance tables
by the NBER and Statistics Canada. In cases where there are several complexity observations
matched with one ISIC industry, we use the simple mean. Table B-1 presents the resulting
industry level values of the complexity variable. ed A
categories. This means several SIC industries have identical complexity values. We match these
with our ISIC industries using a crosswalk via the 1987 SIC revision using concordance tables
by the NBER and Statistics Canada. In cases where there are several complexity observations
matched with one ISIC industry, we use the simple mean. Table B-1 presents the resulting
industry level values of the complexity variable. ed A
categories. This means several SIC industries have identical complexity values. We match these
with our ISIC industries using a crosswalk via the 1987 SIC revision using concordance tables
by the NBER and Statistics Canada. This article is protected by copyright. All rights reserved. p
Nunn (2007)’s contract intensity p
Nunn (2007)’s contract intensity ce
Nunn’s contract intensity z is given by the share of differentiated intermediates in total inter-
mediates that an industry sources as follows: zi Q
j
θjiDifj, A
where θji mji~Pj mji, and Dif is a dummy equal to one if industry j is differentiated and
m’s are generic elements of an ICIO matrix. Thus, the measure is built based on the Rauch
classification and we can apply the values calculated using the methodology described above. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. le
To simplify the empirical analysis we then transform this continuous variable into a dummy by
defining each industry j as having high switching costs if zi is at least equal to the median z
of non-differentiated industries. For robustness exercises we replace this measure with capital
intensity data readily available from the OECD. This article is protected by copyright. All rights reserved.
|
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Colistin Selection of the Mcr-1 Gene in Broiler Chicken Intestinal Microbiota
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antibiotics
Article
Colistin Selection of the Mcr-1 Gene in Broiler Chicken
Intestinal Microbiota
Pedro Miguela-Villoldo 1,2 , Miguel A. Moreno 1,2 , Agustín Rebollada-Merino 1,3 ,
Antonio Rodríguez-Bertos 1,3 , Marta Hernández 4 , David Rodríguez-Lázaro 5 , Alejandro Gallardo 6 ,
Alberto Quesada 6,7 , Joaquín Goyache 1,2 , Lucas Domínguez 1,2 and María Ugarte-Ruiz 1, *
1
2
3
4
5
6
Citation: Miguela-Villoldo, P.;
Moreno, M.A.; Rebollada-Merino, A.;
7
*
VISAVET Health Surveillance Centre, Universidad Complutense de Madrid, Avenida Puerta de Hierro, s/n,
28040 Madrid, Spain; pedromig@ucm.es (P.M.-V.); mamoreno@ucm.es (M.A.M.); agusrebo@ucm.es (A.R.-M.);
arbertos@ucm.es (A.R.-B.); jgoyache@ucm.es (J.G.); lucasdo@visavet.ucm.es (L.D.)
Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avenida
Puerta de Hierro, s/n, 28040 Madrid, Spain
Departamento de Medicina y Cirugía Animal, Facultad de Veterinaria, Universidad Complutense de Madrid,
Avenida Puerta de Hierro, s/n, 28040 Madrid, Spain
Laboratorio de Biología Molecular y Microbiología, Instituto Tecnológico Agrario de Castilla y León, Ctra.
Burgos Km. 119, 47071 Valladolid, Spain; ita-HerPerMa@itacyl.es
Área de Microbiología, Departamento de Biotecnología y Ciencia de los Alimentos, Facultad de Ciencias,
Universidad de Burgos, Plaza Misael Bañuelos s/n, 09001 Burgos, Spain; drlazaro@ubu.es
Departamento de Bioquímica, Biología Molecular y Genética, Facultad de Veterinaria, Universidad de
Extremadura, Avda. de la Universidad s/n, 10003 Caceres, Spain; alexgsoler@unex.es (A.G.);
aquesada@unex.es (A.Q.)
INBIO G+C, Universidad de Extremadura, 0004 Caceres, Spain
Correspondence: maria.ugarte@ucm.es; Tel.: +34-913-944-097; Fax: +34-913-943-795
Rodríguez-Bertos, A.; Hernández, M.;
Rodríguez-Lázaro, D.; Gallardo, A.;
Quesada, A.; Goyache, J.;
Domínguez, L.; et al. Colistin
Selection of the Mcr-1 Gene in Broiler
Chicken Intestinal Microbiota.
Antibiotics 2021, 10, 677.
https://doi.org/10.3390/
antibiotics10060677
Academic Editor: Mark A Holmes
Received: 7 May 2021
Accepted: 3 June 2021
Published: 5 June 2021
Abstract: Colistin has a long story of safe use in animals for the treatment and prevention of certain
bacterial diseases. Nevertheless, the first description of the mcr-1 gene showed that colistin resistance
can spread by horizontal gene transfer and changed the landscape. This study aimed to assess the
effect of colistin administration on the dispersion of resistance in the microbiota of day-old broiler
chicks and how the presence of mcr-1 genes influences the spread of colistin resistance determinants.
In this study, 100 one-day-old chicks were divided into four groups of 25 animals (G1, G2, G3, and
G4). Animals from G3/G4 were challenged with mcr-1-carrying Salmonella (day 7), while colistin
(600 mg/L) was administered daily to G2/G4 animals through drinking water (from day 8 to day
15). Two quantitative PCR assays were performed to compare the amount of Salmonella and mcr-1
that were present in the caecal samples. We observed that levels of mcr-1 were higher in G3/G4
animals, especially G4, due to the spread of mcr-1-carrying Salmonella. On day 21, Salmonella levels
decreased in G4, reaching similar values as those for G3, but mcr-1 levels remained significantly
higher, suggesting that colistin may accelerate the spreading process when mcr-1-carrying bacteria
reach the gut.
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Keywords: colistin; mcr-1; Salmonella; in vivo assay; real-time PCR quantification; microbiota; chicks
published maps and institutional affiliations.
1. Introduction
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/
The gut microbiome constitutes a rich and diverse microbial ecosystem that influences
host nutrition and physiology, developing important functions that are beneficial for
host health [1]. The microbiota of new-born chicks, first colonised by bacteria of the
facultative anaerobic Proteobacteria phylum, progressively changes during the first 19
days of life [2]. Commensal Enterobacteriaceae is a major constituent of Proteobacteria and
plays an important role in the protection of the gut against colonisation by pathogens,
such as Salmonella enterica (especially serovars Typhimurium and Enteritidis), obligate
anaerobic spore-forming bacteria, such as Clostridia species, and facultative anaerobic
4.0/).
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https://www.mdpi.com/journal/antibiotics
Antibiotics 2021, 10, 677
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bacteria from the Streptococcus and Staphylococcus genera [3]. However, Proteobacteria,
especially Enterobacteriaceae, play a critical role in the emergence and maintenance of
antimicrobial resistance, as the genes responsible are more prone to be found in this phylum
(and also in Firmicutes) than in other phyla of the gut microbiome [4]. For example, plasmidencoded resistance genes can move between gut bacteria via horizontal gene transfer. Thus,
commensal Enterobacteriaceae become an excellent reservoir for antimicrobial resistance
genes, making them accessible to other pathogenic or commensal bacteria [4,5].
Antimicrobial resistance is a public health concern worldwide and has become an
increasing threat to human and animal health over the last decade, limiting the therapeutic
alternatives available to treat infections caused by multidrug-resistant (MDR) pathogens.
The increase of human infections due to multidrug-resistant Gram-negative bacteria, especially those producing extended-spectrum beta-lactamases and carbapenemases, has forced
the reintroduction of colistin to treat infections caused by these microorganisms in human
medicine, as it is often the only effective antimicrobial against them [6–8]. Consequently,
colistin has been classified among the antibiotic groups with activity against “Critical Priority” or “High Priority” pathogens identified by the World Health Organisation (WHO) [9]
and is considered to be the last line to treat these infections.
The prevalence of colistin resistance in Enterobacteriaceae from food animals has been
increasing annually and is thought to be related to the use of colistin in veterinary medicine
on a global scale [10,11]. Colistin has been used for decades in animals to treat and
prevent infectious diseases [6,12,13], usually administered orally in feed or drinking
water [14,15]. The Committee for Medicinal Products for Veterinary Use (CVMP) of the
European Medicines Agency (EMA) has restricted the administration of colistin sulphate
to poultry to 3 to 7 days to treat gastrointestinal infections [16].
Until 2015, all described colistin resistance mechanisms were related to chromosomal
point mutations, mainly involving the two-component regulatory system (pmrAB/phoPQ)
and its negative regulator mgrB. However, a new plasmid-mediated gene, called mcr-1,
was first described in that year, which meant that horizontal transfer was possible [8,17].
Thus, due to the increase of colistin resistance and the emergence of the mcr-1 gene, the
EMA recommended minimising the use of colistin in animals in the European Union (EU)
in 2016, aiming to limit its use to 5 mg/population correction unit (PCU) at the national
level, with a desirable level of 1 mg/PCU [15]. Since then, colistin resistance levels have
appeared to drop with the decrease in its use. We also observed this trend in a recent study
carried out in Spain [18].
There have been no studies of the effects of colistin administration on the intestinal
microbiota of the neonatal gut and its ability to select colistin-resistant bacteria. Thus, we
aimed to assess the effect of colistin administration on the dispersion of resistance in the
microbiota of day-old broiler chicks and how the presence of mcr-1 genes influences the
spread of colistin resistance determinants using a monophasic Salmonella Typhimurium
strain carrying a mcr-1 gene.
2. Results
2.1. Clinical Signs and Pathological Findings
Four animals (two animals from G1, one from G3, and one from G4) died during the
first week during the adjustment period. Post-mortem examination revealed fibrinous
pericarditis and perihepatitis (septicaemic colibacillosis) as the cause of death. We collected
samples from these animals, as their deaths occurred the day before the first sampling.
Thus, none of the samples were excluded from the experiment. There were no significant
changes found in any animal included in the study during the post-mortem examinations
throughout the experiment.
2.2. Salmonella Counts in the Chick Samples
The amount of Salmonella in the chick caecal samples was estimated by plate count.
Only animals from the Salmonella-challenged groups (G3 and G4) showed growth of
Antibiotics 2021, 10, x FOR PEER REVIEW
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The amount of Salmonella in the chick caecal samples was estimated by plate count.
Only animals from the Salmonella-challenged groups (G3 and G4) showed growth of SalSalmonella
SMID2
plates
(Figure
mean
values
non-statistically
different
monella ononSMID2
plates
(Figure
1). 1).
TheThe
mean
values
werewere
non-statistically
different
(p <
1 to 1.2 × 105 CFU/mg (mean of 2.0 × 104 CFU/mg) for G3
(p 0.05):
< 0.05):
from
<5.4
×
10
1
5
4
from <5.4 × 10 to 1.2 × 10 CFU/mg (mean of 2.0 × 10 CFU/mg) for G3 (without
1 to 3.6 × 105 CFU/mg (mean of 2.3 × 104 CFU/mg)
(without
and
colistin)colistin)
and from
<5.4from
× 101<5.4
to 3.6××10
105 CFU/mg
(mean of 2.3 × 104 CFU/mg) for G4 (colistin
fortreated).
G4 (colistin
treated).
Chicks
from
G4
had
higher
Salmonella
counts
from
D9 time
to D14,
Chicks from G4 had higher Salmonella counts
from D9 to
D14, at
which
G4 at
which
time
G4
reached
the
highest
value.
Finally,
after
the
withdrawal
of
colistin
(D14),
reached the highest value. Finally, after the withdrawal of colistin (D14), both G3 and G4
both
G3 and
G4 showed
similar
CFU
counts
(FigureSalmonella
1). All recovered
Salmonella
showed
similar
CFU counts
on D21
(Figure
1).on
AllD21
recovered
isolates were
mcrisolates
werebymcr-1-positive
by conventional PCR.
1-positive
conventional PCR.
Figure 1. Evolution of Salmonella
counts
over 21 days
in chick counts
caecal samples:
CTRL:
control
group;
Group
1 (G1):
animals
Figure
1. Evolution
of Salmonella
over 21 days
in chick
caecal
samples:
CTRL:
control
Group
1 (G1):
animals
without
colistin nor Group
Salmonella
challenge;
Group
2 (G2): colistin
adwithout colistin nor Salmonellagroup;
challenge;
Group
2 (G2):
colistin
administered;
3 (G3):
Salmonella
challenged;
Group
ministered;
Group
3 (G3): Salmonella
challenged;
Group1 4Salmonella
(G4): colistin
administered
Salmo4 (G4): colistin administered and
Salmonella
challenged.
Dots represent
outliers;
challenge
startedand
at D7
for
nella challenged. Dots represent outliers; 1 Salmonella challenge started at D7 for groups G3 and G4
groups G3 and G4 (sampling from D7 was performed before the challenge was started); 2 Colistin
was provided to chicks
(sampling from D7 was performed before the challenge was started); 2 Colistin was provided to
from groups G2 and G4 from D8
to D14.
chicks
from groups G2 and G4 from D8 to D14.
2.3.
Quantitative
2.3.
QuantitativeqPCR
qPCRofofthe
themcr-1
mcr-1 Gene
Gene
InIntotal,
caecal samples
sampleswere
wereanalysed
analysed
(7, 20,
20, 20,
20,and
20, 20
and
20 samtotal,87
87of
of 100
100 caecal
on on
D7 D7
(7, 20,
samples
from
the the
control
group,
G1, G2,
and
G4,and
respectively).
Overall, 13Overall,
samples13
were
exples
from
control
group,
G1,G3,
G2,
G3,
G4, respectively).
samples
cluded
because
it was it
impossible
to obtain
samplessamples
(at least (at
100least
mg)100
for mg)
DNAfor
were
excluded
because
was impossible
to enough
obtain enough
extraction.
DNA
extraction.
QuantitativePCR
PCRdata
datafor
forthe
the mcr-1
mcr-1 gene of
group
and
Quantitative
of the
the77animals
animalsfrom
fromthe
theCTRL
CTRL
group
and
21
animals
from
G1
(neither
receiving
colistin
nor
challenged
with
Salmonella)
showed
21 animals from G1 (neither receiving colistin nor challenged with Salmonella) showed
high
inter-individualvariability.
variability. Two
Two animals
animals from
the
barely
high
inter-individual
fromthe
theCTRL
CTRLgroup
groupshowed
showed
the
barely
detectable
presence
of
the
mcr-1
gene,
which
remained
below
the
quantification
limit,
and
detectable presence of the mcr-1 gene, which remained below the quantification limit,
and
33to 1.6 × 106 mcr-1
6
four
animals
from
G1
showed
quantifiable
levels
ranging
from
6.7
×
10
four animals from G1 showed quantifiable levels ranging from 6.7 × 10 to 1.6 × 10 mcr-1
genecopies
copiesper
permg
mgcaecal
caecalcontent,
content, establishing
establishing aa slight
1717
gene
slight background.
background.The
Theremaining
remaining
chicks
showedno
noqPCR
qPCRsignals
signalsor
or showed
showed mcr-1
The
chicks
showed
mcr-1 values
valuesbelow
belowthe
thequantitation
quantitationlimit.
limit.
The
values
for
the
mcr-1
levels
from
the
groups
not
challenged
with
Salmonella
(G1
and
G2)
values for the mcr-1 levels from the groups not challenged with Salmonella (G1 and G2)
remainedsimilar
similarthroughout
throughout most
reaching
their
peaks
on D11
(3.2 ×(3.2
105 and
remained
mostofofthe
thestudy,
study,
reaching
their
peaks
on D11
× 105
6
and 2.6 × 10 gene copies per mg caecal content, respectively) (Table 1). Concerning the
Salmonella-challenged groups (G3 and G4), chicks from G3 (colistin untreated) showed
an increase from less than 1.0 × 102 to 1.9 × 105 mcr-1 copies/mg from D9 to D21. The
2.6 × 106 gene copies per mg caecal content, respectively) (Table 1). Concerning the Salmonella-challenged groups (G3 and G4), chicks from G3 (colistin untreated) showed an increase from less than 1.0 × 102 to 1.9 × 105 mcr-1 copies/mg from D9 to D21. The number of
Antibiotics 2021, 10, 677
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mcr-1 copies/mg for chicks from G4 (colistin treated) increased from 1.5 × 103 to 2.1 × 410
for the same period. The greatest differences between the four groups were observed on
D11 and D14, coinciding with the end of colistin administration, with G4 showing significantly higher mcr-1 values, as the median of these values was outside the interquartile
number of mcr-1 copies/mg for chicks from G4 (colistin treated) increased from 1.5 × 103
range of the other groups (Figure 2). In addition, chickens from G1 and G2 showed a nonto 2.1 × 105 for the same period. The greatest differences between the four groups were
significant increase in the median from D9 to D21 (p = 0.23), whereas mcr-1 levels increased
observed on D11 and D14, coinciding with the end of colistin administration, with G4
throughout the study in the challenged groups, G3 and G4.
showing significantly higher mcr-1 values, as the median of these values was outside the
interquartile
range ofPCR
thedata
other
(Figure
2). In
G1 and G2
Table 1. Quantitative
forgroups
mcr-1 gene
copies/mg
peraddition,
day of lifechickens
(expressedfrom
in Log10).
showed a non-significant increase in the median from D9 to D21 (p = 0.23), whereas mcr-1
mcr-1 gene
copies/mg
(expressedgroups,
in Log10)
levels increased throughout
the study
in the challenged
G3 and G4.
1
SC
CA
Day 7
Day 9
Day 11
Day 14
Day 21
Table 1. Quantitative PCR
data
for
mcr-1
gene
copies/mg
per
day
of
life
(expressed
in
Log10).
̅
̅
̅
̅
̅
(D7) (D8)
𝒙
Me
𝒙
Me
𝒙
Me
𝒙
Me
𝒙
Me
CTRL 2
G1
G2
G3
G4
SC
(D7)
CA
(D8)
No
No
No
Yes
Yes
No
No
Yes
No
Yes
CTRL mcr-1 Gene Copies/mg (Expressed in Log10)
No
No 2.07 <2.0 NA NA NA
2
Day 7 1
¯ G1
No
Me
x
G2
No
Day 9
No¯
x
Yes
NA
No
2.49
Yes
<2.0
NA
NA
NA
NA
NA
Me
NA
Day 11
¯
2.49
2.60Me5.51
x
<2.0
<2.0
6.43
NA
NA
NA
Day 14
¯
2.52
3.83
Me 2.47
x
2.75
3.67
<2.0
NA
NA
Day 21
¯3.24
<2.0
Me
x
2.60
<2.0
2.07
<2.0
NA
NA
NA
NA
NA
NA
NA
Yes
NA 5.26
2.90 5.32
5.47 5.29
NA G3 NA
2.60
5.51 <2.02.524.47 3.83
2.47 5.24 3.24
<2.0
Yes
NA 4.23
5.38 6.37
5.68 5.34
NA G4 NA
<2.0
6.43 3.022.755.46 3.67
<2.0 6.01 2.60
<2.0
NA
<2.0
4.47(D8): Colistin
2.90 administration
5.32
5.24
5.47
5.29
SC (D7): NA
Salmonella 5.26
challenge (at
day 7), CA
(colistin treatment
NA
5.46data were
5.38not analysed
6.37 due 6.01
5.68 of animals;
5.34
started atNA
day 8), 𝑥̅ :4.23
Mean, Me:3.02
median; NA:
to the absence
CTRL:
control
animals without
colistin
nor Salmonella
challenge;
G2: Me:
colistin
adminisSC (D7): Salmonella challenge (at day
7), CA
(D8): group;
ColistinG1:
administration
(colistin
treatment
started at day
8), x: Mean,
median;
NA:
1 the CTRL
G3:ofSalmonella
challenged;
colistin
and
Salmonella
challenged;
data were not analysed due to thetered;
absence
animals; CTRL:
controlG4:
group;
G1: administered
animals without
colistin
nor Salmonella
challenge;
G2:
samples
wereG4:
composed
of a differentand
number
of animals
due 1tothe
theCTRL
inability
to obtain
sufficient
colistin administered; G3: Salmonella
challenged;
colistin administered
Salmonella
challenged;
samples
were composed
2 CTRL
of a different number of animals due
to the inability
obtainFive
sufficient
samples
from 13
chicks.
Five animals
group
were
sampled
samples
from 13 to
chicks.
animals
per group
were
sampled
for the per
other
days;
datafor
only
the other days; 2 CTRL data only available
for
D7.
After
D7,
chicks
were
considered
from
different
groups
(G1
to
G4),
depending
on the on
available for D7. After D7, chicks were considered from different groups (G1 to G4), depending
experimental conditions.
the experimental conditions.
Figure 2. Quantitative qPCR data of mcr-1 gene copies/mg obtained per day of life of the chicks for each studied group:
Group 1 (G1): animals without colistin nor Salmonella challenge; Group 2 (G2): colistin administered; Group 3 (G3):
Salmonella challenged; Group 4 (G4): colistin administered and Salmonella challenged. Dots represent outliers; 1 Salmonella
challenge started at D7 for groups G3 and G4 (sampling from D7 was performed before starting the challenge); 2 Colistin
was provided to chicks from groups G2 and G4 from D8 to D14.
Antibiotics 2021, 10, 677
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2.4. Comparison of Salmonella Spp. and mcr-1 Quantitative qPCR Data
Quantitative PCR data for Salmonella quantification for the groups, including chicks
inoculated with mcr-1-carrying Salmonella, showed median values for G3 (without colistin)
of <3.3 × 102 , 5.1 × 103 , 1.5 ×1 03 , 1.3 × 104 , and 1.1 × 104 copies per mg for days 7, 9,
11, 14, and 21, respectively. For G4 (colistin administered), the corresponding median
values were <3.3×102 , 1.7 × 103 , 1.7 × 106 , 4.7 × 104 , and 1.6 × 104 copies/mg (Figure 3).
Quantification of mcr-1 and Salmonella showed similar values on D9 (p < 0.05, Table 2),
indicating that most copies of the mcr-1 genes in the samples came from the inoculated
S. Typhimurium strain. The levels of mcr-1 and Salmonella increased thereafter in G4,
during the colistin administration period, whereas the levels remained approximately the
same in G3. After the withdrawal of colistin, Salmonella levels in G4 decreased, reaching
G3-equivalent values on D21. However, mcr-1 levels of both groups remained significantly
Antibiotics 2021, 10, x FOR PEER REVIEW
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higher (Table 2), suggesting that mcr-1 may have been transferred to other intestinal
bacterial species (Figure 3).
Figure 3. Comparison between
qPCR3.data
for Salmonella
and mcr-1
throughoutand
themcr-1
studyobtained
for the challenged
groups
Figure
Comparison
between
qPCRobtained
data for Salmonella
throughout
the study
for the challenged
groups
(G3 4and
G4):
Groupadministered
3 (G3): Salmonella
Group 4 (G4):
colistin
(G3 and G4): Group 3 (G3): Salmonella
challenged;
Group
(G4):
colistin
and challenged;
Salmonella challenged.
Dots
administered
Salmonella
challenged.
Dots
outliers; 1 Colistin
was
provided
tofor
chicks
represent outliers; 1 Colistin was
provided toand
chicks
from group
G4, from
D8represent
to D14; * Salmonella
challenge
started
on D7
from
group
G4,
from
D8
to
D14;
*
Salmonella
challenge
started
on
D7
for
groups
G3
and
G4
(samgroups G3 and G4 (sampling from D7 was performed before starting the challenge).
pling from D7 was performed before starting the challenge).
Table 2. T-test p-values obtained from a comparative analysis of mcr-1 and Salmonella qPCR quantitative data per sampling day.
3. Discussion
p-Values
Food-producing animals have been highlighted
as potential reservoirs for the dissemination
of
colistin-resistance
determinants,
especially
2015, when Liu
qPCR Data
Group
D7
D9
D11 since lateD14
D21 et al.
identified the plasmid-mediated colistin resistance gene, mcr-1, in China [17]. Horizontal
mcr-1
G3 vs. G4
ND
0.41
0.03 *
0.02 *
0.86
gene transfer
have
a critical
role 0.03
in the
Salmonella
G3 vs. processes
G4
ND been proposed
0.69 to play0.02
*
* spreading
0.69of the
mcr-1
gene
[19,20].
In
the
present
study,
colistin
administration
had
a
significant
effect
mcr-1 vs. Salmonella
G3
ND
0.06
0.44 *
<0.01 *
<0.01
* on
the spread G4
of mcr-mediated
in <0.01
the chicks’
microbiota.
Its effect
mcr-1 vs. Salmonella
ND colistin resistance
0.94
*
<0.01 *
<0.01 *was
greater
when
mcr-1-carrying
bacteria
were
introduced
into
the
gut
environment
in the
* Statistically significant differences (p < 0.05); G3: Salmonella challenged; G4: colistin administered and Salmonella challenged.; Sampling
colistin,
mcr-1
colistin
resistance
appeared
become
widedays: D7: day 7; D9: day 9; D11: presence
day 11; D14:ofday
14; D21: in
daywhich
21.; ND:case
The t-test
was
not applied
for data from
D7 due to the
different
number of samples available fromspread.
each group.
We used a Salmonella enterica serovar Typhimurium strain because neonatal chicks
are highly susceptible to colonisation by Salmonella serovars [21] and it is easily identified
in chicks because it is not a component of their early microbiota. We studied the effect of
colistin by administering it for seven days, as recommended by the EMA for treatment of
enteric infections caused by susceptible non-invasive E. coli [15], and focused on the mcr1 gene because it is the mobile gene most frequently related to colistin resistance world-
Antibiotics 2021, 10, 677
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3. Discussion
Food-producing animals have been highlighted as potential reservoirs for the dissemination of colistin-resistance determinants, especially since late 2015, when Liu et al.
identified the plasmid-mediated colistin resistance gene, mcr-1, in China [17]. Horizontal
gene transfer processes have been proposed to play a critical role in the spreading of the
mcr-1 gene [19,20]. In the present study, colistin administration had a significant effect
on the spread of mcr-mediated colistin resistance in the chicks’ microbiota. Its effect was
greater when mcr-1-carrying bacteria were introduced into the gut environment in the presence of colistin, in which case mcr-1 colistin resistance appeared to become widespread. We
used a Salmonella enterica serovar Typhimurium strain because neonatal chicks are highly
susceptible to colonisation by Salmonella serovars [21] and it is easily identified in chicks
because it is not a component of their early microbiota. We studied the effect of colistin
by administering it for seven days, as recommended by the EMA for treatment of enteric
infections caused by susceptible non-invasive E. coli [15], and focused on the mcr-1 gene
because it is the mobile gene most frequently related to colistin resistance worldwide [17].
This scenario has been described in other studies, in which a link was proposed
between colistin use and the spread of the mcr-1 gene [12,18,22]. Comparison of the
two groups to which colistin was administered (G2 and G4) showed colistin treatment
to significantly increase mcr-1 levels in G4 (in which the chicks were challenged with
Salmonella) but not G2 (in which the chicks were not challenged with Salmonella).
This study shows the importance of the presence of an intestinal bacterial population
that carries the mcr-1 gene for colistin resistance for the dispersion associated with its use.
This is clear from the data of the mcr-1 challenged chicks which were not administered
colistin (G3). Chicks from non-challenged groups (G1 and G2) showed a baseline presence
of mcr-1 from the first day of sampling, which may have been due to the pre-existence of
these genes in certain bacterial groups different from Salmonella. However, chicks from
these groups (G1 and G2) showed parallel progression, with no significant differences in
their mcr-1 outcomes. Chicks from G3, despite not having been given colistin, showed an
increase in mcr-1 levels, which became significantly different from those of the animals
from G1 and G2 by D14 and D21. Similarly, in assessing the effect of colistin administration,
chicks challenged with mcr-1-carrying Salmonella (G4) showed significantly more (p-value <
0.01) mcr-1 copies/mg relative to the non-challenged group (G2) (Figure 2), demonstrating
that the presence of gene-carrying Salmonella in the gut is more decisive in gene dispersal
than the effect of the antibiotic, as has been seen in other studies [23].
Salmonella counts in chicks from group G4 were higher than those in animals from
group G3, as observed for the Salmonella qPCR results. Harbouring mcr genes usually suppose a negative fitness cost to the carrier bacteria [24] and maybe this fact was responsible
for lower levels of Salmonella detected in G3 than those from G4, since Salmonella in G4 had
a selective advantage because of colistin treatment. Thus, data from group G4 support the
initial hypothesis about the effect of colistin use on the spread of colistin resistance caused
by mcr family genes. These data are consistent with the high dispersion capacity of the
mcr-1 gene, especially in the presence of colistin, as previously described [12,13].
Therefore, the mcr-1 levels of group G4 (colistin administered–Salmonella challenged)
began to decline after exposure to colistin stopped, but slower than those of Salmonella,
remaining similar to the values of group G3, which suggests that the reduction corresponded to mcr-1 harboured by the Salmonella and that the remaining mcr-1 quantified on
D21 mainly came from other bacterial species of the microbiota that had been selected by
the administration of colistin. Thus, our results highlight the possible horizontal transfer of
genes from Salmonella to other intestinal bacteria, which may have allowed mcr-1 levels
to remain higher for a longer period in both groups (G3 and G4). Although the clonal
spread of mcr-1-carrying Salmonella is common, there is a close association between certain
serotypes of Salmonella enterica, as serovar Typhimurium, and mcr-1 gene and different
types of plasmid also play an important role in the conjugation phenomenon [8,25]. There-
Antibiotics 2021, 10, 677
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fore, further studies are needed to determine the type of plasmid where mcr-1 was located
and confirm this hypothesis.
4. Methods
4.1. Ethical Approval
Experimental procedures were approved by the University Complutense of Madrid
Animal Care and Ethics Committee (date of approval: 31/07/2019; registration number:
99/107262.9/19) in compliance with the regulations of the Community of Madrid (PROEX
152/19). Animal experiments took place in the biosafety level 3 (BSL-3) facilities of the
VISAVET Health Surveillance Centre and animals were housed according to the European
legislation on animal welfare (Directive 2010/63/EU).
4.2. Experimental Design
In total, 100 one-day-old broiler chicks (Ross 308) were obtained from a commercial
hatchery and housed at the VISAVET Surveillance Centre facilities for the 21 days of the
experiment under the same environmental conditions described previously by HerreroEncinas et al. [26]. On the first day, when the animals arrived, box litter samples were
collected for Salmonella spp. detection following ISO 6579:2017 standards. All samples
were found negative. Chicks were divided into four groups of 25 animals. All chicks were
housed in different cages until day (D) 7 but under the same environmental and feeding
conditions. Thus, they were considered to be a single group for data analysis (control
group: CTRL). From D7, chicks from each cage were exposed to different conditions to form
four groups: animals without colistin nor Salmonella challenge (G1), colistin administered
(G2), Salmonella challenged (G3), and colistin administered and Salmonella challenged (G4).
On D7, chicks from G3 and G4 were challenged with a Salmonella enterica subsp. enterica
serovar Typhimurium (monophasic variant) strain that was positive for mcr-1 through
their drinking water (3.3 × 105 CFU/mL). Starting from D8, colistin (colistin sulphate
1,025,000 IU, Acolan, Spain) was administered for seven days to chickens of groups G2 and
G4 through their drinking water (600 mg/L) adjusting it to a concentration of 75,000 IU
recommended in poultry by the EMA [15], replacing the water and corresponding dose of
colistin every 24 h.
4.3. Sampling and Sample Preparation
On days 7, 9, 11, 14, and 21, five randomly selected animals from each group (CTRL
and G1 to G4) were sedated intramuscularly with diazepam and euthanised with an overdose of sodium pentobarbital by intraperitoneal injection. Caecum faeces were collected
during the autopsy, and an aliquot of 1.5 g was stored at 4 ◦ C for analysis using microbiological methods in the following 24 h. The remaining caecal faeces collected from each
animal was preserved at −80 ◦ C for molecular analysis.
Salmonella Counting Using Selective Media
A gram of each fresh aliquot was mixed with 2 mL saline (0.85% NaCl). Then, 50 µL
was diluted into 9 mL brain–heart infusion broth (BHI) supplemented with a 10 µg colistin
disk and incubated at 37 ◦ C for 4 h. Then, six 10-fold serial dilutions were carried out in
BHI. ChromID selective medium Salmonella Agar (SMID2) (bioMérieux, Marcy-l’Étoile,
France) was used for Salmonella counting. SMID2 plates were inoculated with 100 µL
of −3 and −4 BHI sample dilutions and incubated at 37 ◦ C for 24 h. After incubation,
all colonies suspected to be Salmonella were counted according to the manufacturer’s
specifications. One colony of each SMID2 plate was streaked onto blood agar plates and
incubated at 37 ◦ C for 24 h for subsequent species confirmation by mass spectrometry
using a Bruker Daltonics UltrafleXtrem MALDI TOF/TOF instrument (Bruker Daltonics,
Bremen, Germany). Conventional PCR was performed to confirm the presence of the mcr-1
gene [27].
Antibiotics 2021, 10, 677
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4.4. Quantitative Assay for Mcr-1
Direct DNA extraction from chick caecal samples was carried out using a commercial
kit (FASTI001-1 FavorPrep Stool DNA Isolation Mini Kit, Favorgen-Europe, Vienna, Austria) following the manufacturer’s specifications (elution volume of 200 µL), coupled with a
specific SYBRGreen (Thermo Fisher Scientific, Vilnius) real-time PCR assay for quantitative
detection of the mcr-1 gene (qPCR), as described previously by Li J et al. [28] and further
validated in our previous work [29]. Samples were considered positive if quantitative
values were >1.00 × 102 fg/µL (equivalent to 1.58 × 103 copies/mg caecal content).
4.5. Quantitative Real-Time PCR Assay for Salmonella
Quantitative real-time PCR was carried out for the quantitative detection of Salmonella
spp. in each sample using a commercial “Salmonella spp. DNA extraction and real-time
PCR detection” kit (Kylt® Salmonella spp. (FS), Oldenburg, Germany). Two microliters of
each DNA elute were also run in triplicate. A sample was considered to be positive when
its cycle threshold (CT) was ≤42.
4.6. Statistical Analysis
The data obtained by qPCR were analysed using a t-test for two related samples after
normalisation by logarithmic transformation into Log10. A Kruskal–Wallis test was used to
analyse differences in SMID2 Salmonella counts among experimental groups. A difference
was considered significant when the p-value was <0.05 for both statistical tests.
4.7. Data Visualisation
All figures included in this study were illustrated with R (core team 2019) [30] using
the ggplot2 package (H. Wickham, 2016) [31].
5. Conclusions
The presence of mcr-1-carrying S. enterica serovar Typhimurium in the gut resulted in
the spread of the mcr-1 gene, probably due to horizontal gene transfer. The administration
of colistin accelerated this process by selecting the colistin-resistant bacteria present in the
gut microbiota, keeping mcr-1 levels constant after the withdrawal of colistin.
Author Contributions: Conceptualization, P.M.-V., M.A.M., L.D., and M.U.-R.; methodology, P.M.-V.,
A.R.-M., A.R.-B., M.H., and D.R.-L.; software, M.H., D.R.-L., and A.Q.; validation, M.H., D.R.-L., and
A.Q.; formal analysis, P.M.-V., M.H., M.A.M., D.R.-L., A.G., A.Q., J.G., L.D., and M.U.-R.; investigation
P.M-V., M.A.M., L.D., and M.U-R.; resources, P.M-V., M.A.M., L.D., and M.U-R.; data curation, P.M.-V.,
M.A.M., L.D., and M.U.-R.; writing—original draft preparation, P.M.-V., M.A.M., L.D., and M.U.-R.;
writing—review and editing, P.M.-V., M.H., M.A.M., D.R.-L., A.G., A.Q., J.G., L.D., and M.U.-R.;
visualization, P.M.-V., M.H., M.A.M., D.R.-L., A.G., A.Q., J.G., L.D., and M.U.-R.; supervision, P.M.-V.,
M.H., M.A.M., D.R.-L., A.G., A.Q., J.G., L.D., and M.U.-R.; project administration, P.M.-V., M.A.M.,
L.D., and M.U.-R.; funding acquisition, P.M.-V., M.A.M., L.D., and M.U.-R. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded by the Spanish Ministry of Economy, Industry, and Competitiveness (AGL2016-74882-C3), the Spanish Ministry of Agriculture, Fishing, and Food, and the
Autonomous Community of Madrid (S2013/ABI-2747). Pedro Miguela-Villoldo was supported
by the FPI Programme (BES-2017-080264) from the Spanish Ministry of Science, Innovation, and
Universities and Estefanía Martínez Fernández by a grant co-funded by the European Social Fund
and Youth Employment Initiative (YEI) (PEJ-2017-TL/BIO- 7114). Work in the laboratory of Alberto
Quesada is also funded by the Junta de Extremadura and FEDER (IB16073 and GR15075) in Spain.
Institutional Review Board Statement: Experimental procedures were approved by the University
Complutense of Madrid Animal Care and Ethics Committee (date of approval: 31/07/2019; registration number: 99/107262.9/19) in compliance with the regulations of the Community of Madrid
(PROEX 152/19). Animal experiments took place in the biosafety level 3 (BSL-3) facilities of the
VISAVET Health Surveillance Centre and animals were housed according to the European legislation
on animal welfare (Directive 2010/63/EU).
Antibiotics 2021, 10, 677
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Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: The authors wish to thank the technicians María García, Estefanía Rivero, Nisrin
Maasoumi, and Estefanía Martínez for their excellent technical assistance at the Foodborne Zoonoses
and Antibiotic Resistance Unit, the staff of the Quality and Biosafety Unit, María Mazariegos, Laura
Delgado, David Duque, and Pedro Alcubilla, for their technical support at the BSL3 facilities and
Red de Investigación en Sanidad Animal (RISA).
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Rakoff-Nahoum, S.; Paglino, J.; Eslami-Varzaneh, F.; Edberg, S.; Medzhitov, R. Recognition of Commensal Microflora by Toll-Like
Receptors Is Required for Intestinal Homeostasis. Cell 2004, 118, 229–241. [CrossRef] [PubMed]
Crhanova, M.; Hradecka, H.; Faldynova, M.; Matulova, M.; Havlickova, H.; Sisak, F.; Rychlik, I. Immune response of chicken gut
to natural colonization by gut microflora and to Salmonella enterica serovar enteritidis infection. Infect. Immun. 2011, 79, 2755–2763.
[CrossRef]
Litvak, Y.; Mon, K.K.Z.; Nguyen, H.; Chanthavixay, G.; Liou, M.; Velazquez, E.M.; Kutter, L.; Alcantara, M.A.; Byndloss, M.X.;
Tiffany, C.R.; et al. Commensal Enterobacteriaceae Protect against Salmonella Colonization through Oxygen Competition. Cell
Host Microbe 2019, 25, 128–139.e125. [CrossRef] [PubMed]
Hu, Y.; Yang, X.; Qin, J.; Lu, N.; Cheng, G.; Wu, N.; Pan, Y.; Li, J.; Zhu, L.; Wang, X.; et al. Metagenome-wide analysis of antibiotic
resistance genes in a large cohort of human gut microbiota. Nat. Commun. 2013, 4, 2151. [CrossRef] [PubMed]
Sommer, M.O.A.; Dantas, G.; Church, G.M. Functional Characterization of the Antibiotic Resistance Reservoir in the Human
Microflora. Science 2009, 325, 1128–1131. [CrossRef]
Catry, B.; Cavaleri, M.; Baptiste, K.; Grave, K.; Grein, K.; Holm, A.; Jukes, H.; Liebana, E.; Lopez Navas, A.; Mackay, D.; et al. Use
of colistin-containing products within the European Union and European Economic Area (EU/EEA): Development of resistance
in animals and possible impact on human and animal health. Int. J. Antimicrob. Agents 2015, 46, 297–306. [CrossRef]
Wang, X.; Wang, Y.; Wang, Y.; Zhang, S.; Shen, Z.; Wang, S. Emergence of the colistin resistance gene mcr-1 and its variant in
several uncommon species of Enterobacteriaceae from commercial poultry farm surrounding environments. Vet. Microbiol. 2018,
219, 161–164. [CrossRef]
Lima, T.; Domingues, S.; Da Silva, G.J. Plasmid-Mediated Colistin Resistance in Salmonella enterica: A Review. Microorganisms
2019, 7, 55. [CrossRef] [PubMed]
World Health Organization. WHO Model Lists of Essential Medicines. Available online: https://www.who.int/groups/expertcommittee-on-selection-and-use-of-essential-medicines/essential-medicines-lists (accessed on 26 April 2021).
Poirel, L.; Madec, J.Y.; Lupo, A.; Schink, A.K.; Kieffer, N.; Nordmann, P.; Schwarz, S. Antimicrobial Resistance in Escherichia coli.
Microbiol. Spectr. 2018, 6. [CrossRef]
Borowiak, M.; Fischer, J.; Hammerl, J.A.; Hendriksen, R.S.; Szabo, I.; Malorny, B. Identification of a novel transposon-associated
phosphoethanolamine transferase gene, mcr-5, conferring colistin resistance in d-tartrate fermenting Salmonella enterica subsp.
enterica serovar Paratyphi B. J. Antimicrob. Chemother. 2017, 72, 3317–3324. [CrossRef]
Sun, J.; Zhang, H.; Liu, Y.H.; Feng, Y. Towards Understanding MCR-like Colistin Resistance. Trends Microbiol. 2018, 26, 794–808.
[CrossRef] [PubMed]
Poirel, L.; Jayol, A.; Nordmann, P. Polymyxins: Antibacterial Activity, Susceptibility Testing, and Resistance Mechanisms Encoded
by Plasmids or Chromosomes. Clin. Microbiol. Rev. 2017, 30, 557–596. [CrossRef]
European Food Safety Authority; European Centre for Disease Prevention and Control. The European Union summary report
on antimicrobial resistance in zoonotic and indicator bacteria from humans, animals and food in 2017. EFSA J. 2019, 17, 5598.
[CrossRef]
Agency, E.M. Updated Advice on the Use of Colistin Products in Animals within the European Union: Development of Resistance
and Possible Impact on Human and Animal Health. Available online: https://www.ema.europa.eu/documents/scientificguideline/updated-advice-use-colistin-products-animals-within-european-union-development-resistance-possible_en-0.pdf
(accessed on 26 April 2021).
EMA/CVMP. Article 35. Community Interest Referral: Initiated in Cases Involving the Interests of the Community or Concerns
Relating to the Protection of Human or Animal Health or the Environment. [European Commission Final Decision]. 2015.
Available online: https://www.ema.europa.eu/en/documents/referral/colistin-oral-article-35-referral-annex-i-ii-iii_en.pdf
(accessed on 26 April 2021).
Liu, Y.Y.; Wang, Y.; Walsh, T.R.; Yi, L.X.; Zhang, R.; Spencer, J.; Doi, Y.; Tian, G.; Dong, B.; Huang, X.; et al. Emergence of
plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: A microbiological and molecular
biological study. Lancet Infect. Dis. 2016, 16, 161–168. [CrossRef]
Miguela-Villoldo, P.; Hernández, M.; Moreno, M.A.; Rodríguez-Lázaro, D.; Quesada, A.; Domínguez, L.; Ugarte-Ruiz, M. National
colistin sales versus colistin resistance in Spanish pig production. Res. Vet. Sci. 2019, 123, 141–143. [CrossRef]
Antibiotics 2021, 10, 677
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
10 of 10
Poirel, L.; Kieffer, N.; Nordmann, P. In Vitro Study of ISApl1-Mediated Mobilization of the Colistin Resistance Gene mcr-1.
Antimicrob. Agents Chemother. 2017, 61, e00127-17. [CrossRef] [PubMed]
Sun, J.; Zeng, X.; Li, X.-P.; Liao, X.-P.; Liu, Y.-H.; Lin, J. Plasmid-mediated colistin resistance in animals: Current status and future
directions. Anim. Health Res. Rev. 2017, 18, 136–152. [CrossRef]
Sadler, W.W.; Brownell, J.R.; Fanelli, M.J. Influence of Age and Inoculum Level on Shed Pattern of Salmonella typhimurium in
Chickens. Avian Dis. 1969, 13, 793–803. [CrossRef]
Skov, R.L.; Monnet, D.L. Plasmid-mediated colistin resistance (mcr-1 gene): Three months later, the story unfolds. Euro Surveill.
Eur. Commun. Dis. Bull. 2016, 21, 30155. [CrossRef]
Ahmed, S.; Hansen, C.; Dahlkilde, A.L.; Herrero-Fresno, A.; Pedersen, K.S.; Nielsen, J.P.; Olsen, J.E. The Effect of Colistin
Treatment on the Selection of Colistin-Resistant Escherichia coli in Weaner Pigs. Antibiotics 2021, 10, 465. [CrossRef] [PubMed]
Nang, S.C.; Morris, F.C.; McDonald, M.J.; Han, M.L.; Wang, J.; Strugnell, R.A.; Velkov, T.; Li, J. Fitness cost of mcr-1-mediated
polymyxin resistance in Klebsiella pneumoniae. J. Antimicrob. Chemother. 2018, 73, 1604–1610. [CrossRef]
Cui, M.; Zhang, J.; Zhang, C.; Li, R.; Wai-Chi Chan, E.; Wu, C.; Wu, C.; Chen, S. Distinct mechanisms of acquisition of mcr-1
-bearing plasmid by Salmonella strains recovered from animals and food samples. Sci. Rep. 2017, 7, 13199. [CrossRef] [PubMed]
Herrero-Encinas, J.; Blanch, M.; Pastor, J.J.; Mereu, A.; Ipharraguerre, I.R.; Menoyo, D. Effects of a bioactive olive pomace extract
from Olea europaea on growth performance, gut function, and intestinal microbiota in broiler chickens. Poultry Sci. 2020, 99,
2–10. [CrossRef] [PubMed]
Rebelo, A.R.; Bortolaia, V.; Kjeldgaard, J.S.; Pedersen, S.K.; Leekitcharoenphon, P.; Hansen, I.M.; Guerra, B.; Malorny, B.; Borowiak,
M.; Hammerl, J.A.; et al. Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3,
mcr-4 and mcr-5 for surveillance purposes. Euro Surveill. Eur. Commun. Dis. Bull. 2018, 23, 17-00672. [CrossRef] [PubMed]
Li, J.; Shi, X.; Yin, W.; Wang, Y.; Shen, Z.; Ding, S.; Wang, S. A Multiplex SYBR Green Real-Time PCR Assay for the Detection
of Three Colistin Resistance Genes from Cultured Bacteria, Feces, and Environment Samples. Front. Microbiol. 2017, 8, 2078.
[CrossRef] [PubMed]
Miguela-Villoldo, P.; Moreno, M.A.; Hernández, M.; Rodríguez-Lázaro, D.; Gallardo, A.; Borge, C.; Quesada, A.; Domínguez,
L.; Ugarte-Ruiz, M. Complementarity of Selective Culture and qPCR for Colistin Resistance Screening in Fresh and Frozen Pig
Cecum Samples. Front. Microbiol. 2020, 11, 572712. [CrossRef] [PubMed]
R Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria,
2019; Available online: https://www.R-project.org/ (accessed on 26 April 2021).
Wickham, H. ggplot2; Use R! Springer International Publishing; Available online: https://link.springer.com/book/10.1007%2F9
78-3-319-24277-4 (accessed on 26 April 2021).
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The effect of increasing levels of palm kernel meal containing α-β-mannanase replacing maize to growing-finishing hybrid duck on growth performance, nutrient digestibility, carcass trait, and VFA
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Journal of The Indonesian Tropical Animal Agriculture/Journal of the Indonesian tropical animal agriculture
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cc-by-sa
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ABSTRAK Penelitian bertujuan untuk mengetahui pengaruh penggunaan bungkil sawit yang diberi perlakuan
enzim terhadap nilai nutrisi, performans produksi, dan kualitas karkass bebek hibrida. Materi dalam
penelitian ini 196 DOD Itik pedaging [(Peking x Khaki Campbell)] dengan KK (410.52 ± 95.25 g). Metode penelitian yang digunakan adalah percobaan lapang dengan Rancangan Acak Lengkap dengan 5
perlakuan dan 4 ulangan. Pakan kontrol (tanpa substitusi BIS), BIS25 ( Pakan basal + substitusi Jagung
dengan 25% BIS penambahan α-β-mannanase), BIS50 ( Pakan basal + substitusi Jagung dengan 50%
BIS penambahan α-β-mannanase), BIS75 ( Pakan basal + substitusi Jagung dengan 75% BIS
penambahan α-β-mannanase), BIS100 ( Pakan basal + substitusi Jagung dengan 100% BIS penambahan
α-β-mannanase). Data hasil penelitian dianalisis variansi menggunakan rancangan acak lengkap (RAL)
dengan perlakuan tingkat penggunaan BIS-enzim. Dilanjutkan dengan uji Duncan’s Multiple Range Test
untuk mengetahui perbedaan rataan antar perlakuan menggunakan SAS University. Hasil penelitian
menunjukan bahwa substitusi jagung dengan BIS yang di tambahakan enzim α-β-mannanase tidak
berpengaruh nyata (P>0,05) terhadap penampilan produksi. Namun, berpengaruh nyata (P<0,05)
terhadap kecernaan dan mampu menurunkan lemak perut pada daging bebek hibrida. Disimpulkan
bahwa penggantian bungkil inti sawit sebanyak 25% dengan penambahan enzim α-β-mannanase mampu
meningkatkan kecernaan dan menurunkan lemak perut bebek hibrida. Kata kunci : bebek hibrida, bungkil inti sawit, enzim α-β-mannanase, jagung, substitus The effect of increasing levels of palm kernel meal containing
α-β-mannanase replacing maize to growing-finishing hybrid duck
on growth performance, nutrient digestibility, carcass trait,
and VFA O. Sjofjan, D. N. Adli*, M. H. Natsir, Y. F. Nuningtyas, I. Bastomi and F. R. Amalia
Department of Feed and Animal Nutrition, Faculty of Animal Science,
Universitas Brawijaya, Jl. Veteran, Malang, East Java 65145 - Indonesia
*Corresponding E-mail: danungnuradli1994@gmail.com O. Sjofjan, D. N. Adli*, M. H. Natsir, Y. F. Nuningtyas, I. Bastomi and F. R. Amalia
Department of Feed and Animal Nutrition, Faculty of Animal Science,
Universitas Brawijaya, Jl. Veteran, Malang, East Java 65145 - Indonesia
*Corresponding E-mail: danungnuradli1994@gmail.com O. Sjofjan, D. N. Adli*, M. H. Natsir, Y. F. Nuningtyas, I. Bastomi and F. R. Amalia
Department of Feed and Animal Nutrition, Faculty of Animal Science,
Universitas Brawijaya, Jl. Veteran, Malang, East Java 65145 - Indonesia
*Corresponding E-mail: danungnuradli1994@gmail.com Received October 28, 2020; Accepted December 22, 2020 Received October 28, 2020; Accepted December 22, 2020 J. Indonesian Trop. Anim. Agric.
pISSN 2087-8273 eISSN 2460-6278
http://ejournal.undip.ac.id/index.php/jitaa
46(1):29-39, March 2021
DOI: 10.14710/jitaa.46.1.29-39 J. Indonesian Trop. Anim. Agric. pISSN 2087-8273 eISSN 2460-6278
http://ejournal.undip.ac.id/index.php/jitaa
46(1):29-39, March 2021
DOI: 10.14710/jitaa.46.1.29-39 J I T A A
Journal of the Indonesian Tropical Animal Agriculture
Accredited by Ditjen Penguatan Risbang No. 60/E/KPT/2016 INTRODUCTION in the second place (BPS, 2020). Thus, conditions
produce agro-industrial waste from palm kernel
meal (PKM) included in poultry diets that had a
low price and nutritional content. The palm kernel
meal is imported into the European land (EU),
cited from (BPS-RI, 2020) the statistics show that
34,064 tons of PKM were imported in 2018/2019. The production of palm kernel meal as animal
feed in Europe at the level of 0.48 tonnes in 2018-
2019 (Wilkinson and Young, 2020). The World Health Organization (WHO)
announced the coronavirus novel (COVID-19) as
a global pandemic outbreak as of March 11, 2020. A total of 4.87 million cases have been confirmed
worldwide, with 1.66 million people recovering
and 321 thousand people die. The pandemic
spread over the world and faced developing
countries e.g. Indonesia. COVID-19 impact to
livestock sector especially poultry production. Intensive poultry production systems demand a
supply of high protein- and easily available in
developing countries. To fulfill the requirement,
the efficiency on poultry production must be
developed (Sjofjan and Adli, 2020). Duck demand
increase due it ease and fulfillment in developing
countries like Indonesia. The statistical data
demand on the poultry market increased
nationally (Adli et al., 2018). The data until the
fourth quarter of 2019, broilers increased about
4.9% compared to the population in 2018 (1.4
billion heads) (BPS-RI, 2020). Generally, poultry
rearing keys were dependent on breed, feed, and
management to the purposes the profitable. The
golden triangle was giving 86% including also
feed intake, live weight, and gain/ratio for each
breed of ducks. Feed costs were giving huge
around 65-75% of total production in the poultry
industry. According to the newest regulation
Permendag/10/2020 to anticipate the COVID-19,
the Indonesian government has imposed a ban on
the import of live animal species and imported by-
product originating from China or transiting into
Indonesian territory (BPS-RI, 2020). The rules
made the researchers or farmers develop an
alternative to reduce the cost of maize as main
feed in farming. One potential feed to replace
maize was palm kernel meal since thus palm
kernel meal had similar energy content and
protein but high in the crude fiber (Wilkinson and
Young, 2020). ABSTRACT In this experiment, we investigated the effect of increasing levels of palm kernel meal containing α-
β-mannanase replacing maize to growing-finishing hybrid duck on growth performance, nutrient
digestibility, and carcass trait. One hundred and ninety- six hybrid ducks [(Peking x Khaki Campbell)]
with 410.52 ± 95.25 g BW) were allotted to 5 dietary treatments with 9 ducks (unsexed) per pen and 4
replicates per treatments. These dietary treatments were: NC (negative control; maize-soybean-meal
based diet), PKM25 (maize replacement with PKM α-β-mannanase 25%), PKM50 (maize replacement
with PKM α-β-mannanase 50%), PKM75 (maize replacement with PKM α-β-mannanase 75%),
PKM100 (maize replacement with PKM α-β-mannanase 100%). There was a curve linear decrease in
the growing phase and finisher phase in feed intake as the level of α-β-mannanase palm kernel meal
increased in the diet. Curvilinear, the result was presented no significant difference (P>0.05) on the
initial body weight, growing and finishing phase body weight. The result of this study showed the used Palm Kernel Meal containing α-β-mannanase Replacing Maize for Ducks (A. Sjofjan et al.) 29 α-β-mannanase in the palm kernel meal presented a significant difference (P < 0.05) on the nutrient
digestibility of dry matter and crude protein. Furthermore, Carcass traits showed the reflection in line
with the final weight and internal organs were better to compare to control 0.5% using α-β-mannanase in
the palm kernel meal. In summary, the increase of the level of palm kernel meal with α-β-mannanase
enzyme was had a positive effect on the hybrid ducks and the replacement of palm kernel meal by 25%
with the addition of the α-β-mannanase enzyme was able to improve digestibility and reduce abdominal
fat of hybrid ducks. Keywords: α-β-mannanase, carcass trait, digestibility, hybrid ducks, palm kernel meal J.Indonesian Trop.Anim.Agric. 46(1):29-39, March 2021 INTRODUCTION Indonesia was at the top level in
the world to produce the palm kernel meal above
40.56 MT in 2019-2020, following with Malaysia Indonesia Imports large quantities of staple
components of feed for broilers both maize and
fishmeal (BPS, 2020), which imported 200
thousand tons in 2020 for maize and 800 tonnes/
year. Reducing imports of feed ingredients is one
way of reducing greenhouse gas emissions to
which sea freight is a major contributor. A
potential local feed Indonesia ingredient that
could be used to replace maize is PKM. Even
though, the PKM had high crude fiber the breed
can be adapted more to the high crude fiber that
was a duck (Jang et al., 2020). Indonesia has developed meat ducks that
have a fast growth rate, namely Hybrid ducks
with a short maintenance period of 45 days. Hybrid ducks are the result of a cross between a
Peking duck (male) and a Khaki Champbell duck
(a female). A cross between Peking ducks and
Khaki Champbell ducks were conducted to
produce Day Old Duck (DOD) of final stock
broiler duck’s quality. Peking ducks have rapid
weight gain, while Khaki ducks Champbell has a
high body weight and high egg production
compared to other local ducks. The major non-
starch polysaccharide (NSP) component in
poultry were α-β-mannanase, which is linear with
polysaccharide (Jang et al., 2020). However, there
have some limitations to giving into duck’s diet. To combat this limitation, an exogenous enzyme
of α-β-mannanase had been combined with PKM
diet as replacing for maize of growing-finishing
ducks. The addition of α-β-mannanase helps the
palm kernel meal to stimulate the releasing of
volatile fatty acid in the caecum of the ducks J.Indonesian Trop.Anim.Agric. 46(1):29-39, March 2021 30 facilitated. At 24 days to 38 days of age (feeding
growing phase); at 38-64 days of age (feeding
finishing phase). The Composition of Feed in the
experiment showed in Table 2 and Table 3. (Sharmila et al., 2014). In addition, the α-β-
mannanase was given to reduce the crude fiber in
accordance to improve nutrient digestibility in the
gut of duck. When the nutrient digestibility the
palm kernel meal in feed should be increase
palatability and continued to increase the feed
intake and give a positive impact on the growth
performance of the ducks. Preparation Palm Kernel Meal containing α-β-
Mannanase The palm kernel meal was sifted to separate
the meal from the remaining shells, then placed
on the floor that had been coated with trash bags
and sacks. Suspensions of α-β-mannanase are
homogenized in a blender then added at 0.010%
per 1 kg of PKM. The last step was putting the
PKM in a sack with holes to allow entry of air and
storing it for 12 hours at room temperature after
which it was sun-dried adapted from (Adli et al.,
2020). The dietary treatments were: NC (negative
control; maize-soybean-meal based diet), PKM25
(maize replacement with PKM α-β-mannanase
25%), PKM50 (maize replacement with PKM α-
β-mannanase 50%), PKM75 (maize replacement
with PKM α-β-mannanase 75%), PKM100 (maize
replacement with PKM α-β-mannanase 100%). A
commercial α-β-mannanase and palm kernel meal
from Pt. Wilmar Cahaya Indonesia Tbk was used
for this experiment. The study was carried out to investigate the
effect of increasing levels of palm kernel meal
containing α-β-mannanase replacing maize to
growing-finishing hybrid duck on growth
performance, nutrient digestibility, and meat
quality. Then, the most suitable level of treatments
that able to apply in the duck farm was chosen. Ducks Rearing Condition One hundred and ninety-six hybrid ducks
[(Peking x Khaki Campbell)] with 410.52 ± 95.25
g BW were allotted to 5 dietary treatments with 9
ducks (unsexed) per pen and 4 replicates per
treatments with completely randomized design
(CRD) for the experimental design. All ducks were kept in an environmentally
controlled room with a fan and air facility. The
housing relatively temperature and humidity
among 29°C and 64%, respectively. The rice hull-
littered floor pens with a height of 3.3 (1.9 x 1.9)
m2 per pen. The lighting program was set at 23
hours light and one-hour darkness. Ducks were
reared under the supervision of a veterinarian. The
hybrid duck was taken from a commercial farmer
from Blitar Regency, East Java, Indonesia. Growth Performances The ducks were individually weighed at the
growing phase in time to determine the
coefficients variation, weekly at days of 24, 31,
38, 45, and final week (64). The body weight gain
(BWG) of duck were determined by weighed
difference amount at the initial and previous
weeks. The feed intake calculated routine by
deviation feed offered to ducks and remained feed
every week where areas, after calculated the dead
broiler remain in the current experiment. Feed/gain determined by feed intake divided by
body weight gain of the ducks during the Palm Kernel Meal containing α-β-mannanase Replacing Maize for Ducks (A. Sjofjan et al.)
31 Feeding Treatment Programmed The nutrient composition of diet ingredients
is presented in Table 1. The ducks (male and
female) were ad libitum feed and watering Table 1. Nutrient Compositions of Diet Ingredients (g / kg, as-fed basis)
Item
Maize
Soybean meal
Palm kernel meal
Dry Matter
93.4
92.2
93.5
Crude protein
8.7
44.5
16
Fat
4
1.5
1.5
Crude fibre
2
3.5
1.67
Ash
1.5
6
6.6
Nitrogen free extract
70.5
30
53.5 Table 1. Nutrient Compositions of Diet Ingredients (g / kg, as-fed basis) Palm Kernel Meal containing α-β-mannanase Replacing Maize for Ducks (A. Sjofjan et al.)
31 Palm Kernel Meal containing α-β-mannanase Replacing Maize for Ducks (A. Sjofjan et al.) 31 Table 2. Nutrient Compositions of Diet Ingredients of Growing Phase Ducks (g / kg, as-fed basis) Nutrient Compositions of Diet Ingredients of Growing Phase Ducks (g / kg, as-fed basis) Ingredients, g/kg
Treatments
NC
PKM 25
PKM50
PKM75
PKM100
Maize
540
515
490
465
440
Rice bran
150
150
150
150
150
Soybean meal
120
120
120
120
120
MBM 50
50
50
50
50
50
F.M. 60
50
50
50
50
50
PKM
-
25
50
75
100
Powder Limestone
30
30
30
30
30
Grit
41
41
41
41
41
Betaine
1
1
1
1
1
Palm oil
10
10
10
10
10
α-β-mannanase
-
1
1
1
1
Premix
5
5
5
5
5
Analyses composition, g/kg
Dry matter
87.05
87.18
87.30
87.43
87.55
Crude protein
16.83
17.01
17.19
17.38
17.56
Fat
6.42
6.35
6.29
6.23
6.17
Crude Fibre
3.63
3.95
4.28
4.60
4.93
Ash
6.20
6.27
6.33
6.39
6.45
Nitrogen Free extract
48.33
47.90
47.48
47.05
46.63
Metabolizable energy
(Kcal/kg)
2683.48
2635.73
2587.98
2540.23
2492.48
Lysine
0.92
0.92
0.93
0.93
0.94
Methionine
0.51
0.52
0.52
0.53
0.53
Met. + Cystine
0.78
0.79
0.79
0.80
0.80
Calcium
3.71
3.72
3.72
3.73
3.73
Total Phosphorus
0.85
0.85
0.86
0.87
0.88
Av. Phosphorus
0.39
0.40
0.41
0.42
0.44
Bulk density, g / L
585
584
580
569
565
1 NC (negative control; maize-soyabean-meal based diet), PKM25 (maize replacement with PKM α-β-
mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement
with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). Bulk
density = the weight of experimental feed per unit volume (g / L). Feeding Treatment Programmed 2: Vitamin A: 2,500,000 UI; Vitamin D3: 600,000 UI; Vitamin E: 4,000 mg; Vitamin K3: 400 mg; Folic acid:
80 mg; Choline: 100,000 mg; Mangan: 14 g; Zn: 40 g; Fe: 32 g; Cu: 48 g; Iodine: 0.5 g; Co: 0.28 g; Se: 0.04
g 1 NC (negative control; maize-soyabean-meal based diet), PKM25 (maize replacement with PKM α-β-
mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement
with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). Bulk
density = the weight of experimental feed per unit volume (g / L). 2: Vitamin A: 2,500,000 UI; Vitamin D3: 600,000 UI; Vitamin E: 4,000 mg; Vitamin K3: 400 mg; Folic acid:
80 mg; Choline: 100,000 mg; Mangan: 14 g; Zn: 40 g; Fe: 32 g; Cu: 48 g; Iodine: 0.5 g; Co: 0.28 g; Se: 0.04
g experiment. Mortalities were recorded per pen as
well in (%) from the beginning until the end of the
experiment (Sjofjan et al., 2020). The 24 ducks
from the group, with range nearest live BW, were separated at the days 64 weeks ducks will be
sacrificed of the experiment. Ducks were
slaughtered ready to cook method to obtain
relatively organ weight, and scalded after. J.Indonesian Trop.Anim.Agric. 46(1):29-39, March 2021 32 Table 3. Nutrient Compositions of Diet Ingredients of Finishing Phase Ducks (g / kg, as-fed basis) e 3. Nutrient Compositions of Diet Ingredients of Finishing Phase Ducks (g / kg, as-fed basis) Ingredients, g/kg
Treatments
NC
PKM 25
PKM50
PKM75
PKM100
Maize
550
515
500
475
450
Rice bran
170
170
170
170
170
Soybean meal
90
90
90
90
90
MBM 50
50
50
50
50
50
F.M. 60
50
50
50
50
50
PKM
-
25
50
75
100
Powder Limestone
30
30
30
30
30
Grit
41
41
41
41
41
Betaine
1
1
1
1
1
Palm oil
10
10
10
10
10
α-β-mannanase
-
1
1
1
1
Premix
5
5
5
5
5
Analyses composition, g/kg
Dry matter
86.96
87.09
87.21
87.34
87.46
Crude protein
15,81
1599
16.17
16.35
16.54
Fat
6.66
6.60
6.54
6.47
6.41
Crude Fibre
3.76
4.08
4.41
4.73
5.06
Ash
6.15
6.21
6.28
6.34
6.40
Nitrogen Free extract
48.91
48.48
48.06
47.63
47.21
Metabolizable energy
(Kcal/kg)
2695.68
2647.93
2600.18
2552.43
2509.68
Lysine
0.84
0.85
0.85
0.86
0.86
Methionine
0.50
0.50
0.51
0.51
0.52
Met. Feeding Treatment Programmed + Cystine
0.75
0.76
0.76
0.77
0.78
Calcium
3.71
3.71
3.71
3.72
3.72
Total Phosphorus
0.86
0.87
0.88
0.89
0.89
Av. Phosphorus
0.39
0.40
0.41
0.42
0.44
Bulk density, g / L
585
584
580
569
565
1NC (negative control; maize-soyabean-meal based diet), PKM25 (maize replacement with PKM α-β-
mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement
with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). Bulk
density = the weight of experimental feed per unit volume (g / L). 2Vitamin A: 2,500,000 UI; Vitamin D3: 600,000 UI; Vitamin E: 4,000 mg; Vitamin K3: 400 mg; Folic acid:
80 mg; Choline: 100,000 mg; Mangan: 14 g; Zn: 40 g; Fe: 32 g; Cu: 48 g; Iodine: 0.5 g; Co: 0.28 g; Se: 0.04 1NC (negative control; maize-soyabean-meal based diet), PKM25 (maize replacement with PKM α-β-
mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement
with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). Bulk
density = the weight of experimental feed per unit volume (g / L). 2Vitamin A: 2,500,000 UI; Vitamin D3: 600,000 UI; Vitamin E: 4,000 mg; Vitamin K3: 400 mg; Folic acid:
80 mg; Choline: 100,000 mg; Mangan: 14 g; Zn: 40 g; Fe: 32 g; Cu: 48 g; Iodine: 0.5 g; Co: 0.28 g; Se: 0.04 Digestibility’s Analysis Where IE=ingested energy; FE=fecal energy
voided by the fed birds; while FEL=fasting energy
loss by the unfed birds. Where IE=ingested energy; FE=fecal energy
voided by the fed birds; while FEL=fasting energy
loss by the unfed birds. Excreta samples were collected and stored in
plastic trays. Then, immediately placed in a
combination of liquid of NA2PO4 2%; NA2H2PO4
2%, 24% Formaldehyde; and 900 ml reverse
osmosis water for digestibility’s analysis. The
data were used to calculate AME, AMEn, TME,
and TMEn values according to the following
formulae: The values corrected to zero N balance,
AMEn and TMEn, are calculated as follows: The values corrected to zero N balance,
AMEn and TMEn, are calculated as follows: AMEn=AME-(8.22×ANR/FI) TMEn=TME-(8.22×FNL/FI) - (8.22×ANR/FI)
Where ANR=apparent N retention; FI=feed
intake; and FNL=fasting N loss by the unfed bird;
The factor 8.22 kcal/g for N retained in the body
has been used according to Mustafa et al., (2003). TMEn=TME-(8.22×FNL/FI) - (8.22×ANR/FI)
Where ANR=apparent N retention; FI=feed
intake; and FNL=fasting N loss by the unfed bird;
The factor 8.22 kcal/g for N retained in the body
has been used according to Mustafa et al., (2003). AME=IE-FE
TME=AME+FEL 33 Palm Kernel Meal containing α-β-mannanase Replacing Maize for Ducks (A. Sjofjan et al.) Figure 1. The effect of increasing levels of palm kernel meal containing α-β-mannanase replacing
maize on the growth performance of ducks. The symbols represent growing body weight (♦) and
finishing body weight (n). Figure 1. The effect of increasing levels of palm kernel meal containing α-β-mannanase replacing
maize on the growth performance of ducks. The symbols represent growing body weight (♦) and
finishing body weight (n). the level of α-β-mannanase palm kernel meal
increased in the diet (Table 4). Curvilinear, the
result was presented no significant difference
(P<0.05) on the initial body weight, growing and
finishing phase body weight. The results of this
study presented α-β-mannanase palm kernel meal
had improvement the daily gain, feed/gain ratio
both growing and finishing phase compared to
control (growing phase at 3.31; 2.97; 2.96 vs. 3.34
(control)) and (finishing phase 3.21; 2.93; 2.93;
2.87 vs. 3.45 control). Compared with the study
from Park et al., (2018) that study the used β-
mannanase in the palm gives an improvement on
the growth performance of white Pekin ducks
compare to control. The trends used β-mannanase
also used in other poultry to increase performance
(Park et al., 2018). The use of α-β-mannanase
inhibits the negative effect of NSP in the duck
intestinal. α-β-mannanase is an endogenous
enzyme in the cell wall that supporting-agent in
breaking down the NSP in the palm kernel meal. In addition, α-β-mannanase increasing of NSP can
reduce lower nutrient digestibility and mortalities. In these studies, the percentage of the mortalities
were at a better level compared with the control
(3.43; 0.00; 1.26; and 3.43 vs. 4.26 (control)). The
previous researcher had shown the palm kernel
meal must be treated by enzyme since a lot of
non-starch polysaccharides (NSPs) e.g. mannan
and xylene, together with anti-nutritional factors. In this study, α-β-mannanase helps degrades into
shorter chains, which, more absorptive well in the
nutrients. AMEn=AME-(8.22×ANR/FI) According to Wilkinson and Young VFA Analysis The VFA analyses were following procedure
by (Sharmila et al., 2014) and (Adli and Sjofjan,
2020). The chyme from ileum were taken
approximately one gram and diluted with distilled
water (1:1 wt/vol) in a tube. Later,
homogenization and centrifugation, 1 mL of clear
supernatant was transferred into ampula, around
0.2 mL metaphosphoric acid solution was added
and subjected to another homogenization before it
was placed on ice for at least 30 min to allow the
protein to settle. Then analyses it on the gas
chromatography (Agilent 69890 N series: USA). Continually, the analyses of proximate of the
feed sample were determined dry matter, ash,
crude fiber, fat, and crude protein was used
association of official analytical chemists method. On the other hand, the amino acid using HPLC
(HP Agilent 1200 series, USA); crude protein
were used (Kjeltech analyses, Foss Detector,
Switzerland) and gross energy using (Parr Oxygen
Bomb 1108; USA). Data Analysis Data of the first experiment were statistically
analyzed using SAS University version 4.0 red hat
(64-bit) and the differences among treatment
means (p <0.05) were determined using Duncan’s
multiple range test (Sjofjan et al., 2020). RESULTS AND DISCUSSION There was a curve linear decrease in the
growing phase and finisher phase in feed intake as J.Indonesian Trop.Anim.Agric. 46(1):29-39, March 2021 34 Table 4. The Effect of Increasing Levels of Palm Kernel Meal containing α-β-mannanase Replacing
Maize on the Growth Performance
Performance
0.10% α-β-mannanase
SEM
P-values
NC vs
PKM
NC
PKM 25
PKM50
PKM75
PKM100
Body weight, g
Initial body weight, g
415
418
417
422
433
2.22
0.31
Growing phase body
weight, g
988
978
976
985
987
0.22
0.22
Finishing phase body
weight, g
1,923
1,823
1,877
1,877
1,923
12.33
0.0015
Daily gain, g
56.5b
59.2a
58.7ab
57.8b
58.9ab
4.55
0.0001
F/G ratio
Growing phase
3.34a
3.31a
2.97b
2.96b
2.95b
3.32
0.0001
Finishing phase
3.45a
3.21a
2.93b
2.93b
2.87b
0.88
0.0012
Mortalities %
4.26
3.43
0.00
1.26
3.43
0.23
0.0015
Growing phase feed
intake, g
1,923
1,824
1,727
1,562
1,453
0.21
0.0012
Finisher phase feed
intake, g
2,023
1,923
1,825
1,725
1,726
0.19
0.0023
ab Mean values in the same row without common superscript differ at P<005
1 NC (negative control; maize-soyabean-meal based diet), PKM25 (maize replacement with PKM α-β-
mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement
with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). Table 4. The Effect of Increasing Levels of Palm Kernel Meal containing α-β-mannanase Replacing
Maize on the Growth Performance Mean values in the same row without common superscript differ at P<005 1 NC (negative control; maize-soyabean-meal based diet), PKM25 (maize replacement with PKM α-β-
mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement
with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). (2020) that palm kernel meal had low digestibility
at the range of 65-75%, to help that some enzyme
was added to bind the phytase. The replacement
of up to 15% for maize by palm kernel meal was
made balancing for digestible amino acids, total
phosphorus, and metabolizable energy (Wilkinson
and Young, 2020). In addition, from Natsir et al. (2018) the feed intake of untreated PKM higher
compare with PKM treated by the enzyme at the
level (3242.34 vs. 3097.91 gram/live bird), the
results due to energy content lower compared to
PKM treated by the enzyme. RESULTS AND DISCUSSION In this study, α-β-
mannanase has stimulated the result of crude
protein and dry matter by using methyl donor in
the cycle. The methyl donor of α-β-mannanase
reduce the phytase content and absorb the
nutrients well. While the β-mannanase must be
transformed into α-β-mannanase first before
inside to mitochondria in the synthesis cycle. The
latter was significantly affected (P<0.05) with
Mustafa et al. (2003) in percent digestibility of
palm kernel meal treated by the enzyme (44.09 vs. 41.20 (untreated)). Mustafa et al. (2003) stated the
PKM treated by an enzyme found increasing it
was due to adequate amino acid levels that
arginine, isoleucine, valine, and methionine. In
addition, from (Pasaribu et al., 2019) the increase
of palm kernel meal using microbial fermented
that produced mannanase enzyme were at 24-
32%. and crude protein. The α-β-mannanase help to
give improvement (42.20; 41.30; 44.11; and 42.13
vs 40.20% (control)) for dry matter. In line with
nutrient digestibility of crude protein were also
increasing (42.20; 41.30; 44.11; 42.13 vs. 40.20%
(control)) for crude protein. In this study, α-β-
mannanase has stimulated the result of crude
protein and dry matter by using methyl donor in
the cycle. The methyl donor of α-β-mannanase
reduce the phytase content and absorb the
nutrients well. While the β-mannanase must be
transformed into α-β-mannanase first before
inside to mitochondria in the synthesis cycle. The
latter was significantly affected (P<0.05) with
Mustafa et al. (2003) in percent digestibility of
palm kernel meal treated by the enzyme (44.09 vs. 41.20 (untreated)). Mustafa et al. (2003) stated the
PKM treated by an enzyme found increasing it
was due to adequate amino acid levels that
arginine, isoleucine, valine, and methionine. In
addition, from (Pasaribu et al., 2019) the increase
of palm kernel meal using microbial fermented
that produced mannanase enzyme were at 24-
32%. Carcass traits showed the reflection in line
with the final weight and internal organs were
better to compare to control 0.5% using α-β-
mannanase in the palm kernel meal. It (Table 6)
was not only affected by final weight and internal
organs but also depend on exogenous factors in
these conditions were using α-β-mannanase in the
palm kernel meal. Moreover, the breed in these
studies were hybrid ducks [(Peking x Khaki
Campbell)] also one factor affected to carcass
trait. RESULTS AND DISCUSSION The increased the
broiler body weight during the experiment due to
metabolism from the enzymatic cycles, the
enzyme that absorbs nutrient content combine
with metabolism to produce and grow the organs
in the ducks. The curve linear of live weight gain
may be impacted by feed consumption day by
day. The curve linear were affected from feeding programmed, one factor when feeding method
designed ad-libitum both of sex (male and
female). However, relatively temperature also
helps to increase feed intake during research the
average temperature 1-35 days were (26.19o
morning and 28.63o afternoon) with humidity
(88.94o morning and 88.69o afternoon). The lower
temperature at the chicken house may help
increase feed intake to eat more the experimental
diets. Contrast findings to a study from Stęczny
and Kokoszyński (2020) interactions between
enzyme and palm kernel meal were no interaction
for 35 days of age (2880 experimental vs. 2886
control group). The digestibility reflected the impact of the
F/G ratio (Table 5). The result of this study
showed the used α-β-mannanase in the palm
kernel meal presented a significant difference
(P>0.05) on the nutrient digestibility of dry matter Palm Kernel Meal containing α-β-mannanase Replacing Maize for Ducks (A. Sjofjan et al.) 35 AMEn were better compared to control (1,754;
1,756; 1765; 1777 vs. 1,752 (control)) and (1,844;
1833; 1,823; 1844 vs 1,855 (control)). Furthermore, additional study is required to
confirm these issues. Compared with Mustafa et
al. (2003) that the no significant difference found
in the AME and TME valued to higher NDF result
that ranged between 36-41%. The value of AME
was 1,870 kcal/kg continued AMEn, TME, and
TMEn values were line 1,743; 2053; and 1874
kcal/kg (Mustafa et al., 2003). Park et al. (2018)
stated with increasing β-mannanase concentration
on the nutrient digestibility also increased
significantly (P<0.0001). The nutrient digestibility
in line increasing due to amino acid histidine and
threonine stimulated the mucin secretion. The
goblet cell mucin secretion function was
developed by the discharge of histamine from cell
E linked with O-linked glycosylation in the
intestine (Park et al., 2018). and crude protein. The α-β-mannanase help to
give improvement (42.20; 41.30; 44.11; and 42.13
vs 40.20% (control)) for dry matter. In line with
nutrient digestibility of crude protein were also
increasing (42.20; 41.30; 44.11; 42.13 vs. 40.20%
(control)) for crude protein. ab Mean values in the same row without common superscript differ at p<005
1 1 NC (negative control; maize-soybean-meal based diet), PKM25 (maize replacement with PKM α-β-
mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement
with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). RESULTS AND DISCUSSION Relatively organ weight in this study was The PKM treated by enzyme didn’t help
apparent metabolizable energy (AME) and
apparent metabolizable energy n-correction due to
ME value in the ducks might be higher than
broiler (Mustafa et al., 2003). In this study, the
AME, AMEn, TME, and TMEn might be different
at growing and finishing periods. The AME and Table 5. The Effect of Increasing Levels of Palm Kernel Meal containing α-β-mannanase Replacing
Maize on the Nutrient Digestibility
Nutrient
Digestibility (%)
0.10% α-β-mannanase
SEM
P-values
NC vs PKM
NC
PKM 25
PKM50
PKM75
PKM100
Dry matter
40.20b
42.20a
41.30b
44.11a
42.13a
0.88
0.002
Crude protein
52.27b
53.39b
54.49a
55.56a
52.24b
1.23
0.004
AME (kcal / kg)
1,855
1,844
1,833
1,823
1,844
123.23
0.003
AMEn (kcal/kg)
1,752
1,754
1,756
1,765
1,777
111.11
0.011
TME (kcal/kg)
2,073
2,074
2,075
2,077
2,073
55.4
0.33
TMEn (kcal/kg)
1,893
1,844
1,906
1,852
1,888
54.5
0.22
ab Mean values in the same row without common superscript differ at p<005
1 NC (negative control; maize-soybean-meal based diet), PKM25 (maize replacement with PKM α-β-
mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement e Effect of Increasing Levels of Palm Kernel Meal containing α-β-mannanase Replacing
Nutrient Digestibility 1 NC (negative control; maize-soybean-meal based diet), PKM25 (maize replacement with PKM α-β-
mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement
with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). 1 NC (negative control; maize-soybean-meal based diet), PKM25 (maize replacement with PKM α-β-
mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement
with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). J.Indonesian Trop.Anim.Agric. 46(1):29-39, March 2021 36 Table 6. The Effect of Increasing Levels of Palm Kernel Meal Containing α-β-mannanase Replacing Maize
on the Carcass Traits and Relatively Organ Weight Table 6. RESULTS AND DISCUSSION The Effect of Increasing Levels of Palm Kernel Meal Containing α-β-mannanase Replacing Maize
on the Carcass Traits and Relatively Organ Weight
0.10% α-β-mannanase
SEM
P-values
NC vs PKM
NC
PKM 25
PKM50
PKM75
PKM100
Gizzard
2.69
2.94
2.83
3.25
2.99
0.88
0.002
Heart
0.63
0.59
0.67
0.60
0.63
1.23
0.004
Liver
2.04
1.90
2.05
2.19
1.92
123.23
0.003
Spleen
0.066
0.063
0.081
0.067
0.067 111.11
0.011
Pancreas
0.32
0.26
0.25
0.30
0.26
55.4
0.33
Abdominal fat
2.2
2.0
2.0
1.6
1.5
54.5
0.22
Caeca length (cm)
15.53
16.16
16.48
17.01
16.56
2.22
0.001
Caeca width (cm)
1.88
1.70
1.56
1.86
1.75
0.11
0.002
Carcass weight (g)
1203.75
1301.50
1260
1293
1192.25
234.5
0.003
Carcass (%)
61.47
65.87
63.70
63.23
61.72
2.22
0.004
Breast meat (%)
27.09
28.82
29.60
26.45
25.14
0.22
0.0015
ab Mean values in the same row without common superscript differ at p<005
1 NC (negative control; maize-soybean-meal based diet), PKM25 (maize replacement with PKM α-β-
mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement
with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). 1 NC (negative control; maize-soybean-meal based diet), PKM25 (maize replacement with PKM α-β-
mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement
with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). 1 NC (negative control; maize-soybean-meal based diet), PKM25 (maize replacement with PKM α-β-
mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement
with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). consistent in line with Chinajariyawong and
Muangkeow (2011) and Huang et al. (2009). The
abdominal fat was linearly decreasing with
increasing levels of α-β-mannanase in the palm
kernel meal compared to control (2.0; 2.0; 1.6;1.5
vs. 2.2 (control)). The lower abdominal fat might
be consistent with increasing crude fiber content. Compared
with
Chinajariyawong
and
Muangkeow (2011) studies the palm kernel meal
adjusted with β-mannanase were decrease the
abdominal fat. In addition, from Barbour et al.,
(2006) the supplemented of palm kernel meal
with enzyme were decreasing of abdominal fat pat
as levels of PKM increased. The higher immune
organs result was supposing to enzymatic
activities either mode of action and immune
stimulation. The factors affected to the
significantly different of the stomach is the ability
of the absorption. Wang et al. RESULTS AND DISCUSSION (2018) stated the
variance of the broiler are affects the absorption
and the capacities of the stomach. The growth
factors correlate with age, while the uses of the
broiler in the relative age cause the growth of the
internal organs the same. In some studies, according to Manafi et al. (2018) it results did not
increase in liver and spleen both male and female
groups (P>0.05), where areas, bursa of fabricius
as immune organ were increased (P<0.05). The number VFA presented no significant
difference (P<0.05) (Table 7). The result of
isobutyric of the treatment were not better than
the control (16.5; 16.2; 16.3; 16.4 vs. 16.0
(control)) and followed by n-butyric were (12.1;
12.2; 11.2; 10.2 vs. 11.9 (control)). The
production of VFAs depended on the caecal in
caecum when crude fiber breakdown the NSPs in
the palm kernel meal. The α-β-mannanase enzyme
extent of the fermentation process depends on the
microbial population in the caecum. Dunkley et
al. (2007) reported that acetic acid was the
primary source of VFA produced in the caecal
parts followed by butyrate and propionate. The
fermentation process to produce acetic acid was
started with i) breakdown of the NSP to simply
sugar (xylan to xylose to xylooligomers) that
might escape from enzymatic digestion, later
enter the caecum (Sharmila et al., 2014). Palm Kernel Meal containing α-β-mannanase Replacing Maize for Ducks (A. Sjofjan et al.) 37 Table 7. The Effect of Increasing Levels of Palm Kernel Meal containing α-β-mannanase Replacing Maize
on the Volatile Fatty Acid Table 7. The Effect of Increasing Levels of Palm Kernel Meal containing α-β-mannanase Replacing Maize
on the Volatile Fatty Acid
VFA
0.10% α-β-mannanase
P-values
NC vs PKM
NC
PKM 25
PKM50
PKM75
PKM100
SEM
Iso butyric
16.0
16.5
16.2
16.3
16.4
0.21
0.0014
n-butyric
11.9
12.1
12.2
11.2
10.2
0.22
0.0012
Iso-valeric
8.5
8.6
8.7
8.8
8.9
0.12
0.0011 ab Mean values in the same row without common superscript differ at p<005 1 NC (negative control; maize-soyabean-meal based diet), PKM25 (maize replacement with PKM α-β-
mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement
with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). 1 NC (negative control; maize-soyabean-meal based diet), PKM25 (maize replacement with PKM α-β-
mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement
with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). ACKNOWLEDGMENTS The author appreciated to LPPM, the
University of Brawijaya for Research Grant
Associate professor for granted Number:
732/UN.10. F05/PN/2020 and Non-Tax Revenue
(PNBP) University of Brawijaya SP DIPA-
023.17.2.677512/2020. Thank you to Pt. Wilmar
Cahaya Indonesia Tbk for providing the palm
kernel meal. Last but not least, thanks to Ms. Ainun Seruni Pizar [Missouri, University, and
Columbia, Missouri, United States of America]
for providing joint premium account Grammarly
granted. Chinajariyawong, C. and N. Muangkeow, N. 2011. Carcass yield and visceral organs of
broiler chickens fed palm kernel meal or
Aspergillus wentii TISTR 3075 fermented
palm kernel meal. WJST. 8(2):175-185. Dunkley, D. K., C.S. Dunkley, N.L. Njongmeta,T.R. Callaway, M.E. Hume,L.F. Kubena, D.J. Nisbet and S.C. Ricke. 2007. Comparison of in vitro fermentation and
molecular microbial profiles of high fiber
substrates incubated with chicken cecal
inocula. Poult. Sci. 86(5):801-810. CONCLUSION Adli, D. N. and O. Sjofjan. 2020. Growth
performance, serum blood biochemistry, and
intestinal properties of Arbor Acres Broiler
fed diets containing mannan-riched fraction
(MRF) and probiotic-enhanced liquid
acidifier. Bul. Pet. 44(2): 34-42. The increase of the level palm kernel meal
with α-β-mannanase enzyme wase had a positive
effect on the hybrid ducks and replacement of
palm kernel meal by 25% with the addition of the
α-β-mannanase enzyme was able to improve
digestibility and reduce abdominal fat of hybrid
ducks. BPS-RI. 2020. Livestock in figures 2020. Badan
Pusat Statistik. Jakarta. Barbour, G. W., M. T. Farran, N. N. Usayran, A. H. Darwish, M.G. Uwayjan, M. G. and V.M. Ashkarian. 2006. Effect of soybean oil
supplementation to low metabolizable
energy diets on production parameters of
broiler chickens. J. Appl. Poult. Res. 15(2):190-197. REFERENCES Adli, D.N. and O. Sjofjan. 2018. Nutrient content
evaluation of dried poultry waste urea
molasses block (DPW-UMB) on In-vitro
analysis. Sains Pet. 16(2):50-53. Huang, Y., J.S. Yoo, H.J. Kim, Y. Wang, Y.J. Chen,
J.H. Cho and I.H. Kim. 2009. Effect of
bedding types and different nutrient densities
on growth performance, visceral organ
weight, and blood characteristics in broiler
chickens. J. Appl. Poult. Res. 18(1):1-7. Adli D. N., O. Sjofjan., M. H. Natsir, Y. F. Nuningtyas, N. Sholikah and A. C. Marbun. 2020. The effect of replacing maize with
fermented palm kernel meal (FPKM) on
broiler performance. LRRD. 32(120). Jang, J. C., K.H. Kim, D.H. Kim, S.K. Jang, J.S. Hong, P.S. Heo and Y.Y. Kim. 2020. Effects
of increasing levels of palm kernel meal J.Indonesian Trop.Anim.Agric. 46(1):29-39, March 2021 38 kernel meal-based diet. J. Anim. and Poult. Sci. 3(1):19-28. containing β-mannanase to growing-
finishing pig diets on growth performance,
nutrient digestibility, and pork quality. Livest. Sci. 10(1): 40-41. Sjofjan O, M.H. Natsir, D.N. Adli, D.D Adelina
and L.M. Triana. 2020. Effect of symbiotic
flour (lactobacillus sp. and fos) to the egg
quality and performance of laying hens. IOP
Conf. Series: Earth and Environmental Sci.,
465, P.012-033. Manafi, M., M. Hedayati and S. Mirzaie. 2018. Probiotic bacillus species and saccharomyces
boulardii improve performance, gut
histology, and immunity in broiler chickens. South Afr. J. Anim. Sci 48(2):379-388. Sjofjan, O. and D.N. Adli. 2020. Effect of dietary
of supplementation mannan-riched fraction
(mrf) and probiotic-enhanced liquid acidifier
on the growth performance, serum blood
biochemistry, and intestinal properties of
broilers. In IOP Conference Series: Earth and
Environmental Sci., 478(1), P. 012066. Mustafa, M. F., A.R. Alimon, M.W. Zahari, I. Idris
and M. H. Bejo. 2003. Nutrient digestibility
of Palm kernel Cake for Muscovy ducks. Asian-Australas J. Anim. Sci. 17(4):514-517. Natsir, M. H., I. Djunaidi, O. Sjofjan, A. Suwanto,
E. Puspitasari, and L.J. Virginia. 2018. The
effect of corn substitution with palm kernel
meal treated by enzyme on production
performance and carcass quality of broiler. Bul. Pet. 42(2):103-108. Sjofjan, O., D.N. Adli, I. Djunaidi and K. Kuncoro. 2020. Utilization of Biogas Liquid
Waste For Starter In The Fermentation of
Rice Husk as A Potential Feed For Poultry. Anim. Prod. 22(1):24-30. Park, J., S. Jung and J. Carey. 2019. Effects of a
commercial beta-mannanase product on
growth
performance,
intestinal
histomorphology,
bone
and
body
composition, and amino acid digestibility in
white Pekin ducks. J. Appl. Poult. Res. 28(1): 63-71. Palm Kernel Meal containing α-β-mannanase Replacing Maize for Ducks (A. Sjofjan et al.) REFERENCES Stęczny, K. and D. Kokoszyński. 2020. Effect of
probiotic preparations (EM) and sex on
morphometric characteristics of the digestive
system and leg bones, and caecal microflora
in broiler chickens. J. Appl. Anim Res. 48(1):45-50. Pasaribu, T., E.B. Laconi and I.P. Kompiang. 2019. Evaluation of the nutrient contents of
palm kernel cake fermented by microbial
cocktails as a potential feedstuff for poultry. J. Indonesian Trop. Anim. Agric. 44(3):295-
302. Wang, Y., Z. Dong, D. Song, H. Zhou, W. Wang,
H. Miao, L. Wang and A. Li. 2018. Effects of
microencapsulated probiotics and prebiotics
on growth performance, antioxidative
abilities, immune functions, and caeca micro
flora in broiler chickens. Food and Agr. Immun 3(8):1-11. Sharmila, A., K. Azhar, M.N.Hezmee and A.A. Samsudin. 2014. Effect of xylanase and
cellulase supplementation on growth
performance, volatile fatty acids and caecal
bacteria of broiler chickens fed with palm Wilkinson, J. M. and R.H.Young. 2020. Strategies
to reduce reliance on soya bean meal and
palm kernel meal in livestock nutrition. J. Appl. Anim. Nutr. 8(2):75-82. 39 Palm Kernel Meal containing α-β-mannanase Replacing Maize for Ducks (A. Sjofjan et al.)
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Assessment of protective forest plantations on gray forest and sod-podzolic soils of the Predkamye region of the Republic of Tatarstan
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1 Introduction of national and other programmes to preserve the
environment, improve the efficiency of fertility, ensure
environmental and food security of the country, reduce
the level of discomfort in places of work and human
habitation [8]. To date, agricultural science has
developed and implemented the principles of adaptive-
landscape farming. The most important principle is the
application of a set of anti-degradation measures:
organisational
and
economic,
forest-reclamation,
agrotechnical, meadow-reclamation, hydrotechnical, and
others. The framework of adaptive-landscape farming is
a system of protective forest plantations [5, 6, 7, 17]. Establishment of protective forest plantations provides
multifunctional and long-term protection of soils from
erosion. Under the influence of soil and wind erosion,
unprotected soils lose fertility and are withdrawn from
agricultural turnover. Due to the created plantations,
species diversity is enriched, and ecological conditions
of agricultural lands are improved [20, 3]. They have a
lasting effect on the indicators and properties of washed
away soils, restoring the lost fertility [12]. Forest strips
on agricultural lands form a framework of erosion
protection, and the efficiency of the complex as a whole
depends to a great extent on their location in the relief [1,
2, 22]. In the presence of an interconnected system of
forest ameliorative plantations, crop yields increase by
8–18%. On average, the impact of 1 ha of forest
plantations extends to 10–12 ha of adjacent fields. According to A.I. Petelko, the scientifically justified
need for forest plantations in the country is 14 million
ha, but there are only 3.2 million ha [13]. The strategy
for the development of protective afforestation in the
Russian Federation until 2025 has been developed. It is
aimed at creating a complete system of ameliorative
protective forest plantations as a mandatory component The role of planted forests in carbon storage is
exceptional. Forests located on agricultural land are of
great importance for the absorption of greenhouse gases. According to Ivanov A.Y., Durmanov N.D. (2021), 1 ha
of forests on the lands of the forest fund can absorb, on
average, about 1 tonne of greenhouse gases per year. Protective and anti-erosion forests on agricultural lands
increase this indicator 7 times up to about 7 tonnes per
year. Assessment of protective forest plantations on gray forest and
sod-podzolic soils of the Predkamye region of the Republic of
Tatarstan
Aigul Mukhametshina, Haris Musin, Nail Mirsiyapov, Salavat Usmanov* and Ildar Khalilov
Kazan state agricultural university, 65, Karl Marks St., Kazan, 420015, Russia Aigul Mukhametshina, Haris Musin, Nail Mirsiyapov, Salavat Usmanov* and Ildar Khalilov
K
i
l
l
i
i
65 K l M k S
K
420015 R
i Aigul Mukhametshina, Haris Musin, Nail Mirsiyapov, Salavat Usmanov* and Ildar Khalilov Aigul Mukhametshina, Haris Musin, Nail Mirsiyapov, Salavat Usmanov* and Ildar Khalilov
Kazan state agricultural university, 65, Karl Marks St., Kazan, 420015, Russia Kazan state agricultural university, 65, Karl Marks St., Kazan, 420015, Russia Abstract. Protective forest plantations increase soil fertility and fulfil an important role of soil protection
from erosion. Much attention is paid to protective afforestation in the Republic of Tatarstan. In the region,
in 2022, protective forest plantations were established on the area of 1801 hectares, of which ravine-beam
forests make up 1763 hectares, including 645 hectares with terracing and 38 hectares of field protection
strips. Protective forest plantations were created in Aznakaevsky municipal district on the area of 600 ha
and in settlements of 114 ha of "compensatory" reforestation and afforestation. The article presents the
results of the survey of existing protective forest plantations in the Predkamya zone of the Republic of
Tatarstan on sod-podzolic and grey forest soils. The area is characterised by strong washing away, erosion
and gully formation, where the area of washed away soils is 66%, erosion-hazardous lands amount to
15.5%. A sanitary condition of plantations, productivity and carbon deposition were assessed. According to
silvicultural and taxation indicators, the average age of the surveyed stands is 48 years; the completeness
ranges from 0.5 to 0.9, bonitet ranges from I to II. In terms of the sanitary condition, trees of the 1st and
2nd categories predominate; in some areas forest management measures are required. Under the canopy of
the plantations, there is a natural regeneration of petiole oak, which is an indigenous species of the Pre-
Kama region of the Republic of Tatarstan. On all surveyed sample areas, the largest amount of carbon stock
is concentrated on PP7, PP8 and PP19: 786.96, 833.7 and 828.09 tonnes, respectively. These are mixed
stands of pine and birch. * Corresponding author: salusmanov7@yandex.ru
© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution
License 4.0 (https://creativecommons.org/licenses/by/4.0/). © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution
License 4.0 (https://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1051/bioconf/202410300065 https://doi.org/10.1051/bioconf/202410300065 BIO Web of Conferences 103, 00065 (2024) BIO Web of Conferences 103, 00065 (2024)
FIES 2023 d by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution
commons.org/licenses/by/4.0/). * Corresponding author: salusmanov7@yandex.ru 1 Introduction At the same time, according to various estimates,
from 40 to 90 million hectares of agricultural land in
Russia are overgrown with forests, which are not yet
accounted for in the national statistics of greenhouse gas
absorption because they do not belong to managed BIO Web of Conferences 103, 00065 (2024)
FIES 2023 https://doi.org/10.1051/bioconf/202410300065 years, the length of ravines has increased by more than
10 thousand kilometres. forests [4]. This is approximately 26 million tonnes of
carbon accumulation annually [9, 19]. It is considered a
promising direction to increase soil productivity of these
lands through carbon farming, application of modern
methods of biotechnology and breeding. In March 1997, the Cabinet of Ministers of the
Republic of Tatarstan adopted Resolution No. 216 and
approved the Comprehensive Programme of Soil
Fertility Improvement and Erosion Protection in the
Republic of Tatarstan for 1997–2005. It provides for a
set of anti-erosion measures. In accordance with the
programme, 7.8 thousand ha of protective plantations
were planted [14]. To date, most of the forest belts are
gradually dying off, subject to diseases and in a
neglected state. There is a need to carry out a set of
forestry measures to preserve and renew the systems of
field protective forest belts [10, 21]. For the effective functioning of existing forest
plantations, it is necessary to assess their current state
and to develop measures to maintain them. In this
regard, it is relevant to conduct research in the
Predkamye zone of the Republic of Tatarstan. The aim of the work is to assess the condition of
protective forest plantations on grey forest and sod-
podzolic soils in the Predkamya zone of the Republic of
Tatarstan. Special attention is paid to protective afforestation in
the region in order to prevent water and wind erosion of
soil, to increase the productivity of agricultural land, to
protect settlements, enterprises and other objects from
unfavourable natural phenomena and man-made impacts. According to the data of the Ministry of Forestry of RT,
in 2022, protective forest plantations were established on
the area of 1801 hectares, of which ravine-beam forests
make up 1763 hectares, including 645 hectares with
terracing, and field protection strips occupy 38 hectares. Protective forest plantations were created in the
Aznakaevsky municipal district within the framework of
climate projects on an area of 600 ha and in settlements
of 114 ha of "compensatory" reforestation and
afforestation. 2 Objects and methods of research Protective forest plantations in the Predkamya zone of
the Republic of Tatarstan were chosen as the object of
research. Sample areas were laid in accordance with
OST 56-69-83 "Sample forest inventory areas. Laying
method"
[11]. Glasometer
and
instrumental
measurements (an altimeter, a full-meter, a measuring
fork) were performed on each sample area. When
carrying out the works, we were guided by the rules of
sanitary safety in forests. They were approved by the
Decree of the Government of the Russian Federation of
20 May 2017, No. 607; Order of the Ministry of Natural
Resources of Russia of 16.09.2016, No. 480 "On
approval of the order of forest pathological surveys and
the form of the act of forest pathological survey"
[15, 16]. A total of 20 sampling areas (hereinafter
referred to as PAs) in the Predkamya zone of the
Republic of Tatarstan were investigated. Tree counting
was carried out at the PPs by thickness gradation (2 cm),
and each tree was assigned to a category of condition
[11]. During the research, 2000 trees were measured and
surveyed. On a 100-metre long trial area, trees were
assessed according to the following indicators. The
number of trees was counted; a trunk diameter was
measured with a measuring fork at a breast height of
1.3 m; a height was determined with a Danilin altimeter,
and natural vegetation was visually described. Plots of
different ages were selected for these purposes. Fig. 1. Protective afforestation over the last 5 years
2470
2415
2450
3559
2515
0
500
1000
1500
2000
2500
3000
3500
4000
ϮϬϭϴ
ϮϬϭϵ
ϮϬϮϬ
ϮϬϮϭ
ϮϬϮϮ
zĞĂƌƐ
Линейная (zĞĂƌƐͿ
>ŝŶĞĂƌ 1 Introduction The volumes of protective afforestation for
the last five years are shown in Fig. 1. 3 Analysis and discussion of the results Characteristics of plantations on grey forest and sod-
podzolic soils Table 1. Characteristics of plantations on grey forest and sod-
podzolic soils
No. n/a
Composition of
plantations
Breed
Age, years
Height, m
Diameter, cm
Bonnitet
Completeness
Area, ha
1
2
3
4
5
6
7
8
9
PP1 7E3Lz
European
spruce,
Siberian larch
30
8
10
12
14
1а 0.8 3.5
PP2 7B3E. Hanging birch,
European
spruce
30
16
6
18
10
1а 0.8 5.8
PP3
10С
Common pine
54
20
22
1
0.6 4.5
PP4
8B1C
1E
Hanging birch
60
22
26 1а 0.6
5
PP5
10Т
Poplar white
50
24
32 II 0.6 3.0
PP6
10Б
Hanging birch
65
23
32
1
0.6 5.3
PP7
9C1B Pine Common
birch Birch
overhanging
50
15
22
10
24
18
1
0.7 7.2
PP8
9C1B Pine Common
birch Birch
overhanging
50
15
22
10
32
16
1
0.5 7.0
PP9
10Б
Hanging birch
50
22
28 1а 0.6
PP10
10Б
Hanging birch
60
20
22 1а 0.6 4.0
PP11 6C4E
European
spruce
Common pine
35
10
12
8
10
1
0.9 8.0
PP12
10Б
Hanging birch
80
24
36 1а 0.6 2.7
PP13 8E1B1
C
European
spruce
Hanging birch
Common pine
40
10
12
1
0.8 2.65
PP14. 10Е
European
spruce
40
11
12
1
0.8 8.5
PP15. 10Б
Hanging birch
60
22
36 1а 0.6 2.2
PP16
10Б Hanging birch
60
22
32 1а 0.6 7.3
PP17 10Os. Poplar
trembling
60
21
28 II 0.6 1.9
PP18
10Б
Hanging birch
60
21
28 1а 0.6 2.2
PP19. 5B5S Hanging birch
Common pine
60
21
24
32
28
1а 0.7 7.2
PP20 8T2B
Poplar
Hanging birch
50
22
23
32
28
II 0.6 2.9 PP-3 is a clean composition roadside forest belt of 5
rows; a row spacing width is 2 m, in a row of 1.3 m. A
composition of plantations is 10С. Taxation parameters
are Cf. d =22, Cf. h =20 m. The forest belt is located at a
distance of 30 m from the road. On the site, there are
self-seeded pine trees of different ages (3–8 years old). In order to form a stable multi-age plantation, it is
advisable to carry out measures to promote natural
regeneration or mineralised strips. Weakened pine trees
(2nd category) account for 35.0 %. 3 Analysis and discussion of the results Preservation and rational use of lands are the main
conditions for ensuring stable development of the agro-
industrial complex and economy of the Republic of
Tatarstan. Over the last decade, the area of agricultural
land subject to erosion has increased. A high degree of
agricultural land ploughing is observed in Arsk and
Baltasinsk districts: 85–86%. On average, this indicator
for the region is 77.0%. The greatest increase is observed
in the zone of Predvolzhye (34.7%) and Predkamye
(30.6%). Fig. 1. Protective afforestation over the last 5 years For the study, sample areas were established in the
protective forest plantations in the Predkamya zone of
the Republic of Tatarstan (Sabinsky, Arsky, Atninsky
municipal districts). Territorially, the study area is
located in the catchment area of the Volga slope rivers
flowing parallel to each other from north-east to south-
west: Ashit, Kazanka and Meshi. The area is
characterised by strong washout, erosion and gully
formation, where the area of washed out soils is 66%,
erosion-prone lands are 15.5%. In this zone, light grey
forest soils and sod-podzol soils are most widespread on In the most pronounced form, the effect of water
erosion is manifested in the processes of gully formation. The number of active gully tops is about 20 thousand,
and their length is 27.4 thousand km. Over the last 40 2 2 BIO Web of Conferences 103, 00065 (2024)
FIES 2023 https://doi.org/10.1051/bioconf/202410300065 watershed plateaus and upper parts of slopes. There are
also grey and dark grey forest and sod soils. the sanitary condition, trees of the 1st category
predominate (90%) being without signs of damage. At
this site, weakened trees account for 10%; there are
minor mechanical damages. The results of the survey and the silvicultural and
taxonomic characterisation of the plantations at the PP
are given below (Table 1). PP-2 is a mixed composition roadside forest belt of
6-row YehBhEhEhEhBhB, a row spacing width is 2 m,
in a row of 1.3 m. A composition of plantations is 5E5B. Taxation indices of birch are Cf. d =18, Cf. h =16 m,
spruce is Cf. d =10, Cf. h =6 m. The forest belt is located
at a distance of 25 m from the road. The slow-growing
species European spruce lags behind the birch. In this
forest belt, the trees of the 1st category predominate
(95 %), "weakened" trees of the 2nd category are 5.0 %. Table 1. PP-1 is a mixed composition roadside forest belt
representing a mix in row 8Ex4Ltsx8Ex4Lts, 5 rows. A
row spacing width is 2.5 m, in a row of 1.3 m. A
plantation composition is 7E3Lts. Taxation parameters
are an average diameter of spruce (hereafter Cf. d) Cf. of
d = 12 cm, an average height (hereafter Cf. h) is Cf. h =
8 m, a larch is Cf. d = 14, Cf. h = 10 m. The introduced
larch forms a clump, where the forest environment under
the canopy is formed more rapidly due to needle fall. It
is located at a distance of 25 m from the road. In terms of 3 Analysis and discussion of the results PP 11 is a mixed composition roadside forest belt,
with row spacing 2 m wide, in a row of 1 m. A
composition is 6C4E. Taxation parameters of European
spruce are Cf. d = 8, Cf. h = 10 m. Taxation indices of
common pine are Cf. d = 10, Cf. h = 12 m. The condition
of the stands is satisfactory. Damage is localised along
the road: yellowed needles, pine trees are drying out. There are trees of 5th and 6th categories. Natural
regeneration is at the expense of birch. LNP is not
formed; litter is in the form of fallen needles. PP 11 is a mixed composition roadside forest belt,
with row spacing 2 m wide, in a row of 1 m. A
composition is 6C4E. Taxation parameters of European
spruce are Cf. d = 8, Cf. h = 10 m. Taxation indices of
common pine are Cf. d = 10, Cf. h = 12 m. The condition
of the stands is satisfactory. Damage is localised along
the road: yellowed needles, pine trees are drying out. There are trees of 5th and 6th categories. Natural
regeneration is at the expense of birch. LNP is not
formed; litter is in the form of fallen needles. PP 9 is a clear-cut field-protective forest belt. A
width of row spacing is 3 m, in a row of 3 m. A
composition of plantations is 10Б. Taxation indices of
common pine are Cf. d = 22, Cf. h = 28 m. The condition
of stands is satisfactory, trees of the 1st category
"without signs of weakening" prevail (85.0%). There is
the self-seeding of common pine, birch, oak. It is
necessary to take measures to promote natural
regeneration. PP 12 is a pure composition of a field protective
forest belt, with row spacing 3 m wide, in the row of 1.5
m. A composition is 10B. Taxation indices of birch are
Cd. d = 36, Cd. h = 24 m. The condition of plantations is
satisfactory; trees of the 2nd "weakened" category
prevail (75.0%). The forest environment is formed. Oviposition of gypsy moths is found on almost all
counted trees. Fruiting bodies of the true moth were also
found on trees of the 5th and 6th categories. Natural
regeneration takes place due to maple, linden, birch. Forest management measures with preservation of
natural regeneration are required. 3 Analysis and discussion of the results In our opinion, the
deterioration of the condition of plantations is due to
their close proximity to the roadway. PP-4 is a mixed composition roadside forest belt of
old-growth 6-row. A row spacing width is 3 m, in a row
of 1.5 m. Taxation parameters are Cf. d = 26, Cf. h = 22
m. A composition of plantations is 8B1C1E + shrub
Acacia yellow. BxExBxBxSxBxBxB is reconstructed. Trees of the 1st category "without signs of weakening"
predominate (95%). PP 5 is a pure field-protective forest belt representing
an 8-row old-growth; the row spacing is 3 m wide, 1.5 m
in a row. A composition of plantations is 10Т. Cf. d =
32, Cf. h =24 m. A forest litter thickness is up to 25 cm. A forest environment has been formed. Natural
regeneration is due to aspen, sharp-leaved maple, small-
leaved linden, birch, oak, oak, poplar of shoot origin. The predominant category of trees is "without signs of
weakening" (65%), and category 3 is "severely
weakened" (35.0%). Sanitary cuttings with removal of
faulted trees are required. PP 6 is a 6-row pure field protection forest belt. A
width of row spacing is 3 m, in the row of 1.5 m. A
composition is 10B. Taxation parameters are Cf. d = 32,
Cf. h = 26 m. Forest environment is formed;
characteristic forest plants are present. The condition of
plantations is satisfactory, single adult silkworms were
found. Clutches were not found. Natural regeneration is
due to birch, oak, oak, oak, maple. Trees of the 1st
category "without signs of weakening" are 70.0%, and
30.0% of the 2nd category are "weakened". Sanitary
cuttings with removal of faunal trees and promotion of
natural regeneration are required. PP 7 is a mixed composition field protective forest
belt. A width of row spacing is 3 m, in the row of 1.5 m. A composition of plantations is 8C2B. Taxation indices
of common pine are Cf. d = 24, Cf. h = 22 m. Birch is
lagging behind in growth. Taxation indices of birch are
Cf. d = 18, Cf. h = 10 m. The condition of the stands is
satisfactory; trees of the 1st category "without signs of 3 https://doi.org/10.1051/bioconf/202410300065 BIO Web of Conferences 103, 00065 (2024) BIO Web of Conferences 103, 00065 (2024)
FIES 2023 weakening" predominate (80%). Natural regeneration is
carried out by birch and oak. 3 Analysis and discussion of the results It is necessary to carry out
measures to promote natural regeneration. 5th and 6th categories. The natural regeneration of trees
is due to oyster maple, petiole oak and small-leaved
linden. Measures to promote natural regeneration are
required. Fig. 2. PP 7 Fig. 4. PP 10 Fig. 2. PP 7 Fig. 2. PP 7 Fig. 3. PP 8 Fig. 4. PP 10 Fig. 4. PP 10 Fig.5. PP 12 Fig. 3. PP 8 PP 8 is a mixed composition field-protective forest
belt. A width of row spacing is 3 m, in the row of 1.5 m. A composition of plantations is 8C2B. Taxation indices
of common pine are Cf. d = 32, Cf. h = 22 m. The birch
lags behind in growth. Taxation parameters of birch are
Cf. d = 10, Cf. h = 16 m. The condition of the stands is
satisfactory; trees of the 1st category "without signs of
weakening"
predominate
(75.0%). The
natural
regeneration of the forest is represented by common
pine, birch, oak (Fig. 3). It is necessary to carry out
measures to promote natural regeneration. PP 8 is a mixed composition field-protective forest
belt. A width of row spacing is 3 m, in the row of 1.5 m. A composition of plantations is 8C2B. Taxation indices
of common pine are Cf. d = 32, Cf. h = 22 m. The birch
lags behind in growth. Taxation parameters of birch are
Cf. d = 10, Cf. h = 16 m. The condition of the stands is
satisfactory; trees of the 1st category "without signs of
weakening"
predominate
(75.0%). The
natural
regeneration of the forest is represented by common
pine, birch, oak (Fig. 3). It is necessary to carry out
measures to promote natural regeneration. Fig.5. PP 12 Fig.5. PP 12 PP 11 is a mixed composition roadside forest belt,
with row spacing 2 m wide, in a row of 1 m. A
composition is 6C4E. Taxation parameters of European
spruce are Cf. d = 8, Cf. h = 10 m. Taxation indices of
common pine are Cf. d = 10, Cf. h = 12 m. The condition
of the stands is satisfactory. Damage is localised along
the road: yellowed needles, pine trees are drying out. There are trees of 5th and 6th categories. Natural
regeneration is at the expense of birch. LNP is not
formed; litter is in the form of fallen needles. 3 Analysis and discussion of the results The condition of plantations is
satisfactory; trees of the 1st category are predominantly
without signs of weakening (85.0%). g
g (
)
PP 14 is a clean composition of frontage protective
forest plantations. A row spacing width is 3 m, in the
row of 1.5 m. A composition is 10E. Taxation indices of
European spruce are Cf. d = 12, Cf. h = 11 m. There is
intraspecific competition, no silvicultural measures
(thinning). An insignificant number of trees damaged by
bark beetle-typograph is up to 5.0%, it is of natural
character. PP 15 is a pure composition field protective forest
belt. A width of row spacing is 3 m, in the row of 2 m. A
composition is 10B. Taxation parameters of birch are
Cd. d = 36, Cd. h = 22 m. Category 1 trees predominate
(85.0%) without signs of weakening. PP 16 is a clean composition of the forest fringe. A
width of row spacing is 3 m, in the row of 2 m. A
composition is 10 B. Taxation parameters of birch are
Cf. d = 32, Cf. h = 22 m. The age is 60 years. Trees of
the 1st category prevail without signs of weakening
(74.0%). Single trees of category 6 are old deadwood. PP 17 is a pure composition of a field-protective
forest belt. A row spacing width is 3 m, in a row of 1.5
m. A composition of plantations is 10 Os. Taxation
parameters of aspen are Cf. d = 28, Cf. h = 21 m. The
age is 60 years. Trees of the 2nd "weakened" category
predominate (85.0%). Trees of category 6 are old
deadwood; sanitary felling is necessary. PP18 is a clear field protective forest belt. A width of
row spacing is 3 m, in the row of 2.0 m. A composition
of plantations is 10Б. Taxation parameters of birch are
Cf. d = 28, Cf. h = 21 m. The age is 60 years. Trees of
the 1st category without signs of weakening prevail
(85.0%). PP19 is a mixed composition field-protective forest
belt. A width of row spacing is 3 m, in the row of 1.5 m. A composition is 5B5C. Taxation parameters of birch
are Cf. d = 32, Cf. h = 21 m. The age is 60 years. Taxation parameters of common pine are Cf. d = 28, Cf. h = 24 m. 3 Analysis and discussion of the results PP 10 is a pure field-protective forest belt, with the
row spacing of 3 m in width, 1.5 m in a row. A
composition of plantations is 10Б. Taxation indices of
birch are Cf. d = 22, Cf. h = 20 m. The condition of the
stands is satisfactory; "weakened" trees of the 2nd
category predominate (92.0%). Ovipositors of gypsy
moths were found on almost all counted trees. Fruiting
bodies of the true moth were also found on trees of the 4 https://doi.org/10.1051/bioconf/202410300065 BIO Web of Conferences 103, 00065 (2024)
FIES 2023 Table 2. Volume of deposited carbon in forest plantations
Tree
species
Area, ha
Total
stock,
m3
Total
phytomass,
tonnes
Carbon
stock,
tonnes
1
2
3
4
5
Trial area 1
Spruce
3.5
171.5
77.17
38.59
Larch
80.5
53.13
26.57
Total
252.0
130.3
65.16
Trial square 2
Spruce
5.8
214.6
96.57
48.29
Birch
545.2
354.38
177.19
Total
759.8
450.95
225.48
Trial area 3
Pine
4.5
832.5
416.2
208.1
Trial area 4
Birch
5.0
700.0
455.0
227.5
Pine
70.0
35.0
17.5
Spruce
45.0
20.25
10.13
Total
815
510.25
255.13
Trial square 5
Poplar
3.0
705.0
317.2
158.6
Trial square 6
Birch
5.3
869.2
564.98
282.49
Trial square 7
Pine
7.2
1180.8
590.4
295.2
Birch
302.4
196.56
98.28
Total
1483,2
786.96
393.48
Trial square 8
Pine
7.0
1358.0
679
339.5
Birch
238.0
154.7
77.35
Total
1596
833.7
416.85
Trial square 9
Birch
1.0
147.0
95.55
47.77
Trial area 10
Birch
4.0
496.0
322.4
161.2
Trial square 11
Spruce
8.0
360
162.0
81.0
Pine
480.0
240
120.0
Total
992
470.4
235.2
Trial area 12
Birch
2.7
523.8
340.47
170.23
Trial square 13
Spruce
2.65
108.6
48.9
24.4
Birch
18.6
12.6
6.0
Pine
10.2
5.1
2.5
Total
137.4
66.6
32.9
Trial square 14
Spruce
8.5
459.3
206.7
103.3
Trial area 15
Birch
2.2
378.4
245.96
122.98
Trial square 16
Birch
7.3
1116.9
725.98
362.99
Trial square 17
Poplar
1.9
362.9
181.45
90.73
Trial square 18
Birch
2.2
345.4
224.51
112.26
Trial square 19
Birch
7.2
733.6
476.84
238.42
Pine
702.5
351.25
175.63
Total
1436.1
828.09
414.05
Trial area 20
Poplar
2.9
426.3
191.83
95.92
Birch
118,9
77.28
38.64
Total
545.2
269.11
134.56 PP 13 is a mixed composition field protective forest
belt. A width of row spacing is 3 m, in the row of 1.5 m. A composition is 8E1B1C. Taxation parameters are Cf. d=12, Cf. h=10 m. 3 Analysis and discussion of the results Category 1 trees without signs of weakening
prevail (80.0%). p
(
)
PP 20 is a mixed composition field-protective forest
belt. A width of row spacing is 3 m, in the row of 1.5 m. A composition of plantations is 8T2B. Taxation
parameters of poplar are Cf. d = 32, Cf. h = 22 m. The
age is 60 years. Taxation parameters of birch are Cf. d =
28, Cf. h = 23 m. Trees of the 2nd "weakened" category
predominate (85.0%). Trees of the 5th and 6th categories
are present in insignificant quantities. It is necessary to
carry out forest management measures. 5 https://doi.org/10.1051/bioconf/202410300065 BIO Web of Conferences 103, 00065 (2024)
FIES 2023 Table 3. Annual volumes of average stemwood growth and
carbon sequestration in sample plots structure. The annual change in stem wood stock was
assessed to calculate the mass of carbon to be deposited
in the living phytomass of forests. The obtained growth
data were used to calculate phytomass fractions and the
amount of carbon deposited per year [18]. In all
surveyed sample plots, the largest amount of carbon
stock was concentrated in PP7, PP8 and PP19: 786.96,
833.7 and 828.09 tonnes, respectively. These are mixed
stands of pine and birch (Table 2). 3 Analysis and discussion of the results carbon sequestration in sample plots
Tree
species
Increase by
stock
Phytomass growth
Carbon
sequestration
m3/year
m3 /ha*year
tonnes per year
tonnes/ha*year
tonnes per year
tonnes/ha*year
Trial area 1
Spruce
5.72
1.63 2.57
0.73
1.29
0.37
Larch
2.68
0.77 1.77
0.51
0.89
0.25
Trial square 2
Spruce
7.15
1.23 3.22
0.56
1.61
0.28
Birch
18.17 3.13 11.81
2.04
5.91
1.02
Trial area 3
Pine
15.4
3.4
7.7
1.7
3.85
0.85
Trial area 4
Birch
11.66 2.33 7.58
1.52
3.79
0.76
Pine
1.16
0.23 0.58
0.12
0.29
0.06
Spruce
0.75
0.15 0.34
0.07
0.17
0.03
Trial square 5
Poplar 14.1
4.7
6.34
2.1
3.17
1.05
Trial square 6
Birch
13.37 2.52 8.69
1.64
4.35
0.82
Trial square 7
Pine
23.6
3.3
11.8
1.6
5.9
0.8
Birch
20.16
2.8 13.10
1.82
6.55
0.91
Trial square 8
Pine
27.16 3.39 13.58
1.94
6.79
0.97
Birch
9.2
1.31 10.31
1.47
5.16
0.74
Trial square 9
Birch
2.94
2.94 1.91
1.91
0.96
0.96
Trial area 10
Birch
8.26
2.07 5.37
1.34
2.69
0.67
Trial square 11
Spruce
10.2
1.2
4.6
0.81
0.6
0.3
Pine
19.2
2.4
9.60
1.20
4.80
0.60
Trial area 12
Birch
6.55
2.43 4.26
1.58
2.13
0.79
Trial square 13
Spruce
2.7
1.0
1.2
0.5
0.6
0.2
Birch
0.5
0.2
0.3
0.1
0.2
0.1
Pine
0.3
0.1
0.1
0.0
0.1
0.0
Trial square 14
Spruce
11.5
1.4
5.2
2.0
2.6
1.0
Trial area 15
Birch
6.31
2.86 4.10
1.86
2.05
0.93
Trial square 16
Birch
18.62 2.55 12.10
1.66
6.05
0.83
Trial square 17
Aspen
6.05
3.18 3.02
1.59
1.51
0.80
Trial square 18
Birch
5.76
2.62 3.74
1.70
1.87
0.85
Trial square 19
Birch
12.23 1.69 7.95
1.10
3.97
0.55
Pine
11.71 1.63 5.85
0.81
2.93
0.41
Trial area 20
Poplar
8.5
2.9
3.8
1.3
1.90
0.65
Birch
1.98
0.68 1.29
0.44
0.64
0.22
Carbon stocks and rates of carbon storage in forest
d
d
f
d
i i
h i According to literature data, a promising species in
carbon sequestration is poplar (Populus) in northern and
continental climates. This species is common in part of
Russia and is found in many Russian cities. It has a
relatively high carbon sequestration potential of 1.8–
6.35 t CO2-eq/ha/year, a high growth rate of 1.524–3.6
m/year, a short productive cycle of 10–15 years [22]. Out of all the 20 surveyed sites, two of them have PP5
and PP20 poplar in their composition. Conclusions The surveyed protective forest plantations on sod-
podzolic and grey forest soils are in a satisfactory
condition. According to silvicultural and taxation indicators, the
average age of the surveyed stands is 48 years; the
completeness is within the range from 0.5 to 0.9, and the
bonitet is within the range from I to II. In terms of the sanitary condition, trees of the 1st and
2nd categories prevail; in some areas, forest management
measures are required. Under the canopy of plantations, there is a natural
regeneration of petiole oak, which is a native species of
the Pre-Kama region of the Republic of Tatarstan, as
well as birch, pine, and oak maple. Established protective forest plantations fulfil their
functions, i.e. reduce erosion and increase soil fertility. In areas with viable undergrowth, it is advisable to
carry out measures to promote natural regeneration and
conservation. Preservation of undergrowth promotes
further formation of stable mixed, mixed-aged, complex
in structure, protective forest plantations. The largest volume of deposition was detected in
mixed stands of birch and pine, poplar. 3 Analysis and discussion of the results According to the
silvicultural
and
taxonomic
characteristics,
the
plantations were classified as a II class of bonitet. At
these sites, it is necessary to carry out sanitary cuttings. The volume of carbon deposition in these areas was 3.17
and 1.90 tonnes/ha per year. References 1. A.T. Barabanov, A.I. Petelko, A.V. Kulik, A.V. Vypova, New technology of placement of flow-
regulating forest strips on slope lands, Izv. NV AUK
2(54) (2019) 2. A.T. Barabanov, Erosion-hydrological assessment
of interaction between natural and anthropogenic
factors in the formation of surface runoff of melt
water
and
adaptive-landscape
farming
(FSC
Agroecol. RAS, Volgograd, 2017), 188 p. Carbon stocks and rates of carbon storage in forest
ecosystems
depend
on
forest
productivity,
their
condition, species composition, age and commodity 6 BIO Web of Conferences 103, 00065 (2024)
FIES 2023 https://doi.org/10.1051/bioconf/202410300065 and dry-steppe zones of the Volga region, Niva
Povolzhye 2(43) (2017) 3. V.I. Erusalimsky, V.A. Rozhkov, Multifunctional
role of protective forest plantations, Bull. of Soil
Inst. Pochv. inst. 88 (2017) 18. E.M. Romanov, T.V. Nureeva, T.F. Miftakhov, A.S. Puryaev, Ecological and raw material role of forests
of the Republic of Tatarstan, Bull. of Volga Reg. State Technol. Univ., Ser.: Forest, Ecology, Nature
Manag. 2, 5–18 (2015) 4. A.Y. Ivanov, M.P. Orlov, K.V. Piksendeev, Y.E. Rovnov et al., Battle for the climate: carbon farming
as Russia's stake, Expert report, ed. by A.Y. Ivanov,
N.D. Durmanov (Izd. dom Higher School of Econ.,
Moscow, 2021), 120 p. ISBN 978-5-7598-2519-7
(in reg.), ISBN 978-5-7598-2281-3 (e-book). 19. V.G. Sergienko, Impact of expected climate change
on the carbon balance and ecosystem productivity in
the forest sector of the Russian Federation, Proc. of
the St. Petersburg Res. Inst. of Forestry 1, 74–90
(2018) 5. K.N. Kulik, Development of agroforestry science in
Russia, Izv. NV AUC 3(35) (2014) 6. K.N. Kulik, A.T. Barabanov, A.S. Manaenkov,
Forecast
of
the
development
of
protective
afforestation in Russia until 2020, Probl. of Forecast
4 (2015) 20. D.K. Suchkov, Role and economic efficiency of
protective forest plantations in the restoration and
transformation of landscapes, Sci. and Agron. J. 1(102), 20–23 (2018) 7. K.N. Kulik, Protective afforestation in the Russian
Federation: problems and development strategy until
2020, Theor. and appl. probl. of agroindustry
complex 1 (2009) 21. G.A. Petrova, A.R. Mukhametshina, H.G. Musin,
R.H. Mukhametshina, H.G. Musin, R.H. Gafiyatov,
Analysis of the Aspen Growth (Populus tremula L.)
obtained by in Vitro in the Republic of Tatarstan,
BIO Web Conf. 37 (2021) 8. K.N. Kulik, Strategy for the development of
protective afforestation in the Russian Federation
until 2025, Bull. of Agricult. Design 3, 5–11 (2015) 22. S.T. Zhang, J.Z. Zhang, Y. Liu, Y.C. Liu, Effects of
farmland vegetation row direction on overland flow
hydraulic characteristics, Hydrol. References Res.. 49(6), 1991–
2001 (2018) 9. P.I. Lakida, Carbon stocks in the phytomass of
forest phytocenoses of the National Park “Pripyat-
Stokhod”, Problems of forestry and silviculture, In:
Collection of scientific papers of IL NAS of Belarus,
Iss. 77 (Inst. of Forestry of the National Acad. of
Sci. of Belarus, Gomel, 2017), pp. 99–107. 10. A.R. Mukhametshina, G.A. Petrova, H.G. Musin,
I.R. Taziev, Analysis of the state of agroforest
landscapes of the Republic of Tatarstan and ways of
their
restoration,
Innovative
technologies
in
agroindustrial complex in the conditions of digital
transformation, Proc. of the Int. Sci. and Pract. Conf. (Volgograd,
09–11
February
2022)
(Volgograd State Agrar. Univ., Volgograd, 2022),
Vol. I, pp. 421–426, EDN JSIVAC 11. OST 56-69-83 Sample forest inventory areas. Method of laying 12. A.I. Petelko, Restoration of fertility of washed away
soils, Prirodoobustroystvo 1 (2017) 13. A.I. Petelko, N.E. Novikov, Protective afforestation,
Vest. AIC Stavropolya 3(15), 175–178 (2014) 14. On the Comprehensive Programme of Soil Fertility
Improvement and Protection against Erosion in the
Republic of Tatarstan for 1997–2005, Resolution of
the Cabinet of Ministers of the Republic of Tatarstan
of 12 March 1997, no. 216 15. Resolution of the Government of the Russian
Federation, of 20 May 2017, no. 607 16. On approval of the procedure for conducting forest
pathological surveys and the form of the act of
forest pathological survey, Order of the Ministry of
Natural Resources and Environment of Russia,
dated 16.09.2016, no. 480 17. P.N. Proezdov, D.A. Mashtakov, A.V. Panfilov,
Theoretical substantiation of adaptive-landscape
systems of farming and agroforestry in the steppe 7
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English
| null |
A new blattoid from the Cretaceous formation of North America
|
Proceedings of the United States National Museum
| 1,906
|
public-domain
| 547
|
By
Anton
HANDLIRSCH, Adjunct Curator of the Royal Imperial Natural History Museum, Vienna, Austria. Adjunct Curator of the Royal Imperial Natural History Museum, Vienna, Austria. During
the
summer
of
1903,
while
members
of
the
U. 5. Geological
Survey
were
investigating
the
Judith
River
beds
of
the
Upper
Cre-
taceous
of
Montana,
the
following
very
interesting
blattoid
was
discovered;
| @Translated from the German by Lucy Peck Bush, librarian and assistant, geo-
logical department, Yale University Museum. View This Item Online: https://www.biodiversitylibrary.org/item/53438
DOI: https://doi.org/10.5479/si.00963801.29-1439.655
Permalink: https://www.biodiversitylibrary.org/partpdf/211254 View This Item Online: https://www.biodiversitylibrary.org/item/53438
DOI: https://doi.org/10.5479/si.00963801.29-1439.655
Permalink: https://www.biodiversitylibrary.org/partpdf/211254 STANTONIA
CRETACEA,
new
species. Description.—
Front
wing
20
mm. long,
nearly
elliptical,
and
three
and
one-third
times
as
long
as
wide. Costal
area
reduced,
reaching
two-fifths
the
length
of
the
wing,
and
without
distinct
veins,
lancet
shaped. Radius
extending
in
an
almost
straight
course
to
the
tip
of
the
wing
and
with
its
eight
more
or
less
compound
branches,
which STANTONIA
CRETACEA. STANTONIA
CRETACEA. are
directed
obliquely
forward,
taking
up
nearly
half
the
surface
of
the
wing. Parallel
with
the
radius
runs
a
second
principal
vein,
from
which
three
simple
and
two
compound
branches
are
sent
off
backward,
part
striking
the
apical
border
and
part
the
inner
margin. [am
not
in
a
position
to
determine
whether
these
veins
pertain
to
the
media @Translated from the German by Lucy Peck Bush, librarian and assistant, geo-
logical department, Yale University Museum. PROCEEDINGS U. S. NATIONAL Museum, VOL. XXIX—No. 1439. Proc. N. M. vol. xxix—05-——46 655 656 EDINGS
OF
THE
NATIONAL
MUSEUM. XXIX, and
the
cubitus
or
only
to
the
latter;
still
it
seems
to
me
the
most
probable
that
the
first
four
veinlets
belong
to
the
media
and
the
last
six
to
the
cubitus. Or,
is
it
possible
that
the
media
has
entirely
disap-
peared? The
anal
area
is
long
and
narrow,
three
and
one-half
times
as
long
as
high,
and
occupies
almost
two-fifths
the
length
of
the
wing;
its
veins
run
parallel
with
the
posterior
margin,
and
nearly
all
end
on
the
suture. The
veins
are
remarkably
stout. I
was
not
able
to
dis
tinguish
accessory
or
cross
veins. This
highly
specialized
blattoid
form
is
the
first
that
has
been
found
in
the
Cretaceous
formation,
and
may
well
be
regarded
as
the
type
of
a
distinct
family. Holotype.—Cat. No. 35389,
U.S.N.M. Holotype.—Cat. No. 35389,
U.S.N.M. Locality.—The
genus
is
named
in
honor
of
Dr. T. W. Stanton,
of
the
U. S. Geological
Survey,
who
collected
the
type
specimen
in
the
Judith
River
beds
of
the
Upper
Cretaceous,
at
Willow
Creek,
Mon-
tana,
where
it
was
found
associated
with
the
fossil
plants
described
by
Dr. F. H. Knowiton
in
Bulletin
No. 257
of
the
U.S. Geological
Survey. Holding Institution Smithsonian Libraries and Archives This file was generated 29 March 2024 at 07:49 UTC Handlirsch, Anton. 1906. "A new blattoid from the Cretaceous formation of
North America." Proceedings of the United States National Museum 29(No. 1439), 655–656. https://doi.org/10.5479/si.00963801.29-1439.655. Copyright & Reuse Copyright & Reuse
Copyright Status: Public domain. The BHL considers that this work is no longer under
copyright protection. This document was created from content at the Biodiversity Heritage Library, the world's
largest open access digital library for biodiversity literature and archives. Visit BHL at
https://www.biodiversitylibrary.org. This file was generated 29 March 2024 at 07:49 UTC
|
https://openalex.org/W3018455884
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|
English
| null |
Reconstructing Genotypes in Private Genomic Databases from Genetic Risk Scores
|
Journal of computational biology
| 2,021
|
cc-by
| 13,113
|
ABSTRACT Some organizations such as 23andMe and the UK Biobank have large genomic databases that
they re-use for multiple different genome-wide association studies. Even research studies that
compile smaller genomic databases often utilize these databases to investigate many related
traits. It is common for the study to report a genetic risk score (GRS) model for each trait
within the publication. Here, we show that under some circumstances, these GRS models can
be used to recover the genetic variants of individuals in these genomic databases—a recon-
struction attack. In particular, if two GRS models are trained by using a largely overlapping
set of participants, it is often possible to determine the genotype for each of the individuals who
were used to train one GRS model, but not the other. We demonstrate this theoretically and
experimentally by analyzing the Cornell Dog Genome database. The accuracy of our recon-
struction attack depends on how accurately we can estimate the rate of co-occurrence of pairs
of single nucleotide polymorphisms within the private database, so if this aggregate information
is ever released, it would drastically reduce the security of a private genomic database. Caution
should be applied when using the same database for multiple analysis, especially when a small
number of individuals are included or excluded from one part of the study. Keywords: genetic risk scores, genomic privacy, GWAS, long-term privacy, reconstruction attack. Reconstructing Genotypes in Private Genomic
Databases from Genetic Risk Scores BROOKS PAIGE,1,2 JAMES BELL,1 AURE´LIEN BELLET,3
ADRIA` GASCO´ N,1,4 and DAPHNE EZER1,4,5 JOURNAL OF COMPUTATIONAL BIOLOGY
Volume 28, Number 5, 2021
Mary Ann Liebert, Inc.
Pp. 435–451
DOI: 10.1089/cmb.2020.0445 JOURNAL OF COMPUTATIONAL BIOLOGY
Volume 28, Number 5, 2021
Mary Ann Liebert, Inc. Pp. 435–451
DOI: 10.1089/cmb.2020.0445 JOURNAL OF COMPUTATIONAL BIOLOGY
Volume 28, Number 5, 2021
Mary Ann Liebert, Inc. Pp. 435–451
DOI: 10.1089/cmb.2020.0445 1The Alan Turing Institute, London, United Kingdom.
2Department of Computer Science, University College London, London, United Kingdom.
3Inria, Parc Scientifique de la Haute Borne Park Plaza, Villeneuve d’Ascq, France.
4University of Warwick, Coventry, United Kingdom.
5Department of Biology, University of York, York, United Kingdom.
Brooks Paige, et al., 2021. Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the
t
f th C
ti
C
Li
(htt //
ti
/li
/b /4 0)
hi h
it
t i t d Brooks Paige, et al., 2021. Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the
terms of the Creative Commons 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. Keywords: genetic risk scores, genomic privacy, GWAS, long-term privacy, reconstructio PAIGE ET AL. PAIGE ET AL. research focus on long-term privacy of genomic databases rests on the longevity of the encryption scheme
(Huang et al., 2015), it is also important to remember that these genomic databases are not just sitting on
a server somewhere, but are also being continually utilized for making new scientific discoveries. Each time
these databases are accessed and the scientific results are published, there is a risk that information will be
leaked and that eventually this would enable an attacker to reconstruct private information held in the database. Genomic researchers are already aware that some forms of aggregate data from their databases should not be
released publicly, because there is a risk that an attacker may be able to determine whether a particular individual
is a member of the database (a membership inference attack). For instance, such attacks have already been
developedforsummarystatisticsabout the frequencyofsingle nucleotidepolymorphisms(SNPs;Cai etal.,2015;
Dwork et al., 2015; Simmons and Berger, 2015). Membership inference attacks have also been developed for the
case where a person is allowed to repeatedly query a database to learn whether at least one individual contains a
particular SNP(ShringarpureandBustamante,2015; Raisaroetal.,2017; vonThenenetal.,2018).Thesekindsof
aggregate statistics about the frequency or presence/absence of a particular SNP in a database might be useful to
release to the broader research community, but it is not an essential output of the research process. However, the main research findings—that is, the SNPs associated with the trait of interest and their
strength of association—are essential to publish since the entire purpose of these genomic research projects
is to uncover the relationship between genetic variants and phenotypic traits. Moreover, knowledge of these
SNPs can lead to new diagnosis procedures or new potential drug targets, so their release is important for
the public interest (Visscher et al., 2017). However, even this information can potentially leak private
information about individuals in the database. For instance, Im et al. (2012) found that information about
individuals in a genomic database is leaked when studies publish whether each SNP is correlated or anti-
correlated to the trait of interest. It is important to quantify how much information is leaked by publishing
these research findings, so that scientists can make informed decisions about when to publish their results
and whether it is worth risking the privacy of the participants. PAIGE ET AL. g
p
y
p
p
In this study, we demonstrate that the kind of research output that is published from genome-wide
association studies (GWAS) has the potential to leak enough information to recover the SNPs of individuals
in the database (a reconstruction attack), under specific circumstances. In particular, we focus on the release
of genetic risk scores (GRS), a common research output for finding genetic associations with continuous
traits (Qi et al., 2011; Belsky et al., 2013; Zhao et al., 2014; Chouraki et al., 2016; Day et al., 2017;
Knowles and Ashley, 2018). We also focus on cases where a database is repeatedly used to perform a
GWAS analysis, but not all the individuals are part of all the analyses. This could be the case because some
individuals drop out of the study or skip specific survey questions. Alternatively, some databases, such as
23andMe, may grow in size over time and allow several GWAS to be performed within a short period. Under these circumstances, we demonstrate that it is possible to completely reconstruct the SNPs of an
individual by using a custom expectation–maximization (EM) algorithm. We also provide suggestions for
avoiding this kind of attack. To be clear, this article focuses on the simpler case in which the exact same trait is investigated in
multiple GWAS studies; however, we expect that some version of this attack may be developed in the near
future for the case of multiple highly correlated traits. 1. INTRODUCTION I
n a survey of genomic privacy experts, the long-term privacy of genomic information was deemed
both the most important and the most challenging problem to overcome (Mittos et al., 2019). If an
individual’s password or ID number gets leaked, it is always possible to change it. However, it is impossible
for a person to change their genetic code and they will pass part of it onto their children, so any information
leaks can have long-term impacts on both the individual and their descendants. Although much of the I 435 436 1.1. Overview of scenarios that will be investigated We demonstrate a series of reconstruction attacks that enable us to infer the genotypes of individuals in
private genomic databases, based on publicly released GRS. These attacks will initially be deployed in a very
favorable scenario, but the scope of the attack will be subsequently expanded, building up to the scenario
shown in Figure 1. It is worth noting that the reconstruction attacks that we will describe do not depend on
(1) how the SNPs were initially filtered or (2) how strongly they associate with the trait of interest. We will begin by investigating a simple scenario: Two GWAS studies are performed to identify SNPs
associated with the same trait, and the two studies use the same set of participants, except that the second
study includes one extra individual. In addition, we will assume that we know the frequencies of each SNP
and the frequencies that pairs of SNPs co-occur in the same individual. We assume that both studies publish
the coefficients associated with the GRS models that they infer as part of the analysis. Next, we will consider the case in which the second study includes more than one additional participant
and we demonstrate that in many circumstances this still allows us to easily reconstruct the individual
genotypes of all the individuals that are found in the second study but not the first (see Section 3.2). 437 RECONSTRUCTION ATTACKS FROM GRS FIG. 1. We investigate the case where two GWAS studies are performed on two datasets that mostly contain the same
individuals. We reconstruct the genotype of those individuals added to the second study, using the GRS from each study
and an estimate of SNP frequencies. GRS, genetic risk score; GWAS, genome-wide association studies; SNP, single
nucleotide polymorphism. FIG. 1. We investigate the case where two GWAS studies are performed on two datasets that mostly contain the same
individuals. We reconstruct the genotype of those individuals added to the second study, using the GRS from each study
and an estimate of SNP frequencies. GRS, genetic risk score; GWAS, genome-wide association studies; SNP, single
nucleotide polymorphism. Afterward, we will demonstrate that we do not need to know the precise frequencies of SNPs and
frequencies of co-occurring SNPs, as long as we have a reasonable estimate of these values from public
databases (see Section 3.3). We also briefly discuss how loosening additional restrictions would impact our
ability to predict individual genotypes. 1.1. Overview of scenarios that will be investigated In particular, we analyze the case where the two sets of SNPs that
are used by the two studies are not identical. These results imply that if two sets of GRS are released on two
genetic datasets with largely overlapping populations, it may be possible to reconstruct the genotypes of
those individuals who participated in one study but not the other (Fig. 1). PAIGE ET AL. where we have defined the symmetric (N + 1) · (N + 1) matrix K as where we have defined the symmetric (N + 1) · (N + 1) matrix K as K = 1
M F>
MFM: K = 1
M F>
MFM:
(4) (4) Now, suppose a second study is run, targeting the same phenotype, which adds a single extra individual
with SNPs represented by the N length vector x0. This corresponds to adding the row />
0 = [ x>
0
1 ] to the
design matrix, and extending y with the additional phenotypic value y0 for the new individual. The updated
estimator (i.e., the GRS values for the second study) is given by ^bM + 1 = (F>
MFM + /0/>
0 ) - 1(F>
MyM + y0/0):
(5) (5) We assume that both GRS models ^bM and ^bM + 1 are released publicly. An attacker aims at using this
knowledge to reconstruct /0 (the genotype of the added individual). Through algebraic re-arrangement (see
Section 5.2), we find that: /0 = 1
C K(^bM + 1 - ^bM)
(6) (6) where C is a scalar, specifically C = 1
M (y0 - />
0 ^bM + 1). Eq. (6) means that /0 is a scalar multiple of
K(^bM + 1 - ^bM). proach, thus, centers on the use of the vector that we define as d1, Our approach, thus, centers on the use of the vector that we define as d1, d1 =
D K(^bM + 1 - ^bM) = C/0‚
(7) (7) corresponding to a rescaled copy of the input SNP data in the design matrix /0, which can be easily
computed from the two parameter vectors if the matrix K is known. As seen in Section 3.1, we can use d1 to
exactly reconstruct the added individual with 100% accuracy. corresponding to a rescaled copy of the input SNP data in the design matrix /0, which can be easily
computed from the two parameter vectors if the matrix K is known. As seen in Section 3.1, we can use d1 to
exactly reconstruct the added individual with 100% accuracy. We additionally consider the case where m additional individuals have been included in the second
study, yielding a new GRS model ^bM + m including these M + m participants. PAIGE ET AL. The extra rows of the design
matrix now form a matrix Fm of size m · (N + 1), where each row is an individual that was added to the
second study and each column is an SNP (and the last column contains only 1). The corresponding analog
to Eq. (7) for multiple individuals, which we derive in Section 5.2, is dm =
D K(^bM + m - ^bM) = F>
mCm‚
(8) (8) where Cm is a vector of length m. For sufficiently small m (relative to N), exact reconstruction of all m
added individual genomes is also possible in this setting, following the algorithm we will introduce in
Section 3.2. where Cm is a vector of length m. For sufficiently small m (relative to N), exact reconstruction of all m
added individual genomes is also possible in this setting, following the algorithm we will introduce in
Section 3.2. The previous examples have focused on cases in which the participants in the first study are a subset of
the individuals in the second study. In Section 5.7, we consider the case in which the first study has some
participants that are not found in the second study and vice versa. We show that the same strategies for
reconstructing the genome can be used as in the previous scenario that we discussed, in which multiple
participants are added to the second study. 2. METHODS GRS models describe the relationship between a particular phenotype of interest and particular SNPs. These models are fit in a two-stage process: First, a reduced set of SNPs is selected from a potentially very
large pool of candidates; then, this reduced set is used as the independent variables in a linear regression
analysis. The set of SNPs is selected by first filtering for those that significantly correlate to the trait of
interest, after controlling for other covariates. These SNPs are then further filtered to ensure that they are
far apart from one another, to decrease the correlation between them. In this setting, we suppose that M
individuals have taken part in a study, and N SNPs have passed the filtration steps to be used in a linear
model. Let yM be the vector of M real-valued phenotypes, and XM be an M · N binary matrix, where
XM[i‚ j] = 1 if individual i has SNP j. To include an intercept term in the linear model, we define the design
matrix FM to be the M · (N + 1) matrix FM = XM
1M
½
:
(1) (1) The GRS model parameter bM is just the coefficient vector of the linear model yM = FMbM + e‚
(2) yM = FMbM + e‚ (2) where e is independent Gaussian noise. Given FM and phenotypes yM, the maximum likelihood estimate of
this parameter has a closed form where e is independent Gaussian noise. Given FM and phenotypes yM, the maximum likelihood estimate of
this parameter has a closed form ^bM =
D 1
M K - 1(F>
MyM)‚
(3) (3) 438 – For i = 1‚ . . . ‚ N: Kii estimates the probability that SNP i has value 1 (i.e., the frequency of the SNP in
the population).
– For i = 1‚ . . . ‚ N - 1 and j > i: Kij = Kji estimates the probability that both SNP i and SNP j are 1
simultaneously (i.e., the frequency of SNP i and SNP j co-occurring in the same individual).
– For i = 1‚ . . . ‚ N and j = N + 1: Kij = Kji also estimates the probability that SNP i has value 1, that is,
Ki‚ N + 1 = KN + 1‚ i = Kii.
– Finally, KN + 1‚ N + 1 = 1. or i = 1‚ . . . ‚ N and j = N + 1: Kij = Kji also estimates the probability that SNP i has value 1, that i
+ 1 = KN + 1‚ i = Kii. 3.1. Complete reconstruction of one individual’s genotype when SNP frequency information
is known 3.1. Complete reconstruction of one individual’s genotype when SNP frequency inform
is known The first, most straightforward case is when only one participant is added between the first and second
studies, that is, where ^bM is the GRS for the first study (containing M participants), and ^bM + 1 is the GRS for
the second study as described in Eqs. (3) and (5). Both of these are vectors of length N + 1, where the first N
indices correspond to the relationship between each SNP and the trait and the last element is the intercept of
the linear model. For now, we also assume we are in the setting where the matrix K is known, for example,
because the SNP frequency information has been publicly released. ^
^ Given K, ^bM + 1, and ^bM, we can use d1 (a vector of length N + 1) to precisely determine the genotype of
the individual who was added to the database. For each i = 1‚ . . . ‚ N, the ith entry of d1 is either equal to 0 if
/0 contains a 0 (i.e., the individual does not have the SNP at that index) or to C if /0 contains a 1 (i.e., the
individual has the SNP at that index). In other words, it is possible to exactly read off the SNPs of the added
individual in this setting. Indeed, we tested this strategy on the Cornell Dog Database and found that we
were able to reconstruct the genotype of the dog that was added to the second study with 100% accuracy, on
both common and uncommon SNPs (Fig. 2A). A
B
C
FIG. 2. (A) We have perfect accuracy in reconstructing the genotype when K is known (using 200 random SNPs
to estimate average breed weight in the Cornell Dog Database). (B) We can reconstruct all the genotypes of multiple
dogs that are added to the second study and (C) this works in practice by using the data from the Cornell Dog Database,
as in (A). B
C A C C B A FIG. 2. (A) We have perfect accuracy in reconstructing the genotype when K is known (using 200 random SNPs
to estimate average breed weight in the Cornell Dog Database). RECONSTRUCTION ATTACKS FROM GRS 439 Thus, knowledge of SNP frequencies and pairwise co-frequencies from the original study are all that is
required to compute K. In the following Sections 3.1 and 3.2, we consider adding one and multiple
individuals at once, respectively, in the setting where this matrix K can be estimated exactly. However,
although ^bM, ^bM + 1 and M are likely to be published along with the study, an attacker would often need to
estimate K from other publicly available data. Most studies will report some information about the study
population (such as whether the study focused on individuals from a specific continent), which can help
with estimating K. From this information, we can estimate the value of K in similar populations as those
used in the study using publicly available data, for example, from the HapMap project. Our additional
experiments in Section 3.3 use a custom EM algorithm to find maximum likelihood estimates of /0 when
the matrix ^K K is estimated from independent public data. The derivation of this EM algorithm is given
in Section 5.4.3, and a formal analysis of the reconstruction error of /0 given the error in ^K is found in
Section 5.2.1. 2.1. Estimation of K As it turns out, the entries of matrix K correspond to simple population-level statistics of the SNPs, which
could either be inadvertently released (under the assumption they would be safe to share), or could be
estimated from another sample from the same population. In fact, the entries of K depend only on the SNP
frequencies and SNP co-occurrence frequencies in the dataset: – For i = 1‚ . . . ‚ N: Kii estimates the probability that SNP i has value 1 (i.e., the frequency of the SNP in
the population). – For i = 1‚ . . . ‚ N - 1 and j > i: Kij = Kji estimates the probability that both SNP i and SNP j are 1
simultaneously (i.e., the frequency of SNP i and SNP j co-occurring in the same individual). – For i = 1‚ . . . ‚ N and j = N + 1: Kij = Kji also estimates the probability that SNP i has value 1, that is,
Ki N + 1 = KN + 1 i = Kii. – Finally, KN + 1‚ N + 1 = 1. 3. RESULTS The key observation from the previous section is that the vectors d1 and dm, derived from the change
in parameter vectors ^b from a first study to a second study, take only a finite number of values thanks to
the fact that the design matrices F contain only zeros and ones. In particular, when m new individuals are
added to the second study, each entry of the vector dm can only take at most 2m values, and a zero value
corresponds to the setting where all individuals have the most common variant for that SNP. This section describes algorithmically how these vectors can be used to recover the genomes of the
additional individuals, as well as empirical tests that use the Cornell Dog Genome dataset as a case study
(Hayward et al., 2016). More details on the experimental setup can be found in Section 5.1. PAIGE ET AL. 440 3.2. Complete reconstruction of multiple individuals’ genotype when SNP frequency
information is known We now consider the case where m additional individuals have been included in the second study,
yielding a new GRS model ^bM + m, including these M + m participants. Consider again Eq. (8) described earlier. The ith row of Fm is a binary vector that represents the com-
bination of the m individuals who have SNP i. This means that, for a fixed value of Cm, the value of the
vector dm at index i is uniquely determined by the combination of individuals who have SNP i (Fig. 2B). In
other words, there will be at most 2m unique values taken by entries of dm, each corresponding to a
combination of the values in vector Cm (Fig. 2C). If we were to learn which values of dm are also found in Cm, then we could infer the complete genotypes
of all the m individuals added to the second study. We would be able to reconstruct m complete genotype
vectors, although it would be impossible to know which of the genotypes corresponded to which of the m
individuals. In fact, in many cases it is extremely straightforward to determine which values in dm cor-
respond to values in Cm. Here, we describe a simple algorithm for finding Cm when there are exactly 2m
unique values in dm. If this is not the case, please refer to the more complete algorithm in Section 5.3. 1. First, extract all unique, nonzero values from dm. Find the sum of all pairs of values in this vector p
3. Find all values that are in Eq. (1), but not in Eq. (2). The values of Cm appear in this list. There is no
way to know which value of Cm corresponds to which index, so for simplicity we can randomly assign
them indices. 3. Find all values that are in Eq. (1), but not in Eq. (2). The values of Cm appear in this list. There is no
way to know which value of Cm corresponds to which index, so for simplicity we can randomly assign
them indices. 4. Each value in Cm corresponds to a specific individual who was added to the second study. Each value
in dm can be described as a sum of a unique combination of values in Cm. PAIGE ET AL. For instance, if dm[i] =
Cm[j] + Cm[k], this means that the SNP at position i is found in individual j and k, but no one else. 4. Each value in Cm corresponds to a specific individual who was added to the second study. Each value
in dm can be described as a sum of a unique combination of values in Cm. For instance, if dm[i] =
Cm[j] + Cm[k], this means that the SNP at position i is found in individual j and k, but no one else. We tested this approach by using the Cornell Dog Database, in a test scenario where the second study
added three different dogs. We were able to uniquely identify the genotypes of all three dogs with 100%
accuracy, with both common and uncommon SNPs (Fig. 2C). 3.1. Complete reconstruction of one individual’s genotype when SNP frequency information
is known (B) We can reconstruct all the genotypes of multiple
dogs that are added to the second study and (C) this works in practice by using the data from the Cornell Dog Database,
as in (A). PAIGE ET AL. 4. DISCUSSION In this study, we demonstrate that private information is leaked when GRS models are published,
specifically in the case where two sets of largely overlapping individuals are used for multiple studies. In
particular, we show that we can recover SNPs from an individual in a private database—a reconstruction
attack. Even though we would not have a name associated with this genotype, it may be possible to identify
the individual once the genotypic data are available to the attacker. For instance, the attacker may have FIG. 4. Accuracy at reconstruction of genomes x0 using EM estimation and a noisy estimate ^K, as compared with a
natural baseline that always predicts the most common variant at each SNP locus. We use this as a baseline, because
without any additional information about bM and bM + 1, the most accurate prediction of the dog’s genotype would be to
predict the most common variant at each locus. Here, we define accuracy as the proportion of SNPs that are correctly
identified in the dog that was found in the second GWAS study, but not the first. Each distribution is constructed from
500 experimental test points, in which we (1) took 10 random splits of the full dog dataset, assigning dogs to either the
public or private dataset; (2) for each split, we tested the reconstruction 50 times, each time adding a different randomly
sampled dog to the second GWAS study. The private dataset always has 1000 individuals; the public test dataset is of
increasing size, improving performance. EM, expectation–maximization. FIG. 4. Accuracy at reconstruction of genomes x0 using EM estimation and a noisy estimate ^K, as compared with a
natural baseline that always predicts the most common variant at each SNP locus. We use this as a baseline, because
without any additional information about bM and bM + 1, the most accurate prediction of the dog’s genotype would be to
predict the most common variant at each locus. Here, we define accuracy as the proportion of SNPs that are correctly
identified in the dog that was found in the second GWAS study, but not the first. 3.3. Accurate estimation of an individual’s genotype when SNP frequency information
is estimated from a public database Previously, we assumed that the attacker had access to the matrix K, which consists of population-level
statistics on frequencies and co-occurrence frequencies of SNPs. Although this could be released volun-
tarily by organizations that are not aware of the risk, we now consider the case where K is not directly
available to the attacker but is instead estimated from a separate public database assumed to correspond to
individuals from the same population. We simulated this scenario by using the Cornell Dog Database by taking one random set of dogs for
building the GRS model, and a second non-overlapping set of dogs for estimating ^K. We compared the
value of ^d1 = ^K(^bM + 1 - ^bM) with the known value of /0. We observe that ^d1 has significantly different
values at indices where /0[i] = 0 and /0[i] = 1; examples for the cases where one and three dogs are added
can be seen in Figure 3. ^ Themainchallengeisthatthevector ^d1 nowincludesadditionalnoise,sowecannotsimplyuseitsentryatindex
N + 1 to estimate C, nor do the entries i with /0[i] = 0 also correspond directly to ^d1[i] = 0. Instead, we develop a
custom EM algorithm to find a maximum likelihood estimate of the constant C and recover /0, that is, to
determine the probability that each /0[i] = 0 or /0[i] = 1, based on the value of ^d1 (see Section 5.4.3 for details). We find that this method can successfully reconstruct the correct value of /0[i] much better than a baseline that
uses the public dataset to independently estimate the most common variant for each of the SNP (Fig. 4). Crucially, we show that our approach is able to reconstruct, with relatively high accuracy, the genotypes
of dogs even when they differ significantly from those in the public dataset (Fig. 5). This shows that our
attack is able to extract information about the particular individuals that differ across the two studies, not
merely about the general population as in the most-common-variant baseline. By definition, dogs that have
genotypes that differ significantly from the general population have a higher proportion of uncommon
SNPs, and the ability to recover these uncommon SNPs is particularly important from a privacy per-
spective. Indeed, uncommon SNPs can be used to identify a particular individual and are also more likely to
be associated with disease phenotypes, which is sensitive information. 3.4. Accurate estimation of an individuals’ genotype when different SNPs are used in each study When GRS models are constructed, the first step is to filter the set of SNPs down to a small set of SNPs
that are (1) significantly correlated to the trait after covariates are considered and (2) far apart from one
another along the genome. If the two studies use two different sets of SNPs to construct the GRS model, it
is still possible to recover whether or not each of the SNPs in the overlap is present in the new individual. This process is highly analogous to the previous cases and is detailed in Section 5.6. 3.3. Accurate estimation of an individual’s genotype when SNP frequency information
is estimated from a public database In general, we find that the larger the RECONSTRUCTION ATTACKS FROM GRS 441 FIG. 3. Example values taken by the noisy vector ^d, given the true value of the corresponding SNP in the genome. (Left) adding one new participant; (right) adding three new participants. These figures are analogous to those in
Figure 2, although in the case where K is not known and instead estimated from an independent public database. FIG. 3. Example values taken by the noisy vector ^d, given the true value of the corresponding SNP in the genome. (Left) adding one new participant; (right) adding three new participants. These figures are analogous to those in
Figure 2, although in the case where K is not known and instead estimated from an independent public database. public dataset available, and the more similar the dataset is to the unknown private dataset, the better we are
able to reconstruct the genome of the added individual. Full details and description of the experimental
setting are given in Section 5.1. We also derive theoretical error bounds for our estimate of /0 based on the
error in ^K in Section 5.4.1. public dataset available, and the more similar the dataset is to the unknown private dataset, the better we are
able to reconstruct the genome of the added individual. Full details and description of the experimental
setting are given in Section 5.1. We also derive theoretical error bounds for our estimate of /0 based on the
error in ^K in Section 5.4.1. This task becomes more challenging when multiple individuals are added simultaneously and K is
unknown; an algorithm for estimating Fm for m > 1, along with additional empirical results, is given in
Section 5.4. 4. DISCUSSION Each distribution is constructed from
500 experimental test points, in which we (1) took 10 random splits of the full dog dataset, assigning dogs to either the
public or private dataset; (2) for each split, we tested the reconstruction 50 times, each time adding a different randomly
sampled dog to the second GWAS study. The private dataset always has 1000 individuals; the public test dataset is of
increasing size, improving performance. EM, expectation–maximization. 442 PAIGE ET AL. FIG. 5. Results of Figure 4 broken down by individual dogs. Here, each point represents a dog and we define
atypicality as the proportion of uncommon variants that the dog has compared with the public database—for instance, if
51% or more of dogs in the public database have a G in a specific locus, but this dog has a T, then this would count
toward the dog’s atypicality. In other words, dogs further to the right are less and less similar to average dogs present in
the public dataset (measured by percentage of different variants). In contrast to the most-common-variant baseline, our
method generalizes well even to dogs that are highly dissimilar to those in the public dataset. Larger public databases
(right) provide more accurate population estimates of ^K, leading to more accurate reconstructions overall. FIG. 5. Results of Figure 4 broken down by individual dogs. Here, each point represents a dog and we define
atypicality as the proportion of uncommon variants that the dog has compared with the public database—for instance, if
51% or more of dogs in the public database have a G in a specific locus, but this dog has a T, then this would count
toward the dog’s atypicality. In other words, dogs further to the right are less and less similar to average dogs present in
the public dataset (measured by percentage of different variants). In contrast to the most-common-variant baseline, our
method generalizes well even to dogs that are highly dissimilar to those in the public dataset. Larger public databases
(right) provide more accurate population estimates of ^K, leading to more accurate reconstructions overall. access to partial genotypic information of the individual and then be able to identify them. Alternatively,
they could use the genotype information to predict ethnicity and other phenotypic traits that could then be
used to uniquely identify the individual. 4.1. Suggestions for good practice We provide a number of simple suggestions for good practice that would help limit this attack. We provide a number of simple suggestions for good practice that would help limit this attack. 1. Aggregate statistics about the frequency of SNPs in the database or the frequency of co-occurrence of
SNPs should never be released. We have shown that this information, combined with GRS, allows to
precisely reconstruct individual genomes in various settings. It may be possible to release noisy
versions of SNP frequency data, but this would be equivalent to releasing ^K (our estimated K from the
public database). With our EM algorithm, we have demonstrated that it is still possible to do some
genotypic reconstruction with a noisy ^K, but this becomes harder as the noise in ^K increases. On the
other hand, providing a very noisy ^K may be of limited utility to the scientific community. 2. If a genetic dataset is intended to serve for multiple complementary analyses, it is important that all
study participants are used in every analysis performed. If there are missing phenotypic data from a
few individuals, they should not be included in any of the analyses performed, or their privacy may be
compromised. 3. When multiple individuals are added in between two studies, then the ability to reconstruct the genomes
dependsonthenumberofSNPs,beinglargerelativetothenumberofindividuals.Inparticular,ifmnewdogs
are added, exact reconstruction is only possible by using the approach in Section 3.2 if the number of SNPs
N> 2m. Thus, we suggest to avoid releasing multiple studies that differ by fewer than log2 N individuals. 4. DISCUSSION We also note that even an incomplete reconstruction attack (in which only a proportion of the SNPs are
correctly identified) is likely to be sufficient to perform a membership inference attack. Investigating the
relationship between the reconstruction attack and the membership inference attack will be a subject of
future research. Importantly, if the attackers were unable to link the genomic data with a particular
individual, the reconstruction attack would still be a breach in privacy that could have serious conse-
quences. For instance, the patient may have only consented to have their genomic data used in particular
kinds of research studies, whereas the attacker may use the reconstructed genomic data for a different
(potentially unethical) purpose. 4.2. Extensions and future work Although we have analyzed the case where the genome is represented by binary values of 0 or 1, often
studies instead count the number of times each allele is present, which would lead to a design matrix F
containing values 0‚ 1‚ or 2. In this scenario, K no longer contains the frequencies of SNPs and their co-
occurrences, but it is something slightly more complicated that we describe in Section 5.8. This does not
dramatically change the approach in this study, except in that the vector dm can take 3m possible values, 443 RECONSTRUCTION ATTACKS FROM GRS rather than 2m. In practice, then, studies that use allele counts are somewhat more robust to attacks; the
multiple dog reconstruction attack would likely be ambiguous if 3m > N, rather than 2m > N. p
g
y
g
A possible countermeasure to our reconstruction attack could consist of randomly perturbing the GRS
models before releasing them, as done in differentially private linear regression (Wang, 2018). However, a
naive application of this strategy could destroy the utility of the models. A formal and empirical analysis of
the effectiveness of such protection against reconstruction attacks, as well as of the usefulness of the
resulting GRS models to genomic researchers, is beyond the scope of this article and left for future work. Another countermeasure is to refrain from releasing precise information about the population structure
of the study population to prevent the attacker from estimating K effectively. This would, however, limit the
utility of the research study, because the researchers would not know to what populations the research applies. Our work has a number of limitations. For instance, we only test our EM algorithm on dog data. Dog
populations may have different population structures than human populations due to selective breeding,
so in the future we aim at investigating how properties of population structure will impact our ability to
estimate K and the accuracy of our reconstruction attack. It may seem unlikely on the surface that two GWAS analyses will include nearly the same participants. One potentially common setting where this could arise is when a single study collects both genotype and
phenotype data from a single set of participants, and it releases multiple models to predict multiple traits. 5.1. Experimental details 5.1.1. Cornell dog database. To experimentally test the reconstruction attacks, we used data from
the Cornell Dog Genome Database, which contains data about SNPs from a wide range of dog breeds and a
number of associated phenotypic traits. The two traits we focused on were average breed weight and
average breed height, because these two phenotypes had the fewest number of missing values. For the
initial investigation, we binarized the genotype matrix—considering all heterogenous alleles to have a
value of 1. (We also repeated the analysis with the original genotype matrix.) Only common SNPs (i.e.,
SNPs that were found in 25%–75% of the dogs) were used, leaving 23,497 SNPs. For each linear model
built, M = 1000 dogs were randomly sampled as the ‘‘private’’ dataset and N = 200 SNPs were randomly
selected. To ensure that the SNPs that were sampled were spatially distributed, the SNPs were randomly
sampled in a stratified way, so one SNP was selected in every 23‚ 497
200 -sized bin. 5.1.2. Experiment with imprecise K. First, two linear models were constructed to predict average
breed weights: one with the M = 1000 randomly sampled dogs and another that contained one additional
randomly sampled dog. This gives ^bM and ^bM + 1. To mimic the process of estimating K from a public
database, we randomly sampled an additional 200, 400, or 800 dogs that were not included as part of the
original set and used this to estimate K, which we denote by ^K. Now, we could calculate ^K(^bM + 1 - ^bM) and
compare this with the known /0 for the additional dog from the second study. These additional dogs are taken
from a third ‘‘test’’ dataset, disjoint from both the public and private data. The plots in Figures 4 and 5 are
produced by re-running the algorithms across 10 random public/private/test splits, where the ‘‘test’’ dataset has
50 dogs that are each individually considered as candidates for the (M + 1)th dog added to the private dataset. 4.2. Extensions and future work In
this case, there may be a small number of individuals who are used in one analysis, but not the other; for
instance, there may be a small subset of participants who skip a particular survey question that was used to
collect phenotype information, and this is, indeed, evident in a recent study (Jiang et al., 2019). In such
settings, it could be very possible for multiple released GRS models to be computed on sets of individuals
that differ by only a few participants. In future work, we aim at extending our analysis and attack to settings
where multiple GRS models are released, each predicting different but highly correlated traits. 5.3. Algorithm for identifying unique genotypes of multiple dogs when K is known Although the simple approach described in the main article will work in many cases, there are a few
special circumstances where a more complex algorithm may be required. In particular, it would not work if
there are combinations of SNPs that are not observed among the individuals added to the database. For
instance, if there is not an SNP location where the first individual has an SNP variant and the others do not,
then we would miss the corresponding value in Cm. However, it is still possible to identify all the values in
Cm through a more complex algorithm: 1. First, extract all unique, nonzero values from dm. 2. Find the sum of all pairs of values in Eq. (1). 3. Find all values that are in Eq. (1), but not in Eq. (2). 4. If there are exactly m values in Eq. (3) and the sum of these values equal the last value of dm
(corresponding to the intercept term), then we have found the correct values of Cm. p
g
p
5. Otherwise, this suggests that there are one or more elements of Cm that are missing from Eq. (3) and
possibly a few values in Eq. (3) that are not in Cm. 5. Otherwise, this suggests that there are one or more elements of Cm that are missing from Eq. (3) and
possibly a few values in Eq. (3) that are not in Cm. 6. Begin by subtracting every pair of values in Eq. (3). These are now also potential values of C
7 S
h f
f
l
f
E
(3)
d (6) h
h l
l
f d
Th 7. Search for a set of m values from Eqs. (3) and (6) that sum to the last element of dm. There may be
more than one set of values for which this is true. 8. If this search is unsuccessful, repeat steps 6–7. Eventually, a set of m values summing to dm should be
found. 8. If this search is unsuccessful, repeat steps 6–7. Eventually, a set of m values summing to dm should be
found. 9. If more than one possible set of values is found for Cm in Eq. (7), it is still possible to compare these
sets and identify which is the most likely to contain the true values of Cm. 5.3. Algorithm for identifying unique genotypes of multiple dogs when K is known For each possible Cm
vector, a set of genotypes can be constructed for the m additional individuals. Using the frequencies of
each SNP, it is possible to calculate the probability of observing each genotype. The set of values that
produces the most likely genotypes for the m individuals is most likely to be the correct one. In addition, this algorithm depends on the fact that it is extremely unlikely that if someone were to sample
three random continuous numbers i, j, and k, it would just so happen that i + j = k. There is an extremely
small chance that a value of Cm would be un-discoverable because of a coincidence of this nature. 5.2. Adding multiple dogs Here, we explain Eqs. (7) and (8). Note that the former is a special case of the latter so we will only
explain the latter in detail. First note that, by definition, ^bM = (F>
MFM) - 1F>
MyM = (MKM) - 1FT
MyM‚
^bM + m = (F>
M + mFM + m) - 1F>
M + myM + m = (MKM + mKm) - 1F>
M + myM + m: ^bM = (F>
MFM) - 1F>
MyM = (MKM) - 1FT
MyM‚ 444 PAIGE ET AL. Substituting these into the left hand side of the following equation gives the right hand side: Substituting these into the left hand side of the following equation gives the right hand side:
(MKM + mKm)^bM + m - MKM ^bM = FT
mym:
(9) (9) (MKM + mKm)^bM + m - MKM ^bM = FT
mym:
(9) (MKM + mKm)^bM + m - MKM ^bM = FT
mym: This equation can be rearranged to give KM(^bM + m - ^bM) = 1
M FT
mym - m
M Km ^bM + m
= 1
M FT
m(ym - Fm ^bM + m): Defining the length m vector Cm = 1
M (ym - Fm ^bM + m) yields the form used in Eq. (8). For the special case
of m = 1, Cm is a scalar and we recover Eq. (7). 5.3. Algorithm for identifying unique genotypes of multiple dogs when K is known RECONSTRUCTION ATTACKS FROM GRS 5.4.1. Analytic bound on k /0 - ^/0 k. For convenience, we only consider the case of adding a
single individual, though the generalization is quite straightforward. If ^K is substituted for K in our
reconstruction Eq. (7), we get an approximation of /0 that we denote ^/0. We would like to bound the
(relative) error between /0 and ^/0. Later, we ignore the constant factors C and ^C for simplicity, noting
that these scaling factors are estimated from the resulting /0 or ^/0 anyway. We, thus, consider
u0 = K(^bM + 1 - ^bM) and ^u0 = ^K(^bM + 1 - ^bM). Using k k on vectors, and also on matrices, we denote the
corresponding operator norm. The relative error between u0 and ^u0 is given by: k u0 - ^u0 k
k u0 k
= k ( ^K ^K
- 1 - ^KK - 1)u0 k
k u0 k
k ^K ^K - 1 - ^KK - 1 k = k ^K( ^K - 1 - K - 1) k :
Note that ^K - 1 - K - 1 = ^K - 1(K - ^K)K - 1 and hence
k ^u0 - u0 k
k u0 k
k K - 1 kk K - ^K k ‚
(10) (10) This means that we can bound the error by two quantities. The term k K - 1 k is bounded earlier by
1= min (eig(K)), which is finite as soon as K is nonsingular. This is not a strong requirement, as in the case
of linear regression it is required for ^bM + 1 and ^bM to exist. Note that in the case of L2-regularized linear
regression (i.e., ridge regression), K is replaced by K + kI, where k is the regularization parameter, and we
can directly bound this term by k. The key term in Eq. (10) is k K - ^K k, the error in estimating K by ^K. Let us assume that the public
database used to obtain ^K follows the same distribution as the private database used to fit the GRS models. Denote by ^M the number of individuals used to estimate ^K. Then, under classic boundedness assumptions
and leveraging matrix concentration inequalities such as matrix Bernstein Tropp (2015), we can show that
E[ k K - ^K k ] = O(1=
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
min ( ^M‚ M)
p
). RECONSTRUCTION ATTACKS FROM GRS This shows that the error in estimating K is small as long as the
private and public databases are large enough. 5.4.2. Modeling the error in ^K. In this section, we define a model to capture the error in ^K, which
leads to the EM algorithm for estimating /0, which is used in the experiments. As our estimated ^K drifts
from the true K, this expression ^K(^bM + 1 - ^bM) would produce a wider range of values than just 0 and C. Let eij*N (0‚ r2) be independent noise, which we assume corrupts each element of Kij; that is, given the
estimated matrix ^K suppose 5.4.2. Modeling the error in ^K. In this section, we define a model to capture the error in ^K, which
leads to the EM algorithm for estimating /0, which is used in the experiments. As our estimated ^K drifts
from the true K, this expression ^K(^bM + 1 - ^bM) would produce a wider range of values than just 0 and C. leads to the EM algorithm for estimating /0, which is used in the experiments. As our estimated K drifts
from the true K, this expression ^K(^bM + 1 - ^bM) would produce a wider range of values than just 0 and C. Let eij*N (0‚ r2) be independent noise, which we assume corrupts each element of Kij; that is, given the
estimated matrix ^K, suppose Let eij*N (0‚ r2) be independent noise, which we assume corrupts each element of Kij; that is, given the
estimated matrix ^K, suppose Kij*N ( ^Kij‚ r2)‚
(11) (11) for some small r2. This is clearly an oversimplification (as we know K is e.g., bounded and symmetric), but
it is a useful starting point that allows derivation of a simple estimation algorithm. For notational brevity, in
this and the following section we define the vector (12) D = ^bM + m - ^bM
(12) which corresponds to the difference between the two GRS model parameter vectors when m additional dogs
are added. Given the true value of K, the system of equations F>
mcm = KD relates the known quantity D and the Gaussian-distributed K with the matrix Fm and the unknown values in
the vector cm 2 Rm. 5.4. Estimating K If the true matrix K is unknown, it can be estimated with public data. We denote this estimator by ^K. For
^K to be an accurate estimate, the data that it is generated from must be drawn from the same (or
a sufficiently similar) population as that used in the private study. We will model this assuming no dis-
crepancy between population distributions; however, when we discuss how to evaluate whether the esti-
mate is good, that assessment should account for this systematic error as well. Later, we will be primarily
concerned with the error due to the subsampling in both the private and public datasets. Also of note, the same analysis given next applies to the scenario in which the researchers do not release
K, but rather release a ‘‘noisy’’ version of K, where the noise is drawn from a normal distribution. They
might consider doing this if they feel that releasing information about SNP frequencies is important for the
research community, but they do not wish to release the real K because this would allow for an exact
reconstruction of genotype. This noisy K could still be used in a reconstruction attack in the same way as an
estimate of K from a public database is used. RECONSTRUCTION ATTACKS FROM GRS 445 PAIGE ET AL. With some algebraic re-arrangement, and since for the true underlying value of K we have KD = F>
mcm,
we can write this as With some algebraic re-arrangement, and since for the true underlying value of K we have KD = F>
mcm,
we can write this as ^KD*N (
X
m
j = 1
Cj/j‚ r2D>DI)
(13) (13) where C1‚ . . . ‚ Cm and r are parameters we need to estimate. The vector ^KD is observed ‘‘data,’’ computed
from the public SNP database and the two released parameter vectors. We can model each of the entries of Fm,
which are zeros and ones, as Bernoulli distributions, whose prior probabilities correspond to the public dataset
estimated frequencies. This suggests a model for ^KD that is akin to a constrained mixture of Gaussians. where C1‚ . . . ‚ Cm and r are parameters we need to estimate. The vector ^KD is observed ‘‘data,’’ computed
from the public SNP database and the two released parameter vectors. We can model each of the entries of Fm,
which are zeros and ones, as Bernoulli distributions, whose prior probabilities correspond to the public dataset
estimated frequencies. This suggests a model for ^KD that is akin to a constrained mixture of Gaussians. For the special case of m = 1, with only a single scalar C and vector /0, this reduces to ^KD*N (C/0‚ r2D>DI):
(14) (14) 5.4.3. Parameter estimation with EM. We now can use this model to derive EM algorithms for
finding maximum likelihood estimates of all parameters, and estimate the posterior distribution over SNP
variants for the added individuals between the two studies. For notational convenience in this section, denote the entries of the m new individuals Fm 2 f0‚ 1gN + 1‚ m
as zi‚ j, for i = 1‚ . . . ‚ N + 1 and j = 1‚ . . . ‚ m, and let zj denote the column vector z1‚ j‚ . . . ‚ zN + 1‚ j. Denote the
prior probabilities for each i as a1‚ . . . ‚ aN + 1, where a1‚ . . . ‚ aN are the (public) population frequencies for
each SNP, and aN + 1 = 1. Let x1‚ . . . PAIGE ET AL. ‚ xN + 1 denote the entries of the fixed (observed) vector x = ^KD, which in
this simplified notation is distributed as p(xjcm‚ Fm‚ r2) = N (xj
X
m
j = 1
Cjzj‚ r2D>DI): p(xjcm‚ Fm‚ r2) = N (xj
X
m
j = 1
Cjzj‚ r2D>DI): posing we know values of C1‚ . . . ‚ Cm‚ r2, to estimate the entries of Fm we want to find p(zjx‚ C‚ r2 Supposing we know values of C1‚ . . . ‚ Cm‚ r2, to estimate the entries of Fm we want to find p(zjx‚ C‚ r2), p(zjx‚ cm‚ r2) / p(xjcm‚ Fm‚ r2)p(z): p(zjx‚ cm‚ r2) / p(xjcm‚ Fm‚ r2)p(z): An EM algorithm to estimate cm‚ r2 would proceed by alternately: An EM algorithm to estimate cm‚ r2 would proceed by alternately: 1. Given cm‚ r2, estimate the posterior distribution p = p(zjx‚ cm‚ r2); 1. Given cm‚ r2, estimate the posterior distribution p = p(zjx‚ cm‚ r2);
2. Given the posterior p, maximize L = Ep[ log p(xjcm‚ Fm‚ r2)] with respect to cm and r2. 1. Given cm‚ r2, estimate the posterior distribution p = p(zjx‚ cm‚ r2);
2. Given the posterior p, maximize L = Ep[ log p(xjcm‚ Fm‚ r2)] with respect to cm and r2. For each zi‚ j, we can analytically compute the distribution For each zi‚ j, we can analytically compute the distribution p(zi‚ j = 1jx‚ cm‚ zk6¼j‚ r2) p(zi‚ j = 1jx‚ cm‚ zk6¼j‚ r2) (15) =
aiN (xijCj + P
k6¼j Ckzi‚ k‚ r2D>D)
aiN (xijCj + P
k6¼j Ckzi‚ k‚ r2D>D) + (1 - ai)N (xij P
k6¼j Ckzi‚ k‚ r2D>D) ‚
(15) the conditional probability of each particular entry taking a value of 1, rather than 0, for each zj given the
values of the other zk, k 6¼ j. Note that each SNP location i can be treated independently; however, each of
the individuals j = 1‚ . . . ‚ m individuals must be considered jointly. the conditional probability of each particular entry taking a value of 1, rather than 0, for each zj given the
values of the other zk, k 6¼ j. Note that each SNP location i can be treated independently; however, each of
the individuals j = 1‚ . . . ‚ m individuals must be considered jointly. 5.4.4. Exact EM algorithm when 1 individual is added. RECONSTRUCTION ATTACKS FROM GRS This breaks down into a sum across the entries in cm, with KD =
X
m
j = 1
Cj/j: KD =
X
m
j = 1
Cj/j: We need to estimate all m constants Cj‚ j = 1‚ . . . ‚ m. j
If K is Gaussian [following Eq. (11)], then the linear transformation KD is Gaussian as well. We denote
each of the rows of K as a vector ki, i = 1‚ . . . ‚ N; then, for each row, the scalar value k>
i D*N (^k>
i D‚ r2D>D)‚ meaning overall the vector KD is distributed N ( ^KD‚ r2D>DI). 446 PAIGE ET AL. RECONSTRUCTION ATTACKS FROM GRS This yields @L
@C =
X
i
pi
r2DTD (xi - C)‚
@L
@r2 =
X
N
i = 1
pi
@
@r2 log N (xijC‚ r2D>D) + (1 - pi) @
@r2 log N (xij0‚ r2D>D)‚ which we set equal to zero and solve to find which we set equal to zero and solve to find ^C =
P
i pixi
P
i pi
‚
(17) ^C =
P
i pixi
P
i pi
‚ (17) ^r2 =
1
ND>D
X
N
i = 1
pi(xi - C)2 + (1 - pi)x2
i :
(18) (18) These updates taken together can be used to define an EM algorithm that optimizes the values of C and
r2, despite the fact that the entries of /0 are unknown; once C and r2 are then known, the vector p will give
probability estimates for each entry of /0. p
y
y
/0
The overall EM algorithm can be summarized by the following iterative updates: The overall EM algorithm can be summarized by the following iterative updates: 1. pi p(zi = 1jx‚ ^C‚ ^r2) =
aiN (xij ^C‚ ^r2D>D)
aiN (xij ^C‚ ^r2D>D) + (1 - ai)N (xij0‚ ^r2D>D),
2. ^C)
P
i pixi
P
i pi ,
3. ^r2)
1
ND>D
PN
i = 1 pi(xi - ^C)2 + (1 - pi)x2
i : 1. pi p(zi = 1jx‚ ^C‚ ^r2) =
aiN (xij ^C‚ ^r2D>D)
aiN (xij ^C‚ ^r2D>D) + (1 - ai)N (xij0‚ ^r2D>D),
P 1. pi p(zi = 1jx‚ ^C‚ ^r2) =
aiN (xij ^C‚ ^r2D>D)
aiN (xij ^C‚ ^r2D>D) + (1 - ai)N (xij0‚ ^r2D>D),
2. ^C)
P
i pixi
P
i pi ,
3. ^r2)
1
ND>D
PN
i = 1 pi(xi - ^C)2 + (1 - pi)x2
i : 1. pi p(zi = 1jx‚ ^C‚ ^r2) =
aiN (xij ^C‚ ^r2D>D)
aiN (xij ^C‚ ^r2D>D) + (1 - ai)N (xij0‚ ^r2D>D), P
i
3. ^r2)
1
ND>D
PN
i = 1 pi(xi - ^C)2 + (1 - pi)x2
i : To initialize the algorithm, we can set pi to some initial probabilities, and find initial values for ^C‚ ^r2; we
experimented with setting both to the prior probabilities per SNP estimated from the public data and to the
vector of all zeros (corresponding to a ‘‘hard’’ initialization at the value of the baseline estimate), and we
found no qualitative difference in performance. PAIGE ET AL. For the special case of m = 1, this yields a
tractable exact EM algorithm. Since there are no other individuals, Eq. (15) reduces to p(zjx‚ C‚ r2), with 5.4.4. Exact EM algorithm when 1 individual is added. For the special case of m = 1, this yields a
tractable exact EM algorithm. Since there are no other individuals, Eq. (15) reduces to p(zjx‚ C‚ r2), with pi = p(zi = 1jx‚ C‚ r2) =
aiN (xijC‚ r2D>D)
aiN (xijC‚ r2D>D) + (1 - ai)N (xij0‚ r2D>D)
(16) (16) the posterior probability of each particular entry taking a value of 1, rather than 0. To maximize
L = Ep[ log p(xjC‚ /0‚ r2)] with respect to C and r2, we first compute the derivatives of L =
X
i
X
zi
p(zij . . . ) log p(xijC‚ zi‚ r2) =
X
N
i = 1
pi log N (xijC‚ r2D>D) + (1 - pi) log N (xij0‚ r2D>D): RECONSTRUCTION ATTACKS FROM GRS 447 RECONSTRUCTION ATTACKS FROM GRS 5.4.5. Stochastic EM (SEM) for multiple individuals. For m > 1, the exact posterior depends on
all individuals and does not have a compact form. However, we can easily approximate the posterior by
Gibbs sampling using Eq. (15), which describes the full conditional distribution p(zi‚ j = 1jx‚ cm‚ zk6¼j‚ r2),
iteratively drawing samples for each individual j. We can use this for parameter estimation of r2 and each
C1‚ . . . ‚ Cm by using the stochastic EM algorithm Celeux and Diebolt (1985), which differs from a standard
EM algorithm in that the expectation step (evaluating the posterior) is replaced by Monte Carlo sampling. In this algorithm, we alternately 1. draw approximate posterior samples of zi‚ j by one or more sweeps of Gibbs sampling, following
Eq. (15); 1. draw approximate posterior samples of zi‚ j by one or more sweeps of Gibbs sampling, following
Eq. (15);
2 2. conditioned on the current sampled values zi‚ j, find values of r2 and C1‚ . . . ‚ Cm which maximize the
likelihood N (xij Pm
j = 1 Cjzi‚ j‚ r2D>DI). 2. conditioned on the current sampled values zi‚ j, find values of r2 and C1‚ . . . ‚ Cm which maximize the
likelihood N (xij Pm
j = 1 Cjzi‚ j‚ r2D>DI). Although this does not converge to an exact parameter value, under suitable conditions the algorithm
converges in distribution to a Gaussian centered on the maximum likelihood estimate of the parameter. A
point estimate can be extracted by averaging across many iterations after convergence. In contrast to the EM updates, the updates for values of Cj and r2 given actual sampled values of zj are
straightforward and do not scale combinatorially in m. Optimizing cm corresponds to solving a least-squares
problem, that is, min
C1‚ ...‚ Cm
X
N + 1
i = 1
(xi -
X
j = 1
Cjzi‚ j)2 = min
cm k x - Zcm k2
2 ‚ using the vector notation cm = [C1‚ . . . ‚ Cm]> 2 Rm, x = [x1‚ . . . ‚ xN + 1]> 2 RN + 1, and Z 2 [0‚ 1]N + 1‚ m, has
the solution using the vector notation cm = [C1‚ . . . ‚ Cm]> 2 Rm, x = [x1‚ . . . 5.5. Scaling of EM algorithm with size of private dataset Figure 7 demonstrates the change in accuracy of the EM algorithm over a range of different private
database sizes. For this test, a synthetic dataset with 100 SNPs and 1,000,000 individuals is generated;
10,000 are held out as a public database, and 30 individuals are taken as a fixed test dataset of new dogs to
add and are used to estimate EM algorithm accuracy, across increasingly large private database sizes. The
algorithm has stable performance for increasingly large private databases. PAIGE ET AL. PAIGE ET AL. The maximum likelihood estimate of r2 given this estimated ^cm is simply the mean squared error The maximum likelihood estimate of r2 given this estimated ^cm is simply the mean squared error ^r2 =
1
N + 1
X
N
i = 1
(xi - ^c>
mzi)2:
(20) (20) To address permutation invariance in the entries 1‚ . . . ‚ m, we enforce an ordering on the estimated
values of Cj, with C1 C2 . . . Cm. This breaks the symmetry across the indices of the m new indi-
viduals added in the second study, and it is handled by a projection operation at each iteration, in which the
estimated values are sorted in ascending order after each maximization step. g
p
Empirical results quantifying the performance of this algorithm are shown in Figure 6, in an experi-
mental setup similar to that for evaluating EM when a single dog is added to a dataset in the main article,
with unknown K. A private dataset is assumed to contain 1000 individuals, whereas a separate public
dataset of 800 is available; m = 3 new individuals are added to the private dataset to produce two parameter
vectors bM and bM + m. On average, the SEM algorithm predicts the correct SNP 75.5% of the time, relative
to 71.5% for the ‘‘most common variant’’ baseline, a moderate improvement. RECONSTRUCTION ATTACKS FROM GRS ‚ xN + 1]> 2 RN + 1, and Z 2 [0‚ 1]N + 1‚ m, has
the solution using the vector notation cm = [C1‚ . . . ‚ Cm]> 2 Rm, x = [x1‚ . . . ‚ xN + 1]> 2 RN + 1, and Z 2 [0‚ 1]N + 1‚ m, has
the solution ^cm = (Z>Z) - 1Z>x:
(19) ^cm = (Z>Z) - 1Z>x: (19) 448 RECONSTRUCTION ATTACKS FROM GRS 449 FIG. 7. Accuracy at reconstruction of the genome of one additional individual, using EM estimation and a noisy
estimate ^K, measured as the size of the initial private database increases. For very small private databases, accuracy is
very high, as changes in entries of b are clearly attributable to the new individual. Beyond a certain threshold, overall
accuracy is quite stable. Error bars show mean and two standard deviations. FIG. 7. Accuracy at reconstruction of the genome of one additional individual, using EM estimation and a noisy
estimate ^K, measured as the size of the initial private database increases. For very small private databases, accuracy is
very high, as changes in entries of b are clearly attributable to the new individual. Beyond a certain threshold, overall
accuracy is quite stable. Error bars show mean and two standard deviations. and now, from the second experiment, we have and now, from the second experiment, we have and now, from the second experiment, we have and now, from the second experiment, we have (K0 + /0T
0/0
0)^b0 = F0Ty0:
(22) (22) Taking the difference between these expressions, as earlier, gives Taking the difference between these expressions, as earlier, gives K0 ^b0 - K ^b = F0Ty0 - FTy - /0T
0/0
0 ^b0:
(23) (23) Restricting to the overlapping set gives that Restricting to the overlapping set gives that [K0 ^b0] - [K ^b] = [F0Ty0 - FTy - /0T
0/0
0 ^b0]:
(24)
Noting that [K] = [K0] and that [F0Ty0] - [FTy] = [/T
0y0] we get that
[K]([^b0] - [^b]) = [/T
0](y0
0 - /0 ^b0):
(25) (24) (25) Analogous to the previous cases, (y00 - /0 ^b0) is a scalar that we can label C and we get Analogous to the previous cases, (y00 - /0 ^b0) is a scalar that we can label C and we get [/T
0] = 1
C [K]([^b0] - [^b]):
(26) (26) Thus, if K is known, it can be used to deduce whether the additional individual has each of the SNPs in the
overlapping set. If K is not known exactly, it can be estimated from public data just as in the same SNP case. 5.6. Estimating /0 with different SNP sets Here, we analyze what can still be said in the event that the two studies do not use exactly the same set of
SNPs. We will still assume that the sets of SNPs are considered to have a significant overlap. For this purpose we will need a greater variety of notation. A primed variable denotes that it corresponds
to the second set of SNPs, for example, K0 is the co-occurrence matrix from the original M users for the
second experiment. If a vector or matrix is surrounded by square brackets, this denotes the same object but
with rows and columns corresponding to SNPs not in the overlap removed, for example, [K] denotes the
co-occurrence matrix from the first experiment restricted to the overlapping SNPs. As described earlier, from the first experiment, we have As described earlier, from the first experiment, we have K ^b = FTy
(21) (21) K ^b = FTy FIG. 6. Results for running the stochastic EM algorithm when estimating SNPs for three additional dogs simulta-
neously. This experimental setup replicates the experiment for one additional dog, across 5 public/private/test dataset
splits, with 20 different test sets of three additional dogs for each. (Left) Accuracy at predicting SNP presence relative
to the ‘‘most common variant’’ baseline. On average, the SEM algorithm predicts the correct SNP 75.5% of the time,
relative to 71.5% for the baseline. (Right) As in the one-dog example, we see relative improvement in the performance
of our algorithm when considering more atypical dogs. SEM, stochastic EM. FIG. 6. Results for running the stochastic EM algorithm when estimating SNPs for three additional dogs simulta-
neously. This experimental setup replicates the experiment for one additional dog, across 5 public/private/test dataset
splits, with 20 different test sets of three additional dogs for each. (Left) Accuracy at predicting SNP presence relative
to the ‘‘most common variant’’ baseline. On average, the SEM algorithm predicts the correct SNP 75.5% of the time,
relative to 71.5% for the baseline. (Right) As in the one-dog example, we see relative improvement in the performance
of our algorithm when considering more atypical dogs. SEM, stochastic EM. RECONSTRUCTION ATTACKS FROM GRS AUTHOR DISCLOSURE STATEMENT The authors declare they have no competing financial interests. 5.8. Description of K when the genotypes are non-binary – Finally, KN + 1‚ N + 1 = 1. PAIGE ET AL. and the following a + b length vector: ra + b = (ya - Fa ^bM + a)‚ - (yb - Fb ^bM + b)
h
i
(28) (28) Then, this gives us: KM(^bM + a - ^bM + b) = 1
M Fa + bra + b
(29) (29) This means that having two nonoverlapping participant sets is equivalent to the setting in which the first
study is a subset of the second (only m is now a + b). FUNDING INFORMATION This project was funded by the Alan Turing Institute Research Fellowship under EPSRC Research grant
(TU/A/000017); EPSRC/BBSRC Innovation Fellowship (EP/S001360/1), and under the EPSRC grant
EP/N510129/1. It was also partly funded by a grant from CPER Nord-Pas de Calais/FEDER DATA
Advanced data science and technologies 2015–2020. 5.8. Description of K when the genotypes are non-binary 5.8. Description of K when the genotypes are non-binary In many cases, GRS are calculated on genotype matrices that are non-binary. In particular, they may take
on three discrete values 0, 1, and 2, where 0 indicates that the most common variant is homozygous,
1 indicates that the individual is heterozygous for the uncommon variant, and 2 indicates that the individual
is homozygous for the uncommon variant. If this is the case, the description of K will change. However, it is still the case that the entries of K depend
only on the SNP frequencies and SNP co-occurrence frequencies in the dataset, and that knowledge of SNP
frequencies and pairwise co-frequencies from the original study, are all that is required to compute K. – For i = 1‚ . . . ‚ N: Kii = pAa + 4pAA where paa is the frequency of individuals being heterozygou
uncommon variant and pAA is the frequency of individuals being homozygous for the uncommon – For i = 1‚ . . . ‚ N: Kii = pAa + 4pAA where paa is the frequency of individuals being heterozygous for the
uncommon variant and pAA is the frequency of individuals being homozygous for the uncommon variant. uncommon variant and pAA is the frequency of individuals being homozygous for the uncommon variant. – For i = 1‚ . . . ‚ N - 1 and j > i: Kij = Kji = pAa=Bb + 2pAA=Bb + 4pAA=BB where pAa=Bb is the frequency that
both SNPs are simultaneously heterogygous, pAA=Bb is the frequency that one SNP is homozygous for
the rare variant and the other is heterogygous simultaneously, and pAA=BB is the frequency that that
uncommon variants are found to be homozygous simultaneously. yg
y
– For i = 1‚ . . . ‚ N and j = N + 1: Kij = Kji = pAa + 2pAA. – Finally, KN + 1‚ N + 1 = 1. yg
– For i = 1‚ . . . ‚ N and j = N + 1: Kij = Kji = pAa + 2pAA. – For i = 1‚ . . . ‚ N and j = N + 1: Kij = Kji = pAa + 2pAA. – For i = 1‚ . . . ‚ N and j = N + 1: Kij = Kji = pAa + 2pAA. 5.7. Case in which each GWAS study adds two new sets of participants 5.7. Case in which each GWAS study adds two new sets of participants This article mostly explores the case in which one study’s participants are a subset of the other study’s
participants. Here, we demonstrate that this is equivalent to the case where each of the two studies contains
a small number of participants that are not found in the other study. In particular, let us say that the first study has M + a participants and the second study has M + b
participants, where the first M participants are shared between the studies, but there are a participants that
are found in the first study but not the second, and b participants that are found in the second study but not
in the first. Following on from Eq. (9), we see that: KM(^bM + a - ^bM + b) = KM(^bM + a - ^bM) - KM(^bM + b - ^bM)
= 1
M FT
a(ya - Fa ^bM + a) - FT
b(yb - Fb ^bM + b)
h
i
: Let us define the following (N + 1) · (a + b) matrix obtained by concatenating the two genotype matrices: fine the following (N + 1) · (a + b) matrix obtained by concatenating the two genotype matrices: Fa + b = Fa‚ Fb
½
(27) Fa + b = Fa‚ Fb
½
(27) PAIGE ET AL. 450 PAIGE ET AL. REFERENCES Belsky, D.W., Moffitt, T.E., Sugden, K., et al. 2013. Development and evaluation of a genetic risk score for obesity. Biodemography Soc. Biol. 59, 85–100. g
p y
Cai, R., Hao, Z., Winslett, M., et al. 2015. Deterministic identification of specific individuals from GWAS results. Bioinformatics 31, 1701–1707. Celeux, G., and Diebolt, J. 1985. The SEM algorithm: A probabilistic teacher algorithm derived from the EM algorithm
for the mixture problem. Comput. Stat. Q. 2, 73–82. Chouraki, V., Reitz, C., Maury, F., et al. 2016. Evaluation of a genetic risk score to improve risk prediction for
Alzheimer’s disease. J. Alzheimers Dis. 53, 921–932. Day, F.R., Thompson, D.J., Helgason, H., et al. 2017. Genomic analyses identify hundreds of variants associated with
age at menarche and support a role for puberty timing in cancer risk. Nat. Genet. 49, 834–841. Dwork, C., Smith, A., Steinke, T., et al. 2015. Robust traceability from trace amounts. 2015 IEEE 56th Annual
Symposium on Foundations of Computer Science. Berkeley, California. pp. 650–669. Dwork, C., Smith, A., Steinke, T., et al. 2015. Robust traceability from trace amounts. Symposium on Foundations of Computer Science. Berkeley, California. pp. 650–669. RECONSTRUCTION ATTACKS FROM GRS 451 Hayward, J.J., Castelhano, M.G., Oliveira, K.C., et al. 2016. Complex disease and phenotype mapping in th
dog. Nat. Commun. 7, 10460. g
Huang, Z., Ayday, E., Fellay, J., et al. 2015. Genoguard: Protecting genomic data against brute-force attacks. 2015
IEEE Symposium on Security and Privacy. San Jose, California. pp. 447–462. Im, H., Gamazon, E., Nicolae, D., et al. 2012. On sharing quantitative trait gwas results in an era of multiple-omics data
and the limits of genomic privacy. Am. J. Hum. Genet. 90, 591–598. Jiang, L., Zheng, Z., Qi, T., et al. 2019. A resource-efficient tool for mixed model association analysis of large-scale
data. Nat. Genet. 51, 1749–1755. Knowles, J.W., and Ashley, E.A. 2018. Cardiovascular disease: The rise of the genetic risk score. PLoS Med. 15,
e1002546. Mittos, A., Malin, B., and Cristofaro, E.D. 2019. Systematizing genome privacy research: A privacy-enhancing
technologies perspective. Proc. Privacy Enhanc. Technol. 2019, 87–107. Qi, L., Ma, J., Qi, Q., et al. 2011. Genetic risk score and risk of myocardial infarction in hispanics. Circu
374–380. Raisaro, J.L., Trame`r, F., Ji, Z., et al. 2017. Addressing beacon re-identification attacks: Quantification and mitigation
of privacy risks. J. Am. Med. Inform. Assoc. 24, 799–805. p
y
f
Shringarpure, S., and Bustamante, C. 2015. Privacy risks from genomic data-sharing beacons. Am. J. Hum. Genet. 97,
631–646. Simmons, S., and Berger, B. 2015. One size doesn’t fit all: Measuring individual privacy in aggregate genomic data. 2015 IEEE Security and Privacy Workshops. San Jose, California. pp. 41–49. ropp, J.A. 2015. An introduction to matrix concentration inequalities. Found. Trends Mach. Learn. 8, 1–230 Visscher, P.M., Wray, N.R., Zhang, Q., et al. 2017. 10 years of gwas discovery: Biology, function, and translation. Am. J. Hum. Genet. 101, 5–22. von Thenen, N., Ayday, E., and Cicek, A.E. 2018. Re-identification of individuals in genomic data-sharing beacons via
allele inference. Bioinformatics 35, 365–371. Wang, Y.X. 2018. Revisiting differentially private linear regression: Optimal and adaptive prediction & estimation in
unbounded domain. Proceedings of the Conference on Uncertainty in Artificial Intelligence (UAI). Monterey,
California. pp. 93–103. Zhao, X., Xi, B., Shen, Y., et al. 2014. An obesity genetic risk score is associated with metabolic syndrome in Chinese
children. Gene 535, 299–302. E-mail: daphne.ezer@york.ac.uk
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All Supplementary Data from Novel BAFF-Receptor Antibody to Natively Folded Recombinant Protein Eliminates Drug-Resistant Human B-cell Malignancies <i>In Vivo</i>
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Supplementary figure legends 1 1 Day
0
3
6
9
12
16
Vaccination (D2C cells, s.c. at food pad)
Check Ab production
In vaccinated mice
boost
20
establish hybridoma
clones
14
Clone screening
Supplementary Figure 1
A
B
Clone
(h)BAFF-R-expressing L cells
(OD 450 nm)
Parental L cell
(OD 450 nm)
C53
0.552
0.095
C55
1.067
0.102
C67
0.615
0.093
C90
0.645
0.116
C39
0.137
0.091
Hybridoma clones screened by ELISA for antibody production Supplementary Figure 1 Supplementary Figure 1 Supplementary Figure 1 0
3
6
9
12
16
Vaccination (D2C cells, s.c. at food pad)
Check Ab production
In vaccinated mice
boost
20
establish hybridoma
clones
14
Clone screening Check Ab production
In vaccinated mice establish hybridoma
clones A Vaccination (D2C cells, s.c. at food pad) 20 B B Hybridoma clones screened by ELISA for antibody production Hybridoma clones screened by ELISA for antibody production Clone
(h)BAFF-R-expressing L cells
(OD 450 nm)
Parental L cell
(OD 450 nm)
C53
0.552
0.095
C55
1.067
0.102
C67
0.615
0.093
C90
0.645
0.116
C39
0.137
0.091
Hybridoma clones screened by ELISA for antibody production H. Qin, L. W. Kwak Supp. Fig. 1 Anti human BAFF-R monoclonal antibody generation and clone selection. A. L
cell clone D2C, which stably expressed human (h)BAFF-R with a C-terminal GFP tag on the
intracellular domain, was used to immunize BALB/c mice according to the schedule shown. Splenic tissue was harvested on day 20 and B-cell hybridoma clones were established. B. ELISA
results from five hybridoma supernatants using anti-mouse IgG-HRP. Clones 53, 55, 67, and 90
produced BAFF-R- specific mAbs, whereas Clone 39 did not (representative of other negative
clones). 2 2 Supplementary Figure 2 mAb C-53
mAb C-55
mAb C-67
mAb-90
Mino
293T
unstained
2nd Ab only
hybridoma sup at 1/200
hybridoma sup at 1/10
hybridoma sup at 1/50
Anti-mouse IgG-APC
Counts mAb C-53
Mino mAb C-55 mAb C-67 mAb-90 293T
Counts 293T unstained
2nd Ab only
hybridoma sup at 1/200
hybridoma sup at 1/10
hybridoma sup at 1/50 H. Qin, L. W. Kwak Supp. Fig. 2 Verification that selected hybridoma clones bind MCL cells. Binding of
hybridoma Clone 53, 55, 67, and 90 supernatants (1/10, 1/50, and 1/200 dilutions) to Mino (mantle
cell lymphoma) and 293T (negative control) cell lines assessed by flow cytometry performed with
anti-mouse IgG-APC. Supp. Fig. 2 Verification that selected hybridoma clones bind MCL cells. Binding of
hybridoma Clone 53, 55, 67, and 90 supernatants (1/10, 1/50, and 1/200 dilutions) to Mino (mantle
cell lymphoma) and 293T (negative control) cell lines assessed by flow cytometry performed with
anti-mouse IgG-APC. 3 Supplementary Figure 3 Supplementary Figure 3 mAb C-53
mAb C-55
mAb C-67
mAb C-90
H. Qin, L. W. Kwak mAb C-53 mAb C-53 mAb C-55 mAb C-55 mAb C-90 mAb C-67 H. Qin, L. W. Kwak Supp. Fig. 3 Dose-dependent binding of purified mAbs to human BAFF-R. Mouse mAb from
hybridoma Clones 53, 55, 67, 90 supernatant were purified by protein A affinity chromatography. Binding of serially diluted (1 µg/106 cells – 1.6 ng/106 cells) purified mouse mAb to Mino cells
was assessed by flow cytometry with anti-mouse IgG-APC secondary antibody. Supp. Fig. 3 Dose-dependent binding of purified mAbs to human BAFF-R. Mouse mAb from
hybridoma Clones 53, 55, 67, 90 supernatant were purified by protein A affinity chromatography. Binding of serially diluted (1 µg/106 cells – 1.6 ng/106 cells) purified mouse mAb to Mino cells
was assessed by flow cytometry with anti-mouse IgG-APC secondary antibody. 4 Supplementary Figure 4 Supplementary Figure 4 JeKo-1 (MCL)
SU-DHL-6 (DLBCL)
Raji (Burkitt)
RL (FL)
Unstained control
Anti-(m)IgG-APC only
0.05 ug/106 cells C90 mAb + Anti-(m)IgG-APC
2 ug/106 cells C90 mAb + Anti-(m)IgG-APC
0.05 ug/106 cells C55 mAb + Anti-(m)IgG-APC
2 ug/106 cells C55 mAb + Anti-(m)IgG-APC
H Qi
L W K
k JeKo-1 (MCL) SU-DHL-6 (DLBCL) JeKo-1 (MCL) SU-DHL-6 (DLBCL) Unstained control
Anti-(m)IgG-APC only
0.05 ug/106 cells C90 mAb + Anti-(m)IgG-APC
2 ug/106 cells C90 mAb + Anti-(m)IgG-APC
0.05 ug/106 cells C55 mAb + Anti-(m)IgG-APC
2 ug/106 cells C55 mAb + Anti-(m)IgG-APC Unstained control
Anti-(m)IgG-APC only
0.05 ug/106 cells C90 mAb + Anti-(m)IgG-APC
2 ug/106 cells C90 mAb + Anti-(m)IgG-APC
0.05 ug/106 cells C55 mAb + Anti-(m)IgG-APC
2 ug/106 cells C55 mAb + Anti-(m)IgG-APC Raji (Burkitt) RL (FL) Raji (Burkitt) H. Qin, L. W. Kwak Supp. Fig. 4 (h)BAFF-R mAbs recognized non-Hodgkin lymphoma cell lines in vitro. Mouse
mAb Clones 55 and 90 bound additional cell lines at high (2 µg mAb/ 106 cells) and low (0.05 µg
mAb/ 106 cells) doses: JeKo-1 (mantle cell lymphoma), SU-DHL-6 (diffuse large B cell
lymphoma), Raji (Burkitt lymphoma) and RL (follicular lymphoma). Flow cytometry analysis was
performed with anti-mouse IgG-APC. 5 Supplementary Figure 5 Supplementary Figure 5 mAb + anti-mouse IgG-APC
unstained
C90: low conc. C90 high conc. 2nd Ab only
C55: low conc. C55: high conc. Mantle cell lymphoma
(Pt# 10009)
Diffuse large B-cell
lymphoma
(Pt# 10101)
Follicular lymphoma
(Pt# 0505383) Mantle cell lymphoma
(Pt# 10009)
Diffuse large B-cell
lymphoma
(Pt# 10101)
Follicular lymphoma
(Pt# 0505383) Diffuse large B-cell
lymphoma
(Pt# 10101) Diffuse large B-cell
lymphoma
(Pt# 10101) Diffuse large B-cell
lymphoma
(Pt# 10101) Mantle cell lymphoma
(Pt# 10009) Mantle cell lymphoma
(Pt# 10009) Follicular lymphoma
(Pt# 0505383) Follicular lymphoma
(Pt# 0505383) mAb + anti-mouse IgG-APC mAb + anti-mouse IgG-APC unstained
C90: low conc. C90 high conc. 2nd Ab only
C55: low conc. C55: high conc. H. Qin, L. W. Kwak Supp. Fig. 5 (h)BAFF-R mAbs recognized lymphoma patient samples. Mantle cell lymphoma,
diffuse large B cell lymphoma, and follicular lymphoma patient samples were stained with mouse
mAbs C55 and C90 at high (2 µg/106 cells) and low (0.05 µg/106 cells) concentrations. Flow
cytometry analysis was performed with anti-mouse IgG-APC. Supp. Fig. 5 (h)BAFF-R mAbs recognized lymphoma patient samples. Mantle cell lymphoma,
diffuse large B cell lymphoma, and follicular lymphoma patient samples were stained with mouse
mAbs C55 and C90 at high (2 µg/106 cells) and low (0.05 µg/106 cells) concentrations. Flow
cytometry analysis was performed with anti-mouse IgG-APC. 6 mentary Figure 6A
Ko-1 (MCL)
i (BL)
un
Ab
un
Ab
(FL)
(DLBCL) un
Ab
un
Ab
BAFFRBD
C55
C90
BAFFR positive cells mean fluorescence intensity (n=3)
Cell line
BAFFRBD
C55
C90
JeKo-1 (MCL)
2179±109
1249±19
6590±66
Raji (BL)
904±13
1137±18
1240±13
RL (FL)
534±43
598±14
710±20
LY3 (DLBCL)
267±32
297±8
297±7 BAFFRBD H. Qin, L. W. Kwak Supplementary Figure 6B Supplementary Figure 6B (MCL)
(DLBCL)
BAFFRBD
C55
C90
BAFFR positive cells mean fluorescence intensity (n=5)
Patient
BAFFRBD
C55
C90
10022 (MCL)
1008±25
1082±23
2397±190
10101 (DLBCL)
187±7
201±2
226±3
un
Ab
un
Ab MCL)
DLBCL)
BAFFRBD
C55
C90
un
Ab
un
Ab C55 C90 H. Qin, L. W. Kwak Supp. Fig. 6 BAFF-R antibody specific binding was correlated to BAFF-R expression on
NHL lines and primary samples. Alexa fluor 647-conjugated chimeric antibodies and
commercial APC-conjugated BAFF-R antibody binding (A) a panel of NHL cell lines and (B) two
NHL primary patient samples. un: unstained control; Ab: stained sample. C55, C90, and
commercial antibodies were applied at minimal binding dose – 40 ng, 120 ng, and 500 ng per 106
cells, respectively. Mean fluorescence intensity (MFI) is calculated as mean ± s.d. of the integrated
peaks from cell lines (performed in triplicates) and primary samples (performed in pentaplicates). 7 Supplementary Figure 7 A
Raji
RL A
B
Raji
RL A
Raji
RL RL B H. Qin, L. W. Kwak Supp. Fig. 7 Chimeric antibodies required NK cells for cytotoxicity against tumor cells. Calculated specific ADCC lysis on NHL lines (A) Raji, RL, and (B) JeKo-1 cells (target). Cells
were labeled with chromium-51 and incubated with chimeric mAb (C55, C90, or rituximab) and
with or without NK cells (effector) at effector to target ratio of 20:1. 8 8 Supplementary Figure 8 Supplementary Figure 8 Kwak
pplementary Figure 9
R itu x im a b
C 9 0
C 5 5
C o n tro l Ig G
N K o n ly
0
2 0
4 0
6 0
8 0
U 2 6 6
S p e c ific ly s is %
R itu x im a b
C 9 0
C 5 5
C o n tro l Ig G
N K o n ly
0
2 0
4 0
6 0
8 0
J u rk a t
S p e c ific ly s is %
R itu x im a b
C 9 0
C 5 5
C o n tro l Ig G
N K o n ly
0
2 0
4 0
6 0
8 0
S K N O -1
S p e c ific ly s is %
R itu x im a b
C 9 0
C 5 5
C o n tro l Ig G
N K o n ly
0
2 0
4 0
6 0
8 0
R S 4 ;1 1
S p e c ific ly s is %
* *
R itu x im a b
C 9 0
C 5 5
C o n tro l Ig G
N K o n ly
0
2 0
4 0
6 0
8 0
M E C -1
S p e c ific ly s is %
* *
A Supplementary Figure 9
R itu x im a b
C 9 0
C 5 5
C o n tro l Ig G
N K o n ly
0
2 0
4 0
6 0
8 0
R S 4 ;1 1
S p e c ific ly s is %
* *
A A M E C -1 R itu x im a b
C 9 0
C 5 5
C o n tro l Ig G
N K o n ly
0
2 0
4 0
6 0
8 0
U 2 6 6
R itu x im a b
C 9 0
C 5 5
C o n tro l Ig G
N K o n ly
0
2 0
4 0
6 0
8 0
J u rk a t
S p e c ific ly s is %
R itu x im a b
C 9 0
C 5 5
C o n tro l Ig G
N K o n ly
0
2 0
4 0
6 0
8 0
S K N O -1
S p e c ific ly s is % B B H. Supplementary Figure 8 Unlabeled cells
Labeled cells
Labeled cells + 1 hr incubation at 37C
Red: Unlabeled cells
Blue: Labeled cells
Orange: Labeled cells + 1 hr
incubation at 37C
Figure 8
H. Qin, L. W. Kwak Unlabeled cells
igure 8 Labeled cells Labeled cells Unlabeled cells Labeled cells + 1 hr incubation at 37C Red: Unlabeled cells
Blue: Labeled cells
Orange: Labeled cells + 1 hr
incubation at 37C H. Qin, L. W. Kwak Supp. Fig. 8 Limited internalization was observed with BAFF-R mAbs. Mino cells were
incubated with mAb C90 (0.05 g/106 cells) at 4 °C for 20 min followed by incubation at 37 °C
for 1 h. Flow cytometry analysis was performed with anti-mouse IgG-APC. Cells were gated for
surface localized antibodies (OUT) and loss of cell surface staining (IN). 9 upplementary Figure 9
R itu x im a b
C 9 0
C 5 5
C o n tro l Ig G
N K o n ly
0
2 0
4 0
6 0
8 0
R S 4 ;1 1
S p e c ific ly s is %
* *
R itu x im a b
C 9 0
C 5 5
C o n tro l Ig G
N K o n ly
0
2 0
4 0
6 0
8 0
M E C -1
S p e c ific ly s is %
* *
A H. Qin, L. W. Supplementary Figure 8 Qin, L. W. Kwak Supp. Fig. 9 BAFF-R antibody induced cytotoxicity in BAFF-R expressing lines. Antibody-
induced cytotoxicity measured by chromium-51 release after incubation of malignant human B-
cell tumors with C55, C90, or rituximab and effectors (NK cells) at effector to target ratio (E:T)
of 20:1. Percentage of cell specific lysis of target cells: (A) Acute lymphoblastic and chronic
lymphoblastic leukemia lines RS4;11 and MEC-1, respectively, and (B) BAFF-R negative lines
U266 (multiple myeloma), Jurkat (T cell leukemia), and SKNO-1 (acute myeloid leukemia). All
experimental groups were performed in triplicates. **P < 0.01 compared with controls two-tailed
Student’s t-test. 10 Supplementary Figure 10 Supplementary Figure 10 Supplementary Figure 10 H. Qin, L. W. Kwak
**
** **
** H. Qin, L. W. Kwak Supp. Fig. 10 BAFF-R mAb induced ADCC in BAFF-R expressing primary MCL samples. ADCC effects were measured by chromium-51 release after primary MCL patient samples were
incubated with antibodies and effectors (NK cells) at E:T = 20:1. Calculated percentage of cell
specific lysis of target cells are shown. All experimental groups were performed in triplicates. **P
< 0.01 compared with controls two-tailed Student’s t-test. 11 Supplementary Figure 11 Supplementary Figure 11 H Qin L W Kwak
JeKo-1 WT
CD20 KO #3
CD20 KO #18
CD20 KO #19
CD20 KO #31
CD20 KO #34
CD20 KO #49
CD20 KO #50
CD20-APC
RFP
CD20 KO #25
37 kDa
25 kDa
50 kDa
37 kDa A CD20 KO #31
CD20 KO #34
CD20 KO #49
CD20 KO #50
RFP
CD20 KO #25 CD20 KO #31
CD20 KO #34
CD20 KO #49
CD20 KO #50
CD20 KO #25 CD20-APC
37 kDa
25 kDa
50 kDa
37 kDa
B B H. Qin, L. W. Kwak Supplementary Figure 11
C Supplementary Figure 11 C D JeKo-1 WT
CD20 KO #3
CD20 KO #18
CD20 KO #19
CD20 KO #25 1 WT
CD20 KO #3
CD20 KO #18
CD20 KO #19
CD20 KO #25
KO #31
CD20 KO #34
CD20 KO #49
CD20 KO #50 JeKo-1 WT
CD20 KO #3
CD20 KO #18
CD20 KO #19
CD20 KO #25
P RFP CD20 KO #31
CD20 KO #34
CD20 KO #49
CD20 KO #50
RF CD20 KO #31
CD20 KO #34
CD20 KO #49
CD20 KO #50
RF BAFFR-APC Supp. Fig. 11 CD20 knock out clones generated with CRISPR. CD20 knock out clones of JeKo-
1 were generated with a commercial CRISPR/HDR system substituting RFP at the CD20 locus. (A) Clones were screened and sorted by flow cytometry for CD20-/RFP+ expression. (B) Western
blotting with anti-CD20 antibodies was performed on total cell lysate from CD20-/RFP+ clones. β-
actin was blotted as a loading control. (C) Cell viability post-knockout was followed for 20
passages. JeKo-1 wildtype (WT) was used as a control. (D) Clones as in (A) were screened for
BAFF-R/RFP+ expression to confirm that BAFF-R expression had not been affected by the
CRISPR/HDR manipulation of CD20. 12 Supplementary Figure 12A Supplementary Figure 12A Supplementary Figure 12A Day 0
Day3
8
13
18
4 5 6 7
9 10 11 12
14 15 16 17
Ibrutinib
90 days
25 mg/kg ibrutinib was administered by IP injection every other day from Day3 to Day17 Day3
8
13
18
4 5 6 7
9 10 11 12
14 15 16 17
Ibrutinib
90 days
25 mg/kg ibrutinib was administered by IP injection every other day from Day3 to Day17 25 mg/kg ibrutinib was administered by IP injection every other day from Day3 to Day17 H. Qin, L. W. Kwak Supplementary Figure 12B Supplementary Figure 12B Z-138 Z-138 PBS
Ibrutinib
PBS
Ibrutinib PBS
Ibrutinib
PBS
Ibrutinib PBS Ibrutinib Ibrutinib D13 D13 D16 D16 D16 D16 D19 H. Qin, L. W. Kwak Supp. Fig. 12 Ibrutinib treatment failed to protected mice challenged with ibrutinib-resistant
Z-138 cells. (A) Tumor challenge and treatment schedule is shown for NSG mice challenged with
either ibrutinib-sensitive JeKo-1 or ibrutinib-resistant Z-138 tumors. Mice were administered 25
mg/kg ibrutinib in DMSO by IP injection according to the schedule. Tumor control groups
received PBS injections. (B) Bioluminescence images of tumor cells in mice were taken
throughout study; representative days are shown. 13 Supplementary Figure 13 Supplementary Figure 13 Z-138 mouse model
** P < 0.001 compared with controls Z-138 mouse model
** P < 0.001 compared with controls Z-138 mouse model ** P < 0.001 compared with controls H. Qin, L. W. Kwak Supp. Fig. 13 Chimeric antibodies targeting human BAFF-R elicited in vivo therapeutic
effects against Z-138 ibrutinib-resistant tumors. Bioluminescence images of mice challenged
with luciferase-expressing, ibrutinib-resistant Z-138 cells followed by antibody treatments as in
Figure 4. Control group mice received PBS, NK cells alone, or rituximab on the same schedule. 80-day tumor-free and overall survival curves of the mice shown. Tumor free rate and survival
differences between experimental and all control groups were analyzed by log-rank test (groups
of n=5, ** P<0.01). 14 Supplementary Figure 14 A B A H. Qin, L. W. Kwak Supplementary Figure 14C Supplementary Figure 14C Purified B cells
Purified T cells
Purified NK cells
Gated myeloid cells
Unstained control
Anti-(m)IgG-APC only
0.05 ug/106 cells C90 mAb + Anti-(m)IgG-APC
0.05 ug/106 cells C55 mAb + Anti-(m)IgG-APC
0.05 ug/106 cells (m)IgG + Anti-(m)IgG-APC
H. Qin, L. W. Kwak Purified T cells
Unstained control
Anti-(m)IgG-APC only
0.05 ug/106 cells C90 mAb + Anti-(m)IgG-APC
0.05 ug/106 cells C55 mAb + Anti-(m)IgG-APC
0.05 ug/106 cells (m)IgG + Anti-(m)IgG-APC Purified B cells Purified T cells
Unstained cont
Anti-(m)IgG-APC
0.05 ug/106 cell
0.05 ug/106 cell
0.05 ug/106 cell Purified T cells Gated myeloid cells Purified NK cells Gated myeloid cells H. Qin, L. W. Kwak Supp. Fig. 14 Characterization of BAFF-R binding against normal B cells. PBMC from
healthy donors were co-stained with APC-conjugated C90 chimeric antibody and (A) a
lymphocyte marker panel (anti-CD20-PE, anti-CD3-PacificBlue, and anti-CD56-FITC) or (B) a
myeloid cell marker panel (anti-CD45-PE, anti-CD15-PerCP-Cy5.5, and anti-CD14-PacificBlue). Each specific immune cell sub-population was gated and analyzed for binding with BAFF-R
antibodies. (C) B cells, T cells, and NK cells were isolated from PBMC with commercial specific
cell type isolation kits and stained with C55 and C90 (0.05 µg mAb/106 cells). Flow cytometry
analysis was performed with anti-mouse IgG. Myeloid cells from PBMC were gated for CD66b+
and analyzed for mAb C55 and C90 staining. 15 Supplementary Figure 15 Supplementary Figure 15 Supplementary Figure 15 H. Qin, L. W. Kwak
R itu x im a b
C 9 0
C 5 5
C o n tro l Ig G
N K o n ly
0
2 0
4 0
6 0
8 0
B c e lls
S p e c ific ly s is %
* * R itu x im a b
C 9 0
C 5 5
C o n tro l Ig G
N K o n ly
0
2 0
4 0
6 0
8 0
B c e lls
S p e c ific ly s is %
* * R itu x im a b
C 9 0
C 5 5
C o n tro l Ig G
N K o n ly
0
2 0
4 0
6 0
8 0
B c e lls
S p e c ific ly s is %
* * H. Qin, L. W. Kwak Supp. Fig. 15 BAFF-R mAb elicited ADCC effects on normal human B cells. Antibody-
dependent cell-mediated cytotoxicity (ADCC) effects were measured on purified normal B cells
by chromium-51 release after incubation with rituximab, C90, C55, Control IgG, and NK cells
only at E:T = 20:1. Calculated percentage of cell specific lysis of target cells are shown. All
experimental groups were performed in triplicates. **P < 0.05 compared with NK cell only and Control IgG by two-tailed Student’s t-test. Control IgG by two-tailed Student’s t-test. 16
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Editorial
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EURASIP Journal on Advances in Signal Processing
| 2,002
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cc-by
| 1,959
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Vahid Tarokh Department of EECS MIT, Cambridge, MA 02139, USA
Email: vahid@mit.edu Department of EECS MIT, Cambridge, MA 02139, USA
Email: vahid@mit.edu Department of EECS MIT, Cambridge, MA 02139, USA
Email: vahid@mit.edu Xiang-Gen Xia They propose to directly assign channel sym-
bols to transmit antennas at different states by exploiting the
properties of the state transitions in the trellis. The paper by
D. Tujkovic, M. Juntti, and M. Latva-aho considers space-
time turbo code designs. They propose a design method for
recursive space-time trellis codes and parallel-concatenated
space-time turbo coded modulation that can be applied to
an arbitrary existing space-time trellis code. This method en-
ables a large, systematic increase in coding gain while pre-
serving the maximum transmit diversity gain and bandwidth
efficiency property of the space-time trellis code considered. The next six papers are on decoding methods of space-
time coded transmissions. The paper by B. A. Bjerke and
J. G. Proakis considers the equalization of a multiple trans-
mit and receive antenna wireless system. They consider un-
coded, convolutionally coded, turbo coded MIMO channels,
and also precoded MIMO channels. The paper by E. Erez
and M. Feder proposes new decoding techniques for diver-
sity channels employing space-time codes when the chan-
nel coefficients are unknown to both transmitter and re-
ceiver. They propose an efficient implementation of the gen-
eralized maximum-likelihood ratio test algorithm and an
energy weighted decoder. The paper by S. K. Jayaweera and
H. V. Poor considers low complexity multiuser detection q
In space-time coding, space-time code design is one of
the most important topics. Unlike the conventional sin-
gle antenna AWGN channel, the multiple antenna channel
transfer function is a matrix instead of a scalar, even in the
simplest propagation scenario. So, the space-time code de-
sign is more challenging than in single antenna systems. Concerning a multiple antenna channel, that is, a multi-
input multi-output (MIMO) system, two cases can arise: the
MIMO channel can be known or unknown at the receiver. Since the channel is a complex matrix, signal processing may
play an even more important role than it does in a single an-
tenna system. When we deal with space-time matrices, the
code size may be huge and therefore fast decoding algorithms
are critically important. As a result, space-time coding has
become one of the most active research areas in wireless com-
munications. The EURASIP JASP has dedicated two special
issues to this subject. The first issue has 11 papers that spread
over space-time code designs, decoding methods for space-
time coded transmissions, and MIMO systems. EURASIP Journal on Applied Signal Processing 2002:3, 209–210
c⃝2002 Hindawi Publishing Corporation EURASIP Journal on Applied Signal Processing 2002:3, 209–210
c⃝2002 Hindawi Publishing Corporation Dirk Slock Mobile Communications Department, EURECOM Institute, 2229 route des Cretes, BP 193, 06904 Sophia Antipolis Cedex, France
Email: dirk.slock@eurecom.fr Xiang-Gen Xia Department of ECE, University of Delaware, Newark, DE 19716, USA
Email: xxia@ee.udel.edu Space-time coding for multiple transmit and multiple receive
antenna systems has recently attracted considerable atten-
tion in the wireless communications area. It has been shown
theoretically that for certain channels the capacity of a mul-
tiple antenna system increases linearly with the number of
antennas, which implies that multiple antenna systems have
huge potential applications in broadband wireless communi-
cations. In a multiple antenna system, a key building block is
multiple antenna coding and modulation, called space-time
coding. In single antenna systems, the coding and modula-
tion only deals with one-by-one complex symbols or symbol
sequences. However, in multiple antenna systems, the space-
time coding deals with complex symbol matrices or matrix
sequences. The first three papers are on space-time code designs. The
paper by S. M. Haas, J. H. Shapiro, and V. Tarokh considers
space-time coding issues for wireless optical channels. The
space-time codes are used to overcome turbulence-induced
fading in an atmospheric optical heterodyne communica-
tion system. They propose a new criterion for the space-
time code design for such channels. The paper by Z. Safar
and K. J. R. Liu considers a systematic space-time trellis code
design method by jointly considering diversity and coding
advantages. They propose to directly assign channel sym-
bols to transmit antennas at different states by exploiting the
properties of the state transitions in the trellis. The paper by
D. Tujkovic, M. Juntti, and M. Latva-aho considers space-
time turbo code designs. They propose a design method for
recursive space-time trellis codes and parallel-concatenated
space-time turbo coded modulation that can be applied to
an arbitrary existing space-time trellis code. This method en-
ables a large, systematic increase in coding gain while pre-
serving the maximum transmit diversity gain and bandwidth
efficiency property of the space-time trellis code considered. The first three papers are on space-time code designs. The
paper by S. M. Haas, J. H. Shapiro, and V. Tarokh considers
space-time coding issues for wireless optical channels. The
space-time codes are used to overcome turbulence-induced
fading in an atmospheric optical heterodyne communica-
tion system. They propose a new criterion for the space-
time code design for such channels. The paper by Z. Safar
and K. J. R. Liu considers a systematic space-time trellis code
design method by jointly considering diversity and coding
advantages. Xiang-Gen Xia The next six papers are on decoding methods of space-
time coded transmissions. The paper by B. A. Bjerke and
J. G. Proakis considers the equalization of a multiple trans-
mit and receive antenna wireless system. They consider un-
coded, convolutionally coded, turbo coded MIMO channels,
and also precoded MIMO channels. The paper by E. Erez
and M. Feder proposes new decoding techniques for diver-
sity channels employing space-time codes when the chan-
nel coefficients are unknown to both transmitter and re-
ceiver. They propose an efficient implementation of the gen-
eralized maximum-likelihood ratio test algorithm and an
energy weighted decoder. The paper by S. K. Jayaweera and
H. V. Poor considers low complexity multiuser detection EURASIP Journal on Applied Signal Processing 210 existing wireless systems. He received one best journal paper award
from the IEEE-SP and one from EURASIP in 1992. He is the coau-
thor of two IEEE-Globecom98 best student paper awards. He has
been an Associate Editor for the IEEE-SP Transactions. methods for space-time coded synchronous multiple-access
systems in the presence of independent Rayleigh fading. They show that optimal space-time codes designed for sin-
gle user channels can still provide full diversity in the mul-
tiuser channel. The paper by J. Liu, J. Li, and E. G. Larsson
considers a differential space-time block code modulation
for DS-CDMA systems. They propose three demodulation
schemes, namely a differential space-time block code Rake
receiver, a differential space-time block code deterministic
receiver, and a differential space-time block code determinis-
tic de-prefix receiver. The paper by Y. Liu and X. Wang pro-
poses a space-time differential decoding technique based on
multiple-symbol detection and decision-feedback by making
use of the second-order statistics of the fading. The paper by
J. Zhang and P. M. Djuri´c considers joint estimation and de-
coding of space-time trellis codes by employing a new emerg-
ing tool in statistical signal processing, sequential importance
sampling. Vahid Tarokh received his Ph.D. degree in
Electrical Engineering from the University of
Waterloo, Ontario, Canada in 1995. From
August 1995 to May 1996, he was em-
ployed by the Coordinated Science Labora-
tory, of the University of Illinois Urbana-
Champaign, as a visiting Professor. He then
joined the AT&T Labs-Research, where he
was employed as a Senior Member of Tech-
nical Staff, Principal Member of Technical
Staff, and the Head of the Department of Wireless Communica-
tions and Signal Processing until August 2000. Xiang-Gen Xia In the fall of 2000, Dr. Tarokh joined the Department of Electrical Engineering and Com-
puter Sciences of MIT as an Associate Professor, where he is cur-
rently employed. Dr. Tarokh received numerous awards including
the 1987 Gold Tablet of The Iranian Math Society, the 1995 Gov-
ernor General of Canada’s Academic Gold Medal, the 1999 IEEE
Information Theory Society Prize Paper Award, (jointly with A. R. Calderbank and N. Seshadri), and more recently the 2001 Alan T. Waterman Award. The final two papers are on MIMO systems. The pa-
per by A. Grant considers information theoretic properties
of flat fading channels with multiple antennas when per-
fect channel knowledge at the receiver is assumed. Expres-
sions for maximum information rates and outage probabili-
ties are derived. It is shown that the use of orthogonal mod-
ulating signals is asymptotically optimal in terms of infor-
mation rate. The paper by T.-L. Tung and K. Yao considers
the problem of channel estimation and optimal power allo-
cation for a multiple-antenna OFDM system. They develop
a least-squares channel estimation approach, derive a per-
formance bound, investigate the optimal training sequence
for initial channel acquisition, and derive the optimal power
allocation solution that maximizes the bandwidth efficiency
under power and QoS constraints. Xiang-Gen Xia received his B.S. degree in
mathematics from Nanjing Normal Univer-
sity, Nanjing, China, and his M.S. degree in
mathematics from Nankai University, Tian-
jin, China, and his Ph.D. degree in Electrical
Engineering from the University of Southern
California, Los Angeles, USA in 1983, 1986,
and 1992, respectively. He was a Lecturer at
Nankai University, China during 1986–1988,
a Teaching Assistant at University of Cincin-
nati, USA during 1988–1990, a Research Assistant at the University
of Southern California, USA during 1990–1992, and a Research Sci-
entist at the Air Force Institute of Technology during 1993–1994. He was a Senior/Research StaffMember at Hughes Research Labo-
ratories, Malibu, California, during 1995–1996. In September 1996,
he joined the Department of Electrical and Computer Engineer-
ing, University of Delaware, Newark, Delaware, USA, where he is
currently an Associate Professor. His current research interests in-
clude communication systems including equalization and coding;
SAR and ISAR imaging of moving targets, wavelet transform and
multirate filterbank theory and applications; time-frequency anal-
ysis and synthesis; and numerical analysis and inverse problems in
signal/image processing. Dr. Xia has over 80 refereed journal articles
published, and 4 U.S. patents awarded. Xiang-Gen Xia He is the author of the book
“Modulated Coding for Intersymbol Interference Channels” (New
York, Marcel Dekker, 2000). Dr. Xia received the National Science
Foundation (NSF) Faculty Early Career Development (CAREER)
Program Award in 1997, the Office of Naval Research (ONR) Young
Investigator Award in 1998, and the Outstanding Overseas Young
Investigator Award from the National Nature Science Foundation
of China in 2001. He also received the Outstanding Junior Faculty
Award of the Engineering School of the University of Delaware in
2001. He is currently an Associate Editor of the IEEE Transactions
on Mobile Computing, the IEEE Transactions on Signal Processing
and the EURASIP Journal of Applied Signal Processing. He is also
a Member of the Signal Processing for Communications Technical
Committee in the IEEE Signal Processing Society. Dirk Slock
Vahid Tarokh
Xiang-Gen Xia Dirk Slock received the engineer’s degree
from the University of Gent, Belgium in
1982. In 1984 he was awarded a Fulbright
scholarship for Stanford University, USA,
where he received his M.S. in Electrical En-
gineering, M.S. in Statistics, and Ph.D. in
Electrical Engineering in 1986, 1989, and
1989, respectively. While at Stanford, he de-
veloped new fast recursive least-squares al-
gorithms for adaptive filtering. In 1989–91,
he was a member of the research staffat the Philips Research Labo-
ratory, Belgium. In 1991, he joined the Eurecom Institute where
he is now Associate Professor. At Eurecom, he teaches statistical
signal processing and speech coding for mobile communications. His research interests include DSP for mobile communications:
antenna arrays for (semi-blind) equalization/interference cancel-
lation and spatial division multiple access, space-time processing
and audio coding. More recently, he has been focusing on receiver
design, downlink antenna array processing, and speech coding for
third generation systems, and introducing spatial multiplexing in
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“Challenge” or “Hindrance”? The Effect of Leader–Member Exchange on Employee Resilience in Public Sector Organizations
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“Challenge” or “Hindrance”? The Effect of Leader–
Member Exchange on Employee Resilience in Public
Sector Organizations Yan-Han ZHU
Southwest University
Yue YUAN Research Article Keywords: leader-member exchange(LMX), employee resilience, challenge appraisals, hindrance
appraisals, experiment Posted Date: March 14th, 2024 DOI: https://doi.org/10.21203/rs.3.rs-4025326/v1 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read
Full License Additional Declarations: No competing interests reported. Page 1/26 Page 1/26 Abstract Background: In complex, volatile, and uncertain societies, employee resilience is crucial for public sector
organizations to navigate crises and maintain steady growth. Methods: Whether and how leader-member exchange (LMX) influence employee resilience? The present
study offers an answer to this question through a multi-method (field and experiment) research conducted
with a sample of public sector organizations’ members in China. Study 1 conducted a comprehensive
meta-analysis to review the available evidence and assess the influence of LMX on employee resilience. Subsequently, Study 2 conducted a questionnaire survey of 118 members in public sector organizations to
investigate the practical impact of LMX on employee resilience. Finally, Study 3 used a scenario-based
simulation experiment to manipulate the quality of LMX and to explore the mediating role of stress
appraisals. Results:The meta-analysis conducted in Study 1, which included 42 independently extracted samples from
36 studies, explored the importance and scarcity of existing research on the influence of LMX on employee
resilience in public sector organizations through two important aspects of leadership and interpersonal
relationships inherent in LMX. Study 2, a questionnaire survey, showed that LMX has a significant direct
positive effect on employee resilience. Furthermore, using an experimental design, Study 3 revealed that
stress appraisals, both challenge and hindrance appraisals, play a mediating role in the relationship
between LMX and employee resilience. Conclusions: The present research revealed that LMX has a significant and positive effect on employee
resilience; challenge and hindrance appraisals significantly mediate the relationship between LMX and
employee resilience. These findings have practical implications for individuals and organizations from a
pragmatic perspective. 1 Introduction Currently, unforeseen events and sudden changes (such as public health crises such as COVID-19 or
natural disasters such as forest fires) inundate the entire organizational milieu [1], causing unprecedented
disruptions to economic activities and people’s daily lives. This is particularly challenging for public sector
organizations, which are tasked with protecting the public interest and serving the needs of the community
[2]. In the face of an increasingly volatile and highly uncertain environment, the social governance system
itself is being challenged [3], requiring active adaptation to changing circumstances and effective
responses to public needs. How to empower members of these organizations to adapt rapidly to change
and respond effectively to crises has become a critical aspect of public management practice. Research
has shown that resilience can enable organizations and their members to withstand risks and play a
crucial role in responding to crises [1]. Consequently, resilience in organizational contexts has attracted
considerable attention and debate among scholars [4]. Page 2/26 Page 2/26 Organizational member resilience refers to the ability of employees to adapt and respond positively to
stress in the workplace[5, 6]. It is characterized by employees’ ability to persevere, recover quickly, grow,
and achieve goals in the face of adversity such as conflict and failure at work [7, 8, 9]. It plays a crucial role
in helping employees to overcome detrimental effects such as role ambiguity and turnover intentions [10,
11] while increasing job satisfaction [6, 12], well-being, and performance [13, 14], among others. Employees
are central to an organization’s ability to adapt to change in uncertain and dynamic environments [15, 16]. Employees of public sector organizations are the concrete implementers of policies and national laws and
regulations, the performers of state public affairs and the exercisers of administrative power, and the
managers of social affairs [17]. They are the critical windows of government to the public and influence
the quality of the relationship between government and citizens [18]. However, there is little research on
employee resilience in public sector organizations and systematic reflection on the triggering factors and
ways to improve it. Improving employee resilience to cope with the unknown is a pressing issue for them. How leaders manage, treat, and evaluate their subordinates has a strong influence on their working lives
[19] and self-concept [20]. On the one hand, in Eastern cultures, leaders represent hierarchy and authority
[21]. 1 Introduction Employees’ attitudes and behaviours are more likely to be influenced by their leaders, especially in
public sector organizations [22, 23]. Interpersonal relationships at work, on the other hand, are important
not only for their instrumental value (e.g. they are often used in business transactions) but also because of
the central role they play in the self-concept of Chinese people [24, 20]. Chinese people often find
themselves embedded in a network of personal relationships that define their existence and influence their
thoughts and emotions [25]. While hierarchy is valued in Chinese culture, relationships are also valued [26]. Leader-member exchange (LMX) refers to the quality of the relationship between employees and their
supervisors [27], and encompasses a relationship-oriented approach to leadership. Therefore, it is
important to pay attention to the interpersonal relationships between employees and their leaders in the
context of public sector organizations. According to LMX theory, employees who experience high-quality
LMX relationships feel greater trust and respect and receive resources and support that help them
overcome barriers to achieving their goals. As a result, employees develop a more positive self-concept,
exhibit higher levels of effort, and demonstrate greater employee resilience [28]. Although research has
begun to explore the role of LMX as a facilitator of employee resilience, the question of how LMX
enhances employee resilience by influencing employees’ appraisals of stress in objective situations
remains unanswered. Further research is needed to explore the role of leaders in cultivating cognitive
resources to promote employee resilience. Stress appraisals (challenge and hindrance) arise from a dual perception of the individual and the
environment. When encountering a stressor, the individual perceives the stimuli as harmful (already
causing damage to oneself), threatening (possibly causing damage or loss), or challenging (possibly
leading to mastery or benefit). This assessment considers the individual’s available resources and coping
options for dealing with stressful situations [29]. As a result, the perception of resource availability
interacts with challenge and hindrance appraisals to shape coping strategies and subsequent outcomes
[30]. The assessment of employee stressors within an organization is primarily influenced by resources. These resources also enhance individuals’ ability to navigate stressful events, protecting them from Page 3/26 psychological dysfunctions such as anxiety and burnout [31]. According to the conservation of resources
(COR) theory, individuals with sufficient resources are better equipped to acquire more resources. 1 Introduction Individuals strive to obtain, cultivate, and safeguard resources as they are more effective in mitigating the
negative impact of resource loss during work [32]. LMX is a positive resource that provides members with
information, support, and self-esteem [33]. It enables members to access tangible and intangible assets
from their supervisors [34]. Based on the COR theory, it is argued that LMX can activate specific
components of both challenge and hindrance appraisals. High-quality LMX fosters emotional and material
support for employees, providing them with opportunities for growth and development. High-quality LMX
motivates employees to positively evaluate the organization, which can alleviate pressures from the
organizational environment and boost employee resilience. Conversely, low-quality LMX fosters a belief
among employees that their efforts are inadequately reciprocated, inducing negative evaluations of the
organizational environment and diminishing employee initiative. In summary, LMX can influence
employees' behaviour by shaping their cognitive appraisal process. Stress appraisals play a crucial
mediating role in the relationship between LMX and employee resilience. psychological dysfunctions such as anxiety and burnout [31]. According to the conservation of resources
(COR) theory, individuals with sufficient resources are better equipped to acquire more resources. To gain insight into employee resilience, we utilized a meta-analytic approach and analyzed 101
independent samples from 2010 onwards (Study 1). The analysis revealed a significant positive
correlation between leadership, interpersonal relationships, and employee resilience. However, there is a
research gap regarding the association between LMX and employee resilience, particularly within the realm
of Chinese public organizations. Our study aims to examine and enhance comprehension of this specific
relationship. In light of the aforementioned findings, we conducted a questionnaire survey to investigate
the correlation between LMX and employee resilience in public sector organizations (Study 2). Our
hypothesis was based on LMX theory and posited a positive relationship between LMX and employee
resilience. This study aims to complement and validate the field of practical public organizations in light of
our meta-analytic results. To examine the mediating role of stress appraisals, an experimental study was
conducted involving employees in public sector organizations (Study 3). This approach was used to
reduce the impact of extraneous variables and gain a more precise understanding of the mediating effects
of challenging and hindering stress appraisals in the relationship between LMX and employee resilience. This study makes several contributions. First, it sheds light on the relationship between LMX and employee
resilience in the context of public sector organizations. 2 Literature Review and Theoretical Hypotheses 2.1 Study 1: Meta-Analysis Research on the relationship between LMX and employee resilience is limited. The meta-analysis of LMX
on employee resilience is challenging to determine the significance of this limited research in revealing
differences between public sector and other organizations. Previous studies have demonstrated that
leadership and interpersonal relationships are critical factors in employee resilience. Although LMX has not
been extensively explored, research on these topics has provided in-depth insights into their significance in
organizations. To gain a comprehensive understanding of the impact of LMX on employee resilience, this
study conducts a meta-analysis of existing research on leadership and interpersonal relationships. The
review examines relevant studies and provides data support for the elicitation of LMX. We first conducted
a review to identify published articles related to employee resilience at the end of January 2024. Specifically, we used “employee resilience” or “subordinate resilience” in the Web of Science,
PsycARTICLES, PsycINFO, JSTOR, SAGE, Springer, Elsevier, and Google Scholar searches. Additionally, the
reference lists of the included articles were thoroughly scanned to ensure comprehensive coverage of the
literature. Only peer-reviewed articles published in English were considered for the analysis, ensuring a
manageable dataset for examination. Research on the relationship between LMX and employee resilience is limited. The meta-analysis of LMX
on employee resilience is challenging to determine the significance of this limited research in revealing
differences between public sector and other organizations. Previous studies have demonstrated that
leadership and interpersonal relationships are critical factors in employee resilience. Although LMX has not
been extensively explored, research on these topics has provided in-depth insights into their significance in
organizations. To gain a comprehensive understanding of the impact of LMX on employee resilience, this
study conducts a meta-analysis of existing research on leadership and interpersonal relationships. The
review examines relevant studies and provides data support for the elicitation of LMX. We first conducted
a review to identify published articles related to employee resilience at the end of January 2024. Specifically we used “employee resilience” or “subordinate resilience” in the Web of Science used “employee resilience” or “subordinate resilience” in the Web of Science, PsycARTICLES, PsycINFO, JSTOR, SAGE, Springer, Elsevier, and Google Scholar searches. Additionally, the
reference lists of the included articles were thoroughly scanned to ensure comprehensive coverage of the
literature. Only peer-reviewed articles published in English were considered for the analysis, ensuring a
manageable dataset for examination. 2 Literature Review and Theoretical Hypotheses To be eligible for inclusion in this meta-analysis, studies had to meet specific criteria. First, they needed to
employ quantitative research methods and provide empirical data, excluding purely theoretical and review
studies. Second, the samples across studies had to be independent. If there was any overlap or crossing
between two studies, only the study with more detailed information or a larger sample size was included. The analysis should primarily focus on individual-level effects rather than unit or organizational effects. For cross-level studies, only individual-level results were considered. After a thorough screening process,
101 articles out of the initial pool of 331 articles were deemed eligible for inclusion in the meta-analysis. For more details, please refer to Appendix. To extracted relevant information from the articles, including the
title, year of publication, authorship, study population, subject area (e.g., Personnel of public sector
organizations), percentage of female participants, and correlation coefficients. Research suggests that
employee resilience leads to positive outcomes for both the individual and the organization [35, 36]. Therefore, it is important to consider strategies that can improve employee resilience. The meta-analysis
revealed that personal traits, job characteristics, and the organizational environment are all factors that
influence employees’ ability to resist stress. It is worth noting that leadership and colleagues have been
identified as influential factors that affect individual behaviour within the organizational context and have
received considerable attention in relevant studies. 1 Introduction The findings highlight that the development of
employee resilience hinges upon the dimensions of LMX quality. Secondly, the research advances our
understanding of stress appraisals as an antecedent of employee resilience. The appraisal mechanisms
for nurturing and cultivating employee resilience remain relatively unexplored in research. The results show
a significant relationship between stress appraisals and employee resilience. High levels of these (e.g.,
high LMX) prompt subordinates to appraise their leader’s actions and guidance more favourably. This
positive appraisal enhances subordinate motivation and promotes resilience. Finally, this study used
different research methods to validate the mediating mechanisms linking LMX to employee resilience in
public sector organizations. In light of these findings, leaders in public sector organizations are
encouraged to develop effective communication skills that align with the valued and trusted goals of Page 4/26 Page 4/26 subordinates. By doing so, leaders can positively influence the appraisal process of subordinates, leading
to increased resilience and potentially improved future performance. 2.1.1 Leadership and Employee Resilience Page 5/26 “Leader” is a critical factor closely intertwined with and exerting influence on organizational members and
goals. It encompasses the process through which leaders utilize their motivated employees and guide
them towards achieving organizational objectives [37]. Leadership embodies both the behaviours exhibited
by leaders and the personal characteristics that are perceived by organizational members as influencing
others [38]. In management, the selection of an appropriate leader can enhance communication and
interaction between leaders and employees, ultimately enhancing leadership effectiveness and
organizational performance. By aggregating relevant analyses from existing studies on leader
relationships, 23 studies were included in this meta-analysis, comprising a total sample size of 7,170
individuals (refer to Appendix for details). “Leader” is a critical factor closely intertwined with and exerting influence on organizational members and
goals. It encompasses the process through which leaders utilize their motivated employees and guide
them towards achieving organizational objectives [37]. Leadership embodies both the behaviours exhibite
by leaders and the personal characteristics that are perceived by organizational members as influencing
others [38]. In management, the selection of an appropriate leader can enhance communication and
interaction between leaders and employees, ultimately enhancing leadership effectiveness and
organizational performance. By aggregating relevant analyses from existing studies on leader
relationships, 23 studies were included in this meta-analysis, comprising a total sample size of 7,170
individuals (refer to Appendix for details). A random effects model was used to examine the overall correlation between leadership and employee
resilience, after taking into account factors such as publication bias, heterogeneity, and the treatment of
previous studies. The findings showed a correlation coefficient of 0.21, with a 95% confidence interval of
[0.079, 0.334]. As per established guidelines, correlation coefficients of r = 0.1, r = 0.2, and r = 0.3 indicate
low, moderate, and strong correlations, respectively. Therefore, it can be concluded that there is a moderate
positive correlation between leadership and employee resilience. Although individual studies showed
inconsistent correlation coefficients for different aspects of leadership and employee resilience, the overall
outcome of the 23 relevant studies indicated a moderate positive correlation. This research examines the
impact of different facets of leadership on employee resilience. The study highlights the importance of
considering other factors that may influence how leaders affect employee behaviour. Employee perception
can alter the mechanisms through which leadership operates, so it is important to include additional
influential factors when examining the relationship between leadership and employee resilience. 2.1.3 Discussion The meta-analysis revealed a significant relationship between leadership, interpersonal relationships at
work and employee resilience. Studies on employee resilience at work have mainly examined the effect of
leadership on resilience (e.g. paradoxical leadership [43]). However, studies explaining how leadership
affects employee resilience are not very consistent. On the other hand, limited research has examined the
influence of interpersonal relationships at work on employee resilience. These studies claim that workplace
relationships can serve as important sources of support that contribute to increased employee resilience. For example, factors such as support from others [35] and relationships with one's supervisor [44] have
been examined in this regard. Thus, it is crucial to consider the combination of leadership and
interpersonal relationships within organisations, with particular emphasis on the important role of LMX. By
focusing on the interplay between leadership and interpersonal relationships, we can further elucidate how
these dynamics influence employee resilience. The interpersonal dynamics within an organization play an essential role in shaping organizational
performance and behaviour. Employees who experience strong support and positive relationships exhibit
different attitudes and behaviours when faced with challenges [45, 46]. At the same time, the leader, as a
central contextual influence within the organization, has the power to profoundly affect the psychological
states of employees and is seen as an essential catalyst for fostering employee resilience [19]. Combined
with relational leadership theory, the quality of LMX determines the work experience of employees [47]. Compared to employee characteristics and personal perceptions, leader-member interactions have a more
pronounced effect on work outcomes. LMX is a critical construct for exploring these relationships. When
leaders and employees strive to cultivate better relationships, they foster positive perceptions of each other,
which in turn leads to mutually beneficial outcomes [48]. Therefore, exploring LMX, particularly within
traditional Chinese culture, helps to unravel central aspects of how leaders shape employee resilience and
shed light on the significant implications of leader influence in different cultural contexts. Study 1 found a positive relationship between leadership, interpersonal relationships at work, and
employee resilience. Few studies in the literature have focused on the critical dyadic relationships that are
pervasive in public sector organizations. Most of the limited current research has focused on business
organizations. Therefore, in order to test generalisability, this study was further explored using quantitative
research methods to investigate whether there is a positive relationship between LMX and employee
resilience in public sector organizations. 2.1.2 Interpersonal relationships and employ Interpersonal relationships enable individuals to access or exchange resources through social interactions,
which can help facilitate positive coping in the face of difficulty. Employees work in teams where frequent
interpersonal interactions play a crucial role and may influence work attitudes, behaviours, and well-being
[39]. Specifically, relational resources in the workplace include relationships with coworkers, customers,
leaders, etc. Conducted by aggregating the relevant analyses of existing interpersonal relationships, 13
relevant pieces of literature were included in the meta-analysis, with a total of 3,338 people (see Appendix). After accounting for publication bias, heterogeneity, and the treatment of previous studies, a random
effects model was used to explore the degree of overall correlation between interpersonal relationships at
work and employee resilience. The results showed a correlation coefficient of 0.36 and a 95% confidence
interval of [0.260,0.452] for the effect size. According to previous suggestions, r = 0.1, r = 0.2 and r = 0.3 are
weak, moderate, and strong correlations respectively. Therefore, it can be concluded that there is a strong
positive correlation between interpersonal relationships at work and employee resilience. This result is
consistent with the findings of most current studies. Interpersonal relationships at work create a sense of
community, trust, and shared values among employees. It is associated with intimacy, open
communication, access to information, and timely help [40]. Workplace relationships help individuals cope communication, access to information, and timely help [40]. Workplace relationships help individuals cope Page 6/26 Page 6/26 with stressful work situations by providing social and emotional support [41], and they play a role in
achieving positive organizational outcomes [42]. 2.2 Leader-Member Exchange(LMX) P
7/26
LMX was posited by Graen and Uhl-Bien (1995)[27] as an alternative to behavioural theories of leadership
that attempt to explain leadership as a function of personal characteristics, dispositions, and attitudes. LMX theory posits that the quality of leader–member relationships is instrumental in determining member Page 7/26 Page 7/26 behaviour in the workplace [49]. It differs between employees, and this variability may be responsible for
differences in member behaviours and work outcomes [50]. LMX is distinct in its usage of a dyadic level of
analysis that proposes that a leader shares differential, unique relationships with each member, and
workplace outcomes are determined by the quality of this relationship [50]. In contrast, traditional
leadership studies implicitly assume that the leader’s style is perceived similarly by different subordinates
and gives rise to similar behaviours across subordinates. However, LMX theory posits that “leadership” is
not a homogenous concept and that each subordinate may view the leader’s style differently [51]. In real
organizational situations, it is often difficult for employees to discern the “style” of the leader [50]. However,
subordinates experience a certain degree of comfort/discomfort with the leader, which informs them about
their relationship with the leader. LMX is a more exhaustive construct than leadership because it conceives
of leadership as a two-way relationship consisting of leader expectations and subordinate reciprocation
[52]. For example, Dulebohn et al. (2012) [51] posit that LMX is a better indicator of subordinate
performance than leadership, as the perception of leadership develops from the quality of the leader-
subordinate relationship. Employees do not learn and imitate leaders unconditionally. The process by
which different types of leadership come into play has an important relationship with employees’
perceptions of their working relationships with leaders at work. 2.3 LMX and Employee Resilience According to LMX theory, limited by resources and energy, leaders establish different relationships with
subordinates and adopt different management styles and strategies. The quality of LMX plays a
significant role in employee behaviours [28, 49]. A relationship that is high in trust, clarity, and reciprocity
provides a sense of comfort and is termed high quality. Relationships that are low in trust, transparency,
and exchange are termed low-quality LMX relationships [27]. With high-quality LMX, leaders can motivate subordinates and encourage positive evaluations of
situations. Such LMX, with high trust, reciprocity, and clarity of communication, makes subordinates
faithful to the mission and committed to the organization [57]. High-quality LMX encourages employees to
adopt thoughtful, creative, and adaptive solutions to stress rather than disorganized, defensive and
maladaptive keys [58]. It helps employees actively cope with changes and setbacks in the workplace,
promotes the growth and development of subordinates [59], and plays a positive role in the development
of employee resilience [58]. Therefore, we propose the following hypothesis: Hypothesis 1 LMX positively associated with employee resilience. 2.4 LMX and Employee Resilience via Stress Appraisals (Challenge and Hindrance) Lazarus & Folkman (1984)[29] define “stress” as a process initiated when the demands of the environment
impose or exceed the resources of the individual. Stress does not exist in the individual or the environment
alone but is a combination of the individual and the environment. Evaluating the individual and the
environment combines two critical evaluation processes: primary and secondary appraisal [53]. A
preliminary appraisal assigns meaning to any event or situation to determine whether it threatens oneself. Page 8/26 Secondary appraisal refers to what one can do and is how individuals assess internal and external coping
resources. Primary and secondary appraisals converge to determine whether a person’s interaction with the
environment is perceived as meaningful to the individual and, if so, whether it is primarily disruptive,
threatening, or challenging. The appraisal process is implicit in the challenge-hindrance stressor
framework. Validation studies have generally supported the assumption that challenge stressors are
appraised as challenging and hindrance stressors are appraised as hindering [54, 55, 56]. Therefore, while
challenge stressors refer to the presence of challenge demands, challenge appraisals refer to an
individual’s subjective interpretation that the demands have potential for personal gain, growth,
development, and well-being. Hindrance stressors refer to the presence of hindrance demands, while
hindrance appraisals refer to an individual’s subjective interpretation that the potential to result in personal
loss, constraints, or harm is demanded. Secondary appraisal refers to what one can do and is how individuals assess internal and external coping
resources. Primary and secondary appraisals converge to determine whether a person’s interaction with the
environment is perceived as meaningful to the individual and, if so, whether it is primarily disruptive,
threatening, or challenging. The appraisal process is implicit in the challenge-hindrance stressor
framework. Validation studies have generally supported the assumption that challenge stressors are
appraised as challenging and hindrance stressors are appraised as hindering [54, 55, 56]. Therefore, while
challenge stressors refer to the presence of challenge demands, challenge appraisals refer to an
individual’s subjective interpretation that the demands have potential for personal gain, growth,
development, and well-being. Hindrance stressors refer to the presence of hindrance demands, while
hindrance appraisals refer to an individual’s subjective interpretation that the potential to result in personal
loss, constraints, or harm is demanded. Lazarus’ (1966) [60] transactional theory of stress suggests that the environment influences and alters
individuals’ cognitive process information about objective stressors. 2.4 LMX and Employee Resilience via Stress Appraisals (Challenge and Hindrance) Integrating Lazarus’ arguments with
our theoretical foundation, we found that certain environmental factors may influence employees’
experience of stress in objective settings. Secondary appraisals of stressors refer to the process by which
individuals appraise stressors as the process by which individuals appraise internal and external coping
resources [29]. Thus, an individual’s perceived resources in an organization can influence the stress they
experience, which further influences employee attitudes and behaviours. In the workplace, leaders
determine the allocation of resources and serve as essential triggers for employee resilience [61]. LMX is
an important organizational context in an individual’s work life and reflects the support of the organization
and leaders for the individual [62]. High-quality LMX can buffer the drain on employee resources from
environmental stressors and help individuals focus on potential growth opportunities and positive stress
evaluations, resulting in positive behaviours [63]. Individuals who make different stress appraisals show
significant differences in attitudes and behaviours [64]. Specifically, the greater the quality of LMX is, the
more subordinates will have adequate resources to cope with the demands of the stressors [27], actively
assess internal and external coping resources in secondary appraisals, and produce challenging stress
appraisals while reducing hindering appraisals. Hypothesis a: LMX is positively associated with challenge appraisals. Hypothesis b: LMX is negatively associated with hindrance appraisals Hypothesis b: LMX is negatively associated with hindrance appraisals. Based on the COR theory, LMX can be considered an environmental condition, i.e., a critical contextual
factor that ensures the transmission of other resources [65]. Furthermore, in the framework of COR theory,
in addition to environmental resources, resources can be divided into three categories, such as objects,
individual characteristics, and energy, and the degree of matching of these four categories of resources
affects the accumulation status of resources [66]. According to the resource gain spiral path, employees
with larger support pools have more resources to invest and will focus on maintaining or expanding their
networks [31]. Well-supported employees are encouraged to demonstrate network-oriented resilience
behaviours (e.g., engaging in collaborative work, seeking support, etc.) and develop supportive networks to
meet challenges [13, 45]. Thus, the conditioned resource of high-quality LMX allows us to perceive more Page 9/26 Page 9/26 support from the organization, view difficulties as opportunities and challenges, and reduce our
perceptions of negative barriers, thus increasing employee resilience as an energy resource. High-quality LMX influences employees to make more challenging appraisals and enhances employee
initiative at work. Thus, when employees are faced with risks, challenges, or setbacks at work, they focus
on how to improve the organization's current situation to improve efficiency and solve problems,
proactively seek solutions, choose positive coping strategies, and work to change the current situation to
achieve their goals, increasing employee resilience in the process [13]. High-quality LMX also prevents
employees from making hindrance appraisals and decreases their withdrawal behaviour in the face of
difficulties, thus increasing employee resilience. High-quality LMX influences employees to make more challenging appraisals and enhances employee
initiative at work. Thus, when employees are faced with risks, challenges, or setbacks at work, they focus
on how to improve the organization's current situation to improve efficiency and solve problems,
proactively seek solutions, choose positive coping strategies, and work to change the current situation to
achieve their goals, increasing employee resilience in the process [13]. High-quality LMX also prevents
employees from making hindrance appraisals and decreases their withdrawal behaviour in the face of
difficulties, thus increasing employee resilience. High-quality LMX emphasizes that employees and leaders have close relationships, know each other well,
can identify each other’s strengths, and can build relatively strong relationships of trust based on emotions
and interests. They receive more frequent job coaching, training opportunities, and advancement
opportunities. 3 Method To evaluate the impact of LMX on employee resilience in public sector organizations and its mechanism, a
contextual experiment and questionnaire were designed and conducted. In Study 2, the questionnaire
method was used to examine the main effect between LMX and employee resilience, which initially
confirmed its validity in public sector organizations. Study 3 explored the underlying mechanisms through
a between-subjects contextual experiment. This experimental method enhances the internal validity of the
research model and complements the strengths of the survey research method. The experimental study
enhances the internal validity of the research model by providing more reliable inferences about the causal
relationships between the variables, particularly in clarifying the direction of causality and circumventing
the endogeneity problem of reciprocal causality (e.g., whether LMX and employee resilience are interactive
causalities). It also allows for repeated validation of the research model. An experimental study can
increase the reliability and persuasiveness of research findings from a survey study by testing their
applicability at a specific time and in a specific context. 3.1 Study 2: Questionnaire Study Hypothesis b: LMX is negatively associated with hindrance appraisals. This makes employees feel more resourceful as members of the leader's “inner circle”. Employees compare resources to stress when making stress appraisals. When resources are adequate,
employees believe that they can perform their jobs and will evaluate the organizational environment
positively, generating challenging stress appraisals. In addition, employees who make challenging
appraisals have more significant work initiative. When faced with risks, challenges, or setbacks at work,
they focus on how to improve the organization’s current situation to improve efficiency and solve problems,
proactively seek solutions, choose proactive coping strategies, and strive to change the status quo to
achieve goals, increasing employee resilience in the process [13]. Therefore, we hypothesized that LMX is
positively associated with challenge appraisals and negatively associated with hindrance appraisals. Low-quality LMX makes it difficult for employees to form an emotional connection with their leaders
beyond the subordinate relationship. Compared to the privileges of “insiders”, this group of staff believes
that they are being unfairly treated by the leadership and are unlikely to receive sufficient leadership
support, fair promotion opportunities, and a lack of interpersonal and material resources necessary to
solve problems. A low level of LMX is also detrimental. With low-quality LMX, more employees perceive
resource scarcity and risk, and they are more likely to make hindrance appraisals. Employees who make
hindrance appraisals are hindered in their pursuit of self-relevant goals, which may lead them to adopt
retreating behaviours and exhibit avoidance tendencies in the face of difficulties rather than proactively
seeking solutions [67], demonstrating lower employee resilience. Therefore, high-quality LMX can motivate employees to take a more positive attitude toward the
environment and stress in the organization. By increasing employees’ challenge appraisals and decreasing
employees’ hindrance appraisals, stress in the organization can be transformed into motivation, which can
enhance employees’ motivation to take initiative, maintain openness and self-confidence, and take a
positive approach to challenging difficulties and break through bottlenecks. Thus, we mobilize individual
and work resources in multiple ways so that employees can fully engage in their work and cope with the
organizational challenge. In other words, we hypothesize the following: Page 10/26 Hypothesis a: Challenge appraisals mediate the relationship between LMX and employee resilience. Hypothesis b: Hindrance appraisals mediate the relationship between LMX and employee resilience. The theoretical framework of this study is summarized in Fig. 1 Research Model. 3.1 Study 2: Questionnaire Study Measure Leader-member exchange (LMX) : LMX was measured using the seven-item LMX-7 scale developed by
Graen and Uhl-Bien (1995) [27]. Respondents rated the extent to which they considered the LMX score to
be an appropriate description of their work on a five-point scale (1 = strongly disagree, 5 = strongly agree). A sample item was “How would you characterize your working relationship with your leader?” The scale
had a Cronbach’s α of 0.87. Employee resilience: The researchers measured the construct using a 9-item scale developed by Näswall et
al. (2013) and Näswall et al. (2019) [6]. The scale consists of a 7-point rating system (ranging from 1 =
strongly disagree to 7 = strongly agree) and measures employee resilience based on the extent to which
respondents consider it an accurate description of their work. An example item is “I effectively collaborate
with others to handle challenges at work.” The scale demonstrated high internal consistency with a
Cronbach’s α of 0.92. Sample and Procedures The survey was conducted online using the platform wjx.cn. In Study 2, we distributed a questionnaire to
full-time staff members of public sector organizations enrolled in the MPA program at a university in
China. The questionnaire explained the study’s relevance and emphasized the respondents’ anonymity and
privacy. Respondents could discontinue answering questions at any point during the survey. The research
team's contact information was provided for any questions or concerns regarding the questionnaire. Respondents provided informed consent by initiating the questionnaire. To ensure method consistency
and improve data validity, LMX and employee resilience were measured at two different time points. Respondents’ personal information was collected during the first time point (T1), followed by the
measurement of LMX. Employee resilience was measured at the second time point (T2). The
respondents’ last four digits of student IDs were separated to ensure effective data matching for the
aforementioned variables. The survey received responses from 118 participants, with 112 full-time staff (95% response rate) included
in the analysis after listwise deletion. Of these valid samples, 59.8% were female and 64.3% were married. All participants held a bachelor’s degree. In terms of age, 60.7% of the participants were younger than 30 Page 11/26 Page 11/26 years, 32.1% were between 31 and 35 years, 6.3% were between 36 and 40 years, and only 0.9% were older
than 41 years. The tenure distribution was as follows: 33.0% had less than 5 years, 52.7% had 6 to 10
years, and 14.3% had 11 or more years. years, 32.1% were between 31 and 35 years, 6.3% were between 36 and 40 years, and only 0.9% were older
than 41 years. The tenure distribution was as follows: 33.0% had less than 5 years, 52.7% had 6 to 10
years, and 14.3% had 11 or more years. 3.1.2 Results Table 1 presents the means and standard deviations of the variables analysed in this study, along with
their correlations. The LMX was found to have a positive correlation with employee resilience (r = 0.18, p <
0.01). The demographic data collected indicated a positive association between gender and age with
tenure, and between job type and position level. However, none of the demographic variables showed any
correlation with LMX or employee resilience, and therefore were not considered as covariates. To test Hypothesis 1, a simple linear regression analysis was conducted. The analysis revealed a
statistically significant relationship between LMX and employee resilience (β=0.21, t = 2.15, R2=0.11, p <
0.05), thus supporting Hypothesis 1. Page 12/26 Page 12/26 Page 12/26 Page 12/26 Table 1 Descriptive Statistics and Correlations
Variable
Mean
SD
1
2
3
4
5
6
7
8
9
1.Gender
1.60
0.49
1
2.Age
1.47
0.66
0.175
1
3.Education
3.12
0.32
0.07
-0.01
1
4.Tenure
1.81
0.67
0.24*
0.70**
0.10
1
5.Job Type
1.66
0.88
0.12
0.17
0.01
0.04
1
6.Position
1.23
0.42
-0.11
0.06
-0.13
0.12
-0.20*
1
7.Marital
Status
1.70
0.52
0.012
0.29**
0.11
0.44**
-0.07
0.12
1
8.LMX
3.54
0.61
-0.14
-0.05
-0.07
-0.12
0.021
0.01
-0.06
1
9.Employee
Resilience
5.66
1.05
-0.04
0.13
-0.04
0.05
0.123
0.03
-0.10
0.18*
1
Note. N = 112. * p < 0.05; ** p <0 .01. Gender:1=male, 2=famale; Age: 1=30 years or below, 2=31-35
years, 3=36-40 years, 4= 41 years or above; Education:1=High school or below, 2=junior college,
3=Bachelor degree, 4=Master degree or above; Job Type:1= general management, 2=
administrative law enforcement, 3= professional technology; Position:1= No managerial
responsibility, 2= grass-roots cadres, 3= middle-level cadres, 4= higher-level cadres; Marital
Status:1= unmarried, 2= married, 3= other. Table 1 Descriptive Statistics and Correlations Participants and Procedures The study included 472 participants who were full-time staff working in public sector organizations in
China. The participants were selected using the exponential, non-discriminative ‘snowball
sampling’ method, which included MPA students enrolled in a part-time MPA Program at a large university
in the southwestern region of China, as well as other working samples. After list-wise deletion, the effective
sample consisted of 448 full-time staff, resulting in a 94.9% response rate. Of these valid samples, 55.9%
were female, and the mean age was 30.6 years (SD=3.2). Scenarios and experimental manipulations Scenarios were created to manipulate LMX quality to resemble encounters in real workplace contexts. The
initial versions of these scenarios underwent pre-testing for realism and clarity by experts, Ph.D. students,
and full-time staff of public sector organizations to validate the textual material. Minor revisions were Page 13/26 made based on their feedback. LMX was manipulated in the scenarios, resulting in a between-subjects
design. Participants were instructed to imagine themselves in the described situation and make their own
decisions. The material provides information about LMX quality in two conditions: low-quality LMX and high-quality
LMX. In the low-quality LMX condition, the working relationship between you and the leader is limited. In
daily work, the leader only provides instructions and refrains from making subjective evaluations. This
approach may limit their understanding of your work potential and disregard your obstacles at work and
urgent needs in life. In a high-quality LMX condition, you feel that your leader is entirely satisfied with your
work and provides valuable information that other colleagues may not know. This fosters a closer
relationship between you and your leader compared to your colleagues. Additionally, the leader provides
support outside of work and assists in problem-solving, even with personal issues. Measures Challenge Appraisals. Three items from the Employee Stress Scale developed by LePine et al. (2016) [64]
were used to measure challenge appraisals. The items were rated on a Likert scale of 1 (strongly disagree)
to 5 (strongly agree). Such as “Working on your job requirements contributes to your personal growth and
well-being”. The Cronbach’s α was 0.91. Hindrance Appraisals. To measure employees’ hindrance appraisals, we utilised three items from LePine et
al. (2016) Employee Stress Scale [64]. The items were rated on a Likert scale ranging from 1 (strongly
disagree) to 5 (strongly agree). Such as “Your job requirements limit your ability to achieve your personal
goals and development”. The Cronbach’s α was 0.94. Employee Resilience. We measured employee resilience using the Employee Resilience Scale developed by
Näswall et al. (2019) [6]. The scale comprises nine questions that were rated on a Likert Scale ranging
from 1 (strongly disagree) to 7 (strongly agree). Such as “You work effectively with others to solve work
challenges” . The Cronbach’s alpha was was 0.95. Attention test. One general question was designed to test whether respondents knew the information and
were careful to fill in the answers in the context. Specifically, participants were asked to answer a
conventional attention test entry, such as “Please select strongly agree for this option” . Descriptive Statistical Analysis Table 2 provides descriptive statistical results for each research variable. Page 14/26 Page 14/26 Table 2 Descriptive statistical analysis for each research variable. Gender
Age
CA
HA
ER
Total
Mean
0.43
30.55
3.26
2.70
4.87
SD
0.50
3.30
1.07
1.03
1.30
Scenario 1
(N=227)
Mean
0.43
30.56
2.63
3.16
4.15
SD
0.50
3.28
0.85
0.97
1.23
Scenario 2
(N=221)
Mean
0.43
30.54
3.91
2.12
5.60
SD
0.50
3.31
0.86
0.81
0.91
Note: CA = Challenge Appraisals; HA = Hindrance Appraisals; ER = Employee Resilience Table 2 Descriptive statistical analysis for each research variable. To test the hypotheses of the experimental study, ANOVA was used to examine the main effects of
challenge appraisals, hindrance appraisals, and employee resilience, with LMX quality (high vs. low) as the
independent variable. The study also tested the mediating effects, with challenge appraisals, hindrance
appraisals, and employee resilience as dependent variables. Analysis of Variance The results of the analysis of variance (ANOVA) indicate a significant main effect of LMX on employee
resilience (F(1,448) = 199.56, p < 0.001, partial η2 = 0.31). Specifically, employees with a high level of LMX
demonstrated significantly greater resilience (M = 5.60, SD = 0.91) than those with a low level of LMX (M =
4.15, SD = 1.23). Additionally, there was a significant main effect of LMX on challenge appraisals (F(1,448)
= 251.41, p < 0.001, partial η2 = 0.36). The study discovered that high-quality LMX was linked to
significantly greater challenge appraisals (M = 3.91, SD = 0.86) in comparison to low-quality LMX (M =
2.63, SD = 0.85). A significant main effect of LMX quality on hindrance appraisals was observed, F(1, 448)
= 154.01, p < 0.001, partial η2 = 0.26. Hypotheses 1 and 2 were supported. Regression Analysis To test hypothesis 3, we used the bootstrapping method (bootstrap sample=5000) recommended by
Preacher et al. (2007) to examine the indirect effects of LMX on employee resilience through challenge
appraisals and hindrance appraisals. The results, shown in Figure 2 and Table 3, indicate that LMX
significantly affects employee resilience through both challenge and hindrance appraisals. Therefore,
hypothesis 3 was supported. Page 15/26 Table 3 Pathway and effect decomposition of LMX impact ER 4 Di
i
Table 3 Pathway and effect decomposition of LMX impact ER
Path
Effect
Boot SE
95% Boot
LLCI
ULCI
1.Total effect of LMX on ER
0.54
0.04
0.46
0.62
2.Direct effect of LMX ON ER
0.41
0.05
0.31
0.51
3.Indirect effect(s) of LMX ON ER
Total
0.77
0.06
0.66
0.89
CA
0.61
0.07
0.48
0.76
HA
0.16
0.06
0.05
0.28
CA minus HA
0.46
0.11
0.22
0.68
Note: LMX = Leader-Member Exchange; CA = Challenge Appraisals; HA = Hindrance Appraisals; ER =
Employee Resilience Note: LMX = Leader-Member Exchange; CA = Challenge Appraisals; HA = Hindrance Appraisals; ER =
Employee Resilience 4 Discussion
Note: LMX = Leader-Member Exchange; CA = Challenge Appraisals; HA = Hindrance Appraisals; ER =
Employee Resilience 4 Discussion The questionnaire results showed a significant positive correlation between LMX and employee resilience
in public sector organizations in China. Public sector organizations in China are characterized by
hierarchical rankings, positional disparities, and role obligations [68]. Society is known for its 'relationship
orientation' and 'authority orientation' [20]. Examining the relationship between LMX and employee
resilience in public sector organizations can enhance overall organizational resilience at both the
leadership and workplace levels. This can enable public sector organizations to effectively navigate
challenges. The results support the hypotheses of previous studies and align with the findings from the
meta-analysis conducted in Study 1. Meng et al. (2019) suggested that a healthy supervisor-subordinate
relationship can empower employees to handle demanding responsibilities, leading to organizational
success. The nature of the LMX has been found to influence the appraisal process (28). Therefore, leaders
should frame goals in a way that fosters subordinates' positive view of the situation, generating
psychological engagement with the job and the organization. Therefore, individuals invest their time and
energy in understanding a problem and typically view it as a challenge rather than a threat [9]. LMX also
impacts employee resilience by influencing their perception of stressors to some extent, which requires
further investigation. Study 3 used a contextual simulation approach based on COR theory to examine the effect of LMX on
employee resilience and the mediating role of stress appraisals. The results indicated a positive
association between LMX and employee resilience. This finding is consistent with Kakkars’ (2019)
research, which indicates that LMX has a more positive impact on employee attitudes and behaviours than
studies on leadership style and workplace interpersonal relationships in terms of employee resilience [28]. In the context of Chinese public sector organizations, employees who perceive a higher LMX can
effectively reduce power distance. This leads to increased communication and exchange between leaders
and employees, ultimately enhancing employee resilience. Additionally, the effect of LMX on employee
resilience is mediated by stress appraisal. Employees may perceive LMX as a challenge, which plays a Page 16/26 Page 16/26 crucial role in demonstrating their resilience. High-quality LMX provides individuals with additional
resources and attention, enabling them to perceive it as a challenging opportunity for growth, development,
and happiness. Thus, when employees encounter risks, challenges, or setbacks at work, they concentrate
on improving the organization's efficiency and problem-solving. 4 Discussion They proactively seek solutions, choose
positive coping strategies, and work to change the status quo to achieve their own goals, thereby
increasing their resilience. This study has significant theoretical and practical implications. The study contributes to the literature on
interpersonal relationships and leadership in the workplace by clarifying the specific dimensions of leader
relationships that influence employee resilience. Previous research has predominantly focused on the
relationship between leadership style and employee resilience [19, 68], with less emphasis on the positive
effects of LMX on employee resilience [28]. This study suggests that the nature of the relationship between
leaders and subordinates, as opposed to simple leadership styles and interpersonal relationships, plays an
essential role in developing employee resilience [28]. Especially in China, “relationships” are the linchpin of
interpersonal harmony, and employees in organizations not only want to be satisfied at the material level
but also need to be satisfied at the social and spiritual levels. We need to pay attention to grassroots
officials as the source of employee resilience. In the practice of human resource management in public
sector organizations, departmental leaders should pay attention to communication with subordinates,
establish good relationships between subordinates and subordinates, cultivate a higher level of care and
trust between leaders and subordinates, and increase the frequency of communication between leaders
and subordinates. Another crucial contribution of this study is identifying stress appraisals as mediating mechanisms that
promote resilience, thus enriching the literature on resilience and stress appraisals. Until now, resilience has
mainly been associated with positive emotion regulation and focus [28]. However, this study highlights the
significant influence of stress appraisals, which include emotional perceptions, on employee resilience. The perceived gains and losses of resources by employees can influence their stress appraisals, which in
turn affects their ability to cope with distress, also known as employee resilience. Cognitive appraisals play
a critical mediating role in the relationship between LMX and employee resilience. This finding suggests
that LMX has dual effects on the process of cultivating employee resilience. On one hand, it directly
influences individual employee resilience through its enhancing or diminishing effects. However, an
individual's resilience can be affected by their perception of the situation. LMX is a resource that can lead
to an overestimation of an individual's ability to handle situational demands, resulting in a challenging
assessment. 4 Discussion This adaptive perception further enhances the sense of self-control, leading individuals to
adopt positive coping strategies and achieve positive task completion. Ultimately, this can increase
employee resilience in the face of adversity. Conversely, low LMX triggers an underestimation of personal
resources, leading to appraisals of inadequacy in meeting situational demands. This tendency to
maladapt reduces the sense of self-control, generates negative coping styles and reduces the resilience of
employees. Consequently, in practice, individuals with limited resources should actively reorient their
perceptions towards challenges, as this reorientation serves to enhance resilience in stress-coping
scenarios. Page 17/26 Limitations and future directions In addition, there are certain limitations in this study. First, despite efforts to control variables and
processes in contextual simulation experiments, there are inherent limitations in simulating real-world LMX
situations. Contextual simulations serve as scientific abstractions, deliberately aiming to isolate and
control the influence of other factors for research purposes. Therefore, it is essential to supplement and
validate the experimental findings with field studies or alternative methods to establish greater consistency
between the results obtained in the experimental context and real-world outcomes. Second, this study did
not consider the potential impact of certain variables beyond cognitive appraisals on the research findings. There may be additional mediating variables and moderating effects in the relationship between LMX
effectiveness and employee resilience. Thus, future studies should delve more deeply into the mechanisms
through which LMX influences employee resilience and incorporate factors such as employee personality
traits and organizational culture. This will enable a more comprehensive investigation of the nuanced
relationships between LMX and employee resilience. 5 Conclusion Based on the COR theory, the present research explores the relationship between LMX and employee
resilience in public sector organizations in China Mainland. It investigates the importance of LMX as a key
factor in developing resilience using questionnaires and experimental studies. The research also highlights
the crucial mediating role of stress appraisals, including both challenges and hindrances. Ethics approval and consent to participate Participants were informed about the purpose, procedures, potential risks, and benefits of the study prior to
their participation. They were made aware that their participation was voluntary and that they had the right
to withdraw their consent at any time without any negative consequences. Additionally, participants were
assured of anonymity and confidentiality throughout the study and that their data would be used solely for
research purposes. Participants were asked any questions or concerns that were addressed by the
researchers before providing informed consent. The Ethics Committee of the Department of Psychology at
Southwest University approved this study (H23223), and the study was conducted according to the
Declaration of Helsinki. Informed consent was obtained from the participants. Acknowledgements Not applicable Competing interests The authors declare that they have no competing interests. Authors’ contributions YY wrote the manuscript and analysed the data under the guidance of ZYH. ZYH contributed to the study
design, data collection and critical revisions. All authors contributed to the article and approved
the submitted version. Consent for publication Not applicable Availability of data and materials Page 18/26 The data were not available due to privacy or ethical restrictions. The data that support the findings of this
study are available upon request from the corresponding author. Funding The National Natural Science Foundation of China(No. 72374174). References 1. Linnenluecke M K. Resilience in business and management research: A review of influential
publications and a research agenda. International Journal of Management Reviews. 2017; 19(1): 4-30. https://doi.org/10.1111/ijmr.12076 1. Linnenluecke M K. Resilience in business and management research: A review of influential
publications and a research agenda. International Journal of Management Reviews. 2017; 19(1): 4-30. https://doi.org/10.1111/ijmr.12076 2. Saragih Y M, Medaline O. Elements of the corruption crime (element analysis of authority abuse and
self-enrich and corporations in Indonesia). IOP Conference Series: Earth and Environmental Science. 2018; 126(1): 012108. https://doi.org/012108. 10.1088/1755-1315/126/1/012108 3. Sjöblom S, Godenhjelm S. Project proliferation and governance-implications for environmental
management. Journal of Environmental Policy & Planning. 2009; 11(3): 169-185. https://doi.org/10.1080/15239080903033762 4. King D D, Newman A, Luthans F. Not if, but when we need resilience in the workplace. Journal of
Organizational Behavior. 2016; 37(5): 782-786. https://doi.org/10.1002/job.2063 5. Kuntz J R C, Näswall K, Malinen S. Resilient employees in resilient organizations: Flourishing beyond
adversity. Industrial and Organizational Psychology. 2016; 9(2): 456-462. https://doi.org/10.1017/iop.2016.39 5. Kuntz J R C, Näswall K, Malinen S. Resilient employees in resilient organizations: Flourishing beyond
adversity. Industrial and Organizational Psychology. 2016; 9(2): 456-462. https://doi.org/10.1017/iop.2016.39 6. Näswall K, Malinen S, Kuntz J, Hodliffe M. Employee resilience: development and validation of a
measure. Journal of managerial psychology. 2019; 34(5): 353-367. https://doi.org/10.1108/JMP-02-
2018-0102 6. Näswall K, Malinen S, Kuntz J, Hodliffe M. Employee resilience: development and validation of a
measure. Journal of managerial psychology. 2019; 34(5): 353-367. https://doi.org/10.1108/JMP-02-
2018-0102 7. Bardoel E A, Pettit T M, De Cieri H, McMillan L. Employee resilience: An emerging challenge for HRM. Asia Pacific Journal of Human Resources. 204; 52(3): 279-297. https://doi.org/10.1111/1744- Page 19/26 Page 19/26 7941.12033 8. Britt T W, Shen W, Sinclair R R, Grossman M R, Klieger D M. How much do we really know about
employee resilience?. Industrial and Organizational Psychology. 2016; 9(2), 378-404. https://doi.org/10.1017/iop.2015.107 9. Youssef C M, Luthans F. Positive organizational behavior in the workplace: The impact of hope,
optimism, and resilience. Journal of management. 2007; 33(5): 774-800. https://doi.org/10.1177/0149206307305562 10. De Clercq D. Getting creative with resources: How resilience, task interdependence, and emotion
sharing mitigate the damage of employee role ambiguity. The Journal of Applied Behavioral Science. 2019; 55(3): 369-391. https://doi.org/10.1177/0021886319853803 11. Bani-Melhem S, Quratulain S, Al-Hawari M A. Does employee resilience exacerbate the effects of
abusive supervision? A study of frontline employees’ self-esteem, turnover intention, and innovative
behaviors. Journal of Hospitality Marketing & Management. 2021; 30(5): 611-629. https://doi.org/10.1080/19368623.2021.1860850 12. Gonçalves L, Sala R, Navarro J B. Resilience and occupational health of health care workers: a
moderator analysis of organizational resilience and sociodemographic attributes. International
Archives of Occupational and Environmental Health. 2022; 95(1): 223-232. https://doi.org/10.1007/s00420-021-01725-8 13. Kuntz J R, Malinen S, Näswall K. Employee resilience: Directions for resilience development. Consulting Psychology Journal: Practice and Research. 2017; 69(3): 223-242. https://doi.org/10.1037/cpb0000097 14. Charoensap-Kelly P, Sheldon P, Antony M G, Provenzani L. Resilience, well-being, and organizational
outcomes of Croatian, Thai, and US workers during COVID-19. Journal of Management &
Organization. 2021; 27(6): 1083-1111. https://doi.org/10.1017/jmo.2021.58 15. Biggs D. Understanding resilience in a vulnerable industry: the case of reef tourism in Australia. Ecology and society. 2011; 16(1): 30. https://www.jstor.org/stable/26268845 16. Tonkin K, Malinen S, Näswall K, Kuntz J C. Building employee resilience through well-being in
organizations. Human Resource Development Quarterly. 2018; 29(2): 107-124. https://doi.org/10.1002/hrdq.21306 17. Zhang W, Chen H. The structure and measurement of the work values of Chinese civil servants: The
case of Hangzhou city government. Public Personnel Management. 2015; 44(4): 559-576. https://doi.org/10.1177/0091026015607107 18. Vigoda-Gadot E, Eldor L, Schohat L M. Engage them to public service: Conceptualization and empirica
examination of employee engagement in public administration. The American Review of Public
Administration. 2013; 43(5), 518-538. https://doi.org/10.1177/0275074012450943 18. Vigoda-Gadot E, Eldor L, Schohat L M. Engage them to public service: Conceptualization and empirical
examination of employee engagement in public administration. The American Review of Public
Administration. 2013; 43(5), 518-538. https://doi.org/10.1177/0275074012450943 19. Nguyen Q, Kuntz J, Näswall K, Malinen S. Employee resilience and leadership styles: the moderating
role of proactive personality and optimism. New Zealand Journal of Psychology (Online). 7941.12033 2016; 45: Page 20/26 13-21.Retrieved from https://www.proquest.com/scholarly-journals/employee-resilience-leadership-
styles-moderating/ docview/1857281132/se-2 20. Luo Z, Wang Y, Marnburg E, Øgaard T. How is leadership related to employee self-concept?. International Journal of Hospitality Management. 2016; 52: 24-32. https://doi.org/10.1016/j.ijhm.2015.09.003 21. Cartwright D. Influence, leadership, control. In J G March (Ed.), Handbook of organizations (pp. 1-47). Chicago: Rand McNally, 1965. 22. Hwang K K. Chinese relationalism: Theoretical construction and methodological considerations. Journal for the Theory of Social Behaviour. 2000; 30(2): 155-178. https://doi.org/10.1111/1468-
5914.00124 23. Chen N Y F, Tjosvold D. Guanxi and leader member relationships between American managers and
Chinese employees: Open-minded dialogue as mediator. Asia Pacific Journal of Management. 2007;
24(2): 171-189. https://doi.org/10.1007/s10490-006-9029-9 23. Chen N Y F, Tjosvold D. Guanxi and leader member relationships between American managers and
Chinese employees: Open-minded dialogue as mediator. Asia Pacific Journal of Management. 2007;
24(2): 171-189. https://doi.org/10.1007/s10490-006-9029-9 24. Hsu C H, Huang S S. Reconfiguring Chinese cultural values and their tourism implications. Tourism
Management. 2016; 54: 230-242. https://doi.org/10.1016/j.tourman.2015.11.011 24. Hsu C H, Huang S S. Reconfiguring Chinese cultural values and their tourism implications. Tourism
Management. 2016; 54: 230-242. https://doi.org/10.1016/j.tourman.2015.11.011 25. Sun L J. The Deep Structure of Chinese Culture. Guilin: Guang Xi Normal University Press, 2004. 25. Sun L J. The Deep Structure of Chinese Culture. Guilin: Guang Xi Normal University Press, 2004. 25. Sun L J. The Deep Structure of Chinese Culture. Guilin: Guang Xi Normal 26. Nie D, Lämsä A M. The leader–member exchange theory in the Chinese context and the ethical
challenge of guanxi. Journal of Business Ethics. 2015; 128: 851-861. https://doi.org/10.1007/s10551-
013-1983-9 27. Graen G B, Uhl-Bien M. Relationship-based approach to leadership: Development of leader-member
exchange (LMX) theory of leadership over 25 years: Applying a multi-level multi-domain perspective. The leadership quarterly. 1995; 6(2): 219-247. https://doi.org/10.1016/1048-9843(95)90036-5 27. Graen G B, Uhl-Bien M. Relationship-based approach to leadership: Development of leader-member
exchange (LMX) theory of leadership over 25 years: Applying a multi-level multi-domain perspective. The leadership quarterly. 1995; 6(2): 219-247. https://doi.org/10.1016/1048-9843(95)90036-5 28. Kakkar S. Leader-member exchange and employee resilience: the mediating role of regulatory focus. Management Research Review. 2019; 42(9): 1062-1075. https://doi.org/10.1108/MRR-03-2018-0116 28. Kakkar S. Leader-member exchange and employee resilience: the mediating role of regulatory focus. Management Research Review. 2019; 42(9): 1062-1075. https://doi.org/10.1108/MRR-03-2018-0116 29. Lazarus R S, Folkman S. Stress, appraisal, and coping. Springer, New York 30. Mukerjee J, Montani F, Vandenberghe C. 7941.12033 Public management in time: a longitudinal examination of the full range of leadership
theory. Journal of Public Administration Research & Theory. 2014; (2): 407-429. https://doi.org/10.1093/jopart/mus060 39. Meng H, Luo Y, Huang L, Wen J, Ma J, Xi J. On the relationships of resilience with organizational
commitment and burnout: a social exchange perspective. The International Journal of Human
Resource Management. 2019; 30(15): 2231-2250. https://doi.org/10.1080/09585192.2017.1381136 40. Sias P M, Cahill D J. From coworkers to friends: The development of peer friendships in the workplace. Western Journal of Communication (includes Communication Reports). 1998; 62(3): 273-299. https://doi.org/10.1080/10570319809374611 41. Sias P M, Gallagher E B, Kopaneva I, Pedersen H. Maintaining workplace friendships: Perceived
politeness and predictors of maintenance tactic choice. Communication Research. 2012; 39(2): 239-
268. https://doi.org/10.1177/0093650210396869 41. Sias P M, Gallagher E B, Kopaneva I, Pedersen H. Maintaining workplace friendships: Perceived
politeness and predictors of maintenance tactic choice. Communication Research. 2012; 39(2): 239-
268. https://doi.org/10.1177/0093650210396869 42. Rai A, Agarwal U A. Workplace bullying and employee silence: A moderated mediation model of
psychological contract violation and workplace friendship. Personnel Review. 2018; 47(1): 226-256. https://doi.org/10.1108/PR-03-2017-0071 42. Rai A, Agarwal U A. Workplace bullying and employee silence: A moderated mediation model of
psychological contract violation and workplace friendship. Personnel Review. 2018; 47(1): 226-256. https://doi.org/10.1108/PR-03-2017-0071 43. Franken E, Plimmer G, Malinen S. Paradoxical leadership in public sector organisations: Its role in
fostering employee resilience. Australian Journal of Public Administration. 2020; 79(1): 93-110. https://doi.org/10.1111/1467-8500.12396 43. Franken E, Plimmer G, Malinen S. Paradoxical leadership in public sector organisations: Its role in
fostering employee resilience. Australian Journal of Public Administration. 2020; 79(1): 93-110. https://doi.org/10.1111/1467-8500.12396 44. Peters J, Pearce J. Relationships and early career teacher resilience: a role for school principals. Teachers and Teaching. 2012; 18(2): 249-262. https://doi.org/10.1080/13540602.2012.632266 44. Peters J, Pearce J. Relationships and early career teacher resilience: a role for school principals. Teachers and Teaching. 2012; 18(2): 249-262. https://doi.org/10.1080/13540602.2012.632266 45. Nilakant V, Walker B, Kuntz J, de Vries H, Malinen S, Näswall K, van Heugten S. Dynamics of
Organizational Response to a Disaster: A Study of Organizations Impacted by Earthquakes. In C. Michael Hall, Sanna Malinen, Russell Wordsworth and Rob Vosslamber (Eds), Business and Post-
Disaster Management: Business, organizational and consumer resilience and the Christchurch
Earthquakes. Routledge, 2016; 35-48. 45. Nilakant V, Walker B, Kuntz J, de Vries H, Malinen S, Näswall K, van Heugten S. Dynamics of
Organizational Response to a Disaster: A Study of Organizations Impacted by Earthquakes. 7941.12033 A dual model of coping with and commitment to
organizational change: the role of appraisals and resources. Journal of Organizational Change
Management. 2021; 34(5): 1144-1161. https://doi.org/10.1108/JOCM-01-2021-0027 30. Mukerjee J, Montani F, Vandenberghe C. A dual model of coping with and commitment to
organizational change: the role of appraisals and resources. Journal of Organizational Change
Management. 2021; 34(5): 1144-1161. https://doi.org/10.1108/JOCM-01-2021-0027 31. Halbesleben J R, Neveu J P, Paustian-Underdahl S C, Westman M. Getting to the “COR” understanding
the role of resources in conservation of resources theory. Journal of management. 2014; 40(5): 1334-
1364. https://doi.org/10.1177/0149206314527130 31. Halbesleben J R, Neveu J P, Paustian-Underdahl S C, Westman M. Getting to the “COR” understanding
the role of resources in conservation of resources theory. Journal of management. 2014; 40(5): 1334-
1364. https://doi.org/10.1177/0149206314527130 32. Halbesleben J R, Wheeler A R. To invest or not? The role of coworker support and trust in daily
reciprocal gain spirals of helping behavior. Journal of Management. 2015; 41(6): 1628-1650. https://doi.org/10.1177/0149206312455246 32. Halbesleben J R, Wheeler A R. To invest or not? The role of coworker support and trust in daily
reciprocal gain spirals of helping behavior. Journal of Management. 2015; 41(6): 1628-1650. https://doi.org/10.1177/0149206312455246 33. Cohen S, Wills T A. Stress, social support, and the buffering hypothesis. Psychological bulletin. 1985;
98(2): 310. https://doi.org/10.1037/0033-2909.98.2.310 33. Cohen S, Wills T A. Stress, social support, and the buffering hypothesis. Psychological bulletin. 1985;
98(2): 310. https://doi.org/10.1037/0033-2909.98.2.310 34. Erdogan B, Enders J. Support from the top: supervisors’ perceived organizational support as a
moderator of leader-member exchange to satisfaction and performance relationships. Journal of 34. Erdogan B, Enders J. Support from the top: supervisors’ perceived organizational support as a
moderator of leader-member exchange to satisfaction and performance relationships. Journal of Page 21/26 Page 21/26 applied psychology. 2007; 92(2): 321. https://doi.org/10.1037/0021-9010.92.2.321 35. Meneghel I, Salanova M, Martínez I M. Feeling good makes us stronger: How team resilience mediates
the effect of positive emotions on team performance. Journal of Happiness Studies. 2016; 17, 239-
255. https://doi.org/10.1007/s10902-014-9592-6 36. Caniëls M C, Baaten S M. How a learning-oriented organizational climate is linked to different
proactive behaviors: The role of employee resilience. Social Indicators Research. 2019; 143: 561-577. https://doi.org/10.1007/s11205-018-1996-y 37. Bass B M, Avolio B J, Dong I J, Berson Y. Predicting unit performance by assessing transformational
and transactional leadership. Journal of Applied Psychology. 2003; 88(2), 207-218. https://doi.org/10.1037/0021-9010.88.2.207 38. Oberfield Z W. 7941.12033 In C. Michael Hall, Sanna Malinen, Russell Wordsworth and Rob Vosslamber (Eds), Business and Post-
Disaster Management: Business, organizational and consumer resilience and the Christchurch
Earthquakes. Routledge, 2016; 35-48. 46. Kuntz J, Connell P, Näswall K. Workplace resources and employee resilience: the role of regulatory
profiles. Career Development International. 2017; 22(4): 419-435. https://doi.org/10.1108/CDI-11-
2016-0208 46. Kuntz J, Connell P, Näswall K. Workplace resources and employee resilience: the role of regulatory
profiles. Career Development International. 2017; 22(4): 419-435. https://doi.org/10.1108/CDI-11-
2016-0208 Page 22/26 47. Brower H H, Schoorman F D, Tan H H. A model of relational leadership: The integration of trust and
leader–member exchange. The leadership quarterly. 2000; 11(2): 227-250. https://doi.org/10.1016/S1048-9843(00)00040-0 48. Cogliser C C, Schriesheim C A, Scandura T A, Gardner W L. Balance in leader and follower perceptions
of leader–member exchange: Relationships with performance and work attitudes. The Leadership
Quarterly. 2009; 20(3): 452-465. https://doi.org/10.1016/j.leaqua.2009.03.010 49. Cropanzano R, Dasborough M T, Weiss H M. Affective events and the development of leader-member
exchange. Academy of Management Review. 2017; 42(2): 233-258. https://doi.org/10.5465/amr.2014.0384 50. Martin R, Guillaume Y, Thomas G, Lee A, Epitropaki O. Leader–member exchange (LMX) and
performance: A meta‐analytic review. Personnel psychology. 2016; 69(1): 67-121. https://doi.org/10.1111/peps.12100 51. Dulebohn J H, Bommer W H, Liden R C, Brouer R L, Ferris G R. A meta-analysis of antecedents and
consequences of leader-member exchange: Integrating the past with an eye toward the future. Journal
of management. 2012; 38(6): 1715-1759. https://doi.org/10.1177/0149206311415280 52. Sosik J J, Godshalk V M, Yammarino F J. Transformational leadership, learning goal orientation, and
expectations for career success in mentor–protégé relationships: A multiple levels of analysis
perspective. The Leadership Quarterly. 2004; 15(2): 241-261. https://doi.org/10.1016/j.leaqua.2004.02.003 53. Folkman S, Lazarus R S, Gruen R J, DeLongis A. Appraisal, coping, health status, and psychological
symptoms. Journal of personality and social psychology, 1986; 50(3): 571. https://doi.org/10.1037/0022-3514.50.3.571 54. Cavanaugh M A, Boswell W R, Roehling M V, Boudreau J W. An empirical examination of self-reported
work stress among US managers. Journal of applied psychology. 2000; 85(1): 65. https://doi.org/10.1037/0021-9010.85.1.65 55. LePine J A, Podsakoff N P, LePine M A. A meta-analytic test of the challenge stressor–hindrance
stressor framework: An explanation for inconsistent relationships among stressors and performance. Academy of management journal. 2005; 48(5): 764-775. https://doi.org/10.5465/amj.2005.18803921 56. Rodell J B, Judge T A. Can “good” stressors spark “bad” behaviors? The mediating role of emotions in
links of challenge and hindrance stressors with citizenship and counterproductive behaviors. 7941.12033 Journal
of Applied Psychology. 2009; 94(6): 1438. https://doi.org/10.1037/a0016752 57. Kuvaas B, Buch R. Leader‐member exchange relationships and follower outcomes: The mediating role
of perceiving goals as invariable. Human Resource Management. 2018; 57(1): 235-248. https://doi.org/10.1002/hrm.21826 57. Kuvaas B, Buch R. Leader‐member exchange relationships and follower outcomes: The mediating role
of perceiving goals as invariable. Human Resource Management. 2018; 57(1): 235-248. https://doi.org/10.1002/hrm.21826 58. Caniëls M C J, Hatak I. Employee resilience: considering both the social side and the economic side of
leader-follower exchanges in conjunction with the dark side of followers’ personality. The International
Journal of Human Resource Management. 2019; 3(5): 1-32. https://doi.org/10.1080/09585192.2019.1695648 58. Caniëls M C J, Hatak I. Employee resilience: considering both the social side and the economic side of
leader-follower exchanges in conjunction with the dark side of followers’ personality. The International
Journal of Human Resource Management. 2019; 3(5): 1-32. https://doi.org/10.1080/09585192.2019.1695648 Page 23/26 59. Yukl G, O'Donnell M, Taber T. Influence of leader behaviors on the leader‐member exchange
relationship. Journal of managerial psychology. 2009; 24(4): 289-299. https://doi.org/10.1108/02683940910952697 60. Lazarus R S. Psychological stress and the coping process. NY: McGraw-Hill, 1996. 61. Zhu Y H, Zhang S, Shen Y. Humble leadership and employee resilience: exploring the mediating
mechanism of work-related promotion focus and perceived insider identity. Frontiers in Psychology. 2019; 10: 673. https://doi.org/10.3389/fpsyg.2019.00673 62. Erdogan B, Kraimer M L, Liden R C. Work value congruence and intrinsic career success: The
compensatory roles of leader‐member exchange and perceived organizational support. Personnel
psychology. 2004; 57(2): 305-332. https://doi.org/10.1111/j.1744-6570.2004.tb02493.x 63. Lian H, Ferris D L, Brown D J. Does power distance exacerbate or mitigate the effects of abusive
supervision? It depends on the outcome. Journal of applied psychology. 2012; 97(1): 107. https://doi.org/10.1037/a0024610 64. LePine M A, Zhang Y, Crawford E R, Rich B L. Turning their pain to gain: Charismatic leader influence
on follower stress appraisal and job performance. Academy of management journal. 2016; 59(3):
1036-1059. https://doi.org/10.5465/amj.2013.0778 65. Hobfoll S E. Conservation of resource caravans and engaged settings. Journal of occupational and
organizational psychology. 2011; 84(1): 116-122. https://doi.org/10.1111/j.2044-8325.2010.02016.x 66. Penney L M, Hunter E M, Perry S J. Personality and counterproductive work behaviour: Using
conservation of resources theory to narrow the profile of deviant employees. Journal of Occupational
and Organizational Psychology. 2011; 84(1): 58-77. https://doi.org/10.1111/j.2044-
8325.2010.02007.x 67. Sassenberg K, Scholl A. If I can do it my way… The influence of regulatory focus on job-related values
and job attraction. Journal of Economic Psychology. 7941.12033 2013; 38: 58-70. https://doi.org/10.1016/j.joep.2012.09.003 68. Chien C L. Beyond authoritarian personality: The culture-inclusive theory of Chinese authoritarian
orientation. Frontiers in Psychology. 2016; 7: 924. https://doi.org/10.3389/fpsyg.2016.00924 69. Gaddy J W, Gonzalez S P, Lathan C A, Graham P K. The perception of authentic leadership on
subordinate resilience. Military Behavioral Health. 2017; 5(1): 64-72. https://doi.org/10.1080/21635781.2016.1243495 Figures Figure 1 Figure 2 Figures Page 24/26 Figure 1
Research Model
Figure 2
The meditation model with unstandardized coefficients Figure 1
Research Model Figure 2 The meditation model with unstandardized coefficients. Page 25/26 Page 25/26 Note: Standard errors and 95% confidence intervals for 5000 samples are in parentheses below.***p <
0.001. Appendix.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 26/26
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Difficulties of caregivers of cow's milk allergy patients in understanding the labeling of processed products
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Amoroso et al. World Allergy Organization Journal 2015, 8(Suppl 1):A62
http://www.waojournal.org/content/8/S1/A62
MEETING ABSTRACT
Open Access
Difficulties of caregivers of cow’s milk allergy
patients in understanding the labeling of
processed products
Marina Amoroso1*, Mariana Forti1, Cleonir Beck1, Glauce Yonamine2, Andrea Gushken1, Mayra Dorna1,
Ana Paula Moschione Castro1, Antonio Carlos Pastorino1, Cristina Jacob1
From 3rd WAO International Scientific Conference (WISC) 2014
Rio de Janeiro, Brazil. 6-9 December 2014
Background
Cow’s Milk Allergy (CMA) is the main food allergy (FA)
in children and its treatment includes restricted milk
diet. The correct identification of terms that mean milk
ensures the efficacy of treatment.The aim of this study
was to identify the main factors involved in the caregivers’ ability to recognize the presence of milk in products labels.
Methods
Cross-sectional and descriptive study was carried out
with CMA patient’s caregivers in follow-up at a pediatric reference center for FA. All of them were previously
instructed about labels. This study included a questionnaire about the knowledge regarding the labels reading.
Caregivers evaluated 20 labels (15 of foods, 3 of medicines and 2 of cosmetics) and should decide if the product was safe and the reason it can or not be offered to
the patients. 15/20 contained words meaning milk protein. Results were expressed in number of labels reading
(20 labels/caregivers)
Results
Twenty-eight caregivers fulfilled the questionnaire. The
caregivers were 78.5% mothers and 21.5% fathers and
about their schooling years 78.2% finished high school
or college degree. The median of patient’s follow-up was
12 months (0.03-144). Twenty caregivers deal with
patients that presented at least one episode of anaphylaxis. All caregivers referred label reading: 25 read every
time they buy the product and only 9 read the labels
after buying, before storing and offering.Labels were correctly read in 75.7 % being lactose, casein and whey protein the most common correctly identified terms. The
most common mistakes were related to the terms lactil
and caramel color. About medicines the most difficulties
was to localize de terms related to milk in the package
leaflet. Main factors regarding wrong comprehension
were small or unclear printing, difficult localization and
ingredients in foreign language
Conclusions
Labels reading is part of food allergy treatment and
demands constant reinforcements stressing the necessity
of continuous label reading. Labels quality must improve
in many aspects in order to help caregivers understanding of milk labels and avoid patient’s accidental intake.
Authors’ details
1
Unit of Allergy and Immunology, Brazil. 2Nutritionist of Division of Nutrition,
Brazil.
Published: 8 April 2015
doi:10.1186/1939-4551-8-S1-A62
Cite this article as: Amoroso et al.: Difficulties of caregivers of cow’s
milk allergy patients in understanding the labeling of processed
products. World Allergy Organization Journal 2015 8(Suppl 1):A62.
1
Unit of Allergy and Immunology, Brazil
Full list of author information is available at the end of the article
© 2015 Amoroso 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/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. 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.
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Enseñanza bimodal en época de pandemia. Solución provisoria y reto para el futuro en la educación superior
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ISSN 1695-5498
HIPERTEXT.NET · REVISTA ACADÉMICA SOBRE DOCUMENTACIÓN DIGITAL Y COMUNICACIÓN INTERACTIVA
Número 26 · Mayo de 2023
Enseñanza bimodal en época de pandemia. Solución
provisoria y reto para el futuro en la educación superior
Terese Mendiguren Galdospin
Koldobika Meso Ayerdi
Jesús Ángel Pérez Dasilva
María Ganzabal Learreta
Universidad del País Vasco/
Euskal Herriko Unibertsitatea
Universidad del País Vasco/
Euskal Herriko Unibertsitatea
Universidad del País Vasco/
Euskal Herriko Unibertsitatea
Universidad del País Vasco/
Euskal Herriko Unibertsitatea
terese.mendiguren@ehu.eus
https://orcid.org/0000-0003-3092-6608
koldo.meso@ehu.eus
https://orcid.org/0000-0002-0400-133X
jesusangel.perez@ehu.eus
https://orcid.org/0000-0002-3383-4859
maria.ganzabal@ehu.eus
https://orcid.org/0000-0002-4231-402X
Bimodal teaching in times of pandemic. Interim solution and challenge for the
future in higher education
RESUMEN
Esta investigación pone el foco en las experiencias
y reflexiones del profesorado de comunicación de la
Universidad del País Vasco en torno a la enseñanza bimodal,
diseñada para el curso 2020/21 en el marco de la pandemia.
Esta estrategia docente tiene por objetivo reducir el flujo
del alumnado en las aulas, al mismo tiempo que asegura
la oferta de clases presenciales. La mitad de las personas
matriculadas se conectaban a clase desde sus hogares,
mientras la otra mitad asistía al aula presencialmente. Estos
turnos se iban alternando semanalmente. El objetivo del
estudio es analizar las dificultades, los errores y los aciertos
en la adaptación de la docencia al contexto de la enseñanza
bimodal. Se ha optado por una metodología cualitativa,
dividida en dos fases de actuación. En primer lugar, se
realizó Focus Group con profesorado de los tres grados de
comunicación y posteriormente se confeccionó una encuesta
que fue remitida quienes imparten sus clases de forma
bimodal. Este tipo de docencia pretende ser un híbrido entre
la estrategia presencial y la virtual, y obtener los beneficios
de ambas. Sin embargo, mantener los dos focos de atención
y satisfacer las necesidades que surgen en ambos contextos
es un reto difícil. Existe un alto grado de preocupación
por cuestiones relacionadas con la adaptación de las
asignaturas a este tipo de enseñanza, y principalmente, por
la pérdida de interacción con el alumnado.
PALABRAS CLAVE
Enseñanza bimodal; Educación superior; Pandemia;
Profesorado; Covid-19; Enseñanza digital.
ABSTRACT
This research focuses on the communication
lecturers´experiences about bimodal education in the
University of the Basque Country. This teaching strategy
aims to reduce the flow of students in classrooms, while
ensuring the supply of face-to-face classes. Half of the
people enrolled connect to class from their homes, while
the other half attend the classroom in person. These shifts
alternate weekly. The objective of the study is to analyze the
difficulties, errors and successes in adapting teaching to the
context of bimodal teaching. A qualitative methodology has
been chosen, divided into two phases of action. In the first
place, a Focus Group was carried out with teachers of the
three communication degrees and later a survey was made
that was sent by those who teach their classes in a bimodal
way. This type of teaching aims to be a hybrid between
the face-to-face and the virtual strategy, and to obtain the
benefits of both. However, maintaining the two focuses of
attention and meeting the needs that arise in both contexts
is a difficult challenge. There is a high degree of concern
for issues related to the adaptation of subjects to this type
of teaching, and mainly, for the loss of interaction with
students.
KEYWORDS
Bimodal education; Higher education; Pandemic; Teachers;
Covid-19; Digital learning.
Mendiguren-Galdospin, T., Meso-Ayerdi, K., Pérez-Dasilva, J. A. y Ganzabal-Learreta, M. (2023). Enseñanza bimodal en
época de pandemia. Solución provisoria y reto para el futuro en la educación superior. Hipertext.net, (26), 83-92.
https://doi.org/10.31009/hipertext.net.2023.i26.13
Esta obra está bajo la licencia Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional (CC BY-NC-ND 4.0)
Terese Mendiguren Galdospin, Koldobika Meso Ayerdi, Jesús Ángel Pérez Dasilva y María Ganzabal Learreta
1. Introducción
A comienzos del 2020, la propagación del Covid-19 llevó a la
OMS a declarar el brote de coronavirus como pandemia global. Ante el descontrol y la muerte de miles de personas,
prácticamente todos los gobiernos del mundo decretaron
el confinamiento de la población y sellaron sus fronteras,
clausuraron los negocios, las actividades culturales, las competiciones deportivas y cerraron los colegios para contener su
propagación (Pérez-Dasilva, Meso-Ayerdi y Mendiguren-Galdospin, 2020). La pandemia del coronavirus afectó a más de
1500 millones de estudiantes en el mundo, según la UNESCO1.
En este contexto, las universidades se vieron obligadas a
suspender, de un día para otro, las clases presenciales y a
continuar con su docencia en formato virtual (Sahu, 2020). La
suspensión de las actividades docentes presenciales para dar
paso a la virtualización supuso una presión elevada para el
profesorado y el alumnado en su adaptación al nuevo escenario (Tejedor et al., 2020).
Fue un proceso no planificado (Corell y García-Peñalvo, 2021)
que generó inquietud en la comunidad universitaria (de la
Calle et al., 2021) y que diversos autores calificaron como
“enseñanza remota de emergencia” (Ferri, Grifoni y Guzzo,
2020; Hodges et al., 2020; Pérez-López, Vázquez-Atochero
y Cambero-Rivero, 2020; Rahiem, 2020; Toquero, 2020). Los
profesores convirtieron sus hogares en aulas improvisadas (Rapanta et al., 2020) y aprendieron a utilizar software
especializado para impartir sus clases de forma virtual (Bao,
2020; Ebner et al., 2020; Mishra, Gupta y Shree, 2020; Motala y
Menon, 2020). El sistema universitario tuvo que sobrevivir a la
primera oleada de la pandemia sin apenas margen de maniobra y esto dejó patentes las brechas digitales existentes: de
acceso a Internet y de competencias tecnológicas, básicamente. Fue en las universidades presenciales en las que se dio
la mayor brecha competencial (García-Peñalvo, 2020).
Varios estudios analizaron las soluciones adoptadas en las
diferentes universidades (Osman, 2020; Radha et al., 2020;
Rapanta et al., 2020; Sobaih, Hasanein y Abu Elnasr, 2020) y
la calidad de la enseñanza recibida (Lee et al., 2021; Novikov,
2020; Pérez-López, Vázquez-Atochero y Cambero-Rivero,
2020; Rahiem, 2020; Xhelili et al., 2021; Xiong, Jiang y Mok,
2020). En este sentido, diversos trabajos coincidieron en señalar que la pandemia agrandó las desigualdades en el caso de
familias con bajo poder adquisitivo o nivel cultural, por lo que
el debate sobre la necesidad de alfabetización mediática de
los jóvenes se volvió indispensable (Frau-Meigs, 2020). Fue el
caso de países subdesarrollados como Pakistán o Sri Lanka,
donde la gran mayoría de los estudiantes no pudieron acceder a Internet por cuestiones técnicas y económicas. (Adnan
y Anwar, 2020; Olaganwatte, 2020; Sobaih, Hasanein y Abu
Elnasr, 2020). Esta situación también se vivió en los países
desarrollados entre los estudiantes de estratos de población
con menos recursos (Drane, Vernon y O’Shea, 2020; Van Lanc84
Hipertext.net, n. 26. 2023 · https://raco.cat/index.php/Hipertext
ker y Parolin, 2020). Otros autores se centraron en los efectos
de la enseñanza remota en la salud de los universitarios como
la ansiedad (Ozamiz-Etxebarria et al., 2020; Wang y Zhao,
2020), la salud mental (Dodd et al., 2021; Haider y Al-Salman,
2020; Sahu, 2020) o la depresión (Fawaz y Samaha, 2021).
La rápida reacción de las instituciones de enseñanza superior
ante este “cisne negro” (Wind et al., 2020) permitió finalizar
el curso académico 2019-2020 sin demasiados problemas.
Pero sin tiempo para descansar, las universidades tuvieron
que prepararse para el siguiente año en un contexto marcado
por la incertidumbre y donde la improvisación ya no servía de
excusa.
2. Cambio de paradigma en la enseñanza
universitaria
Las universidades se vieron en la necesidad de resolver en
poco tiempo y con escasas herramientas una situación inesperada (Hodges et al., 2020). Las reacciones iniciales a la decisión
de estas instituciones fueron variadas: algunos observadores la alabaron como un paso necesario en la lucha contra la
difusión del Covid-19 y otros la criticaron como una reacción
exagerada (Murphy, 2020). Lo cierto es que la irrupción de la
pandemia provocó un cambio súbito en la universidad, desde
un entorno presencial a uno virtual, generando disfunciones y
dudas (Bartolomé-Pina, 2020). En ese sentido, algunos autores plantearon la necesidad de ser cautos a la hora de sacar
conclusiones, ya que aún era pronto para poder valorar los
resultados obtenidos ante estas primeras medidas adoptadas
y también la precipitación a la hora de actuar podía llevar a falsos éxitos que tan solo podrían ser aparentes (Toquero, 2020).
Otros autores, a su vez, pusieron de manifiesto la necesidad
de repensar las instituciones educativas para que no perdieran su razón de ser (Tarabini, 2020), lo que se dio en llamar
“educación pos pandemia” (Iglesias et al., 2020; Esteban-Guitart et al., 2020).
Poco antes del verano de ese mismo año, muchas instituciones se plantearon sí podrían reanudar la enseñanza presencial
y cómo hacerlo (Weeden y Cornwell, 2020).
En esta dirección, por ejemplo, la UNESCO y el IESALC emitieron un conjunto de principios rectores para planear la salida
de la crisis del Covid-19 en la educación superior (Giannini,
2020), entre los que se encontraban la necesidad prepararse
con tiempo para la reanudación de las clases presenciales y
rediseñar los procesos de enseñanza y aprendizaje.
Parece necesario analizar con cuidado los problemas y
alcances que la educación a distancia evidenció durante el
confinamiento (Ordorika, 2020; Chipia y Santiago, 2020). Los
educadores sintieron que el aprendizaje virtual era una buena
alternativa, pero echaban de menos la interacción con el
alumnado (Chellathurai, 2020). De hecho, la virtualidad puede
Enseñanza bimodal en época de pandemia. Solución provisoria y reto para el futuro en la educación superior
ser asociada con un aumento de la carga de trabajo, lo que
establece la necesidad de revisar los procesos formativos “con
el objetivo de no saturar al alumnado de actividades extracurriculares que pueden ocasionar la deserción del mismo”
(Tejedor et al., 2021b, s.n.). Para algunos, los contactos directos cara a cara entre los estudiantes y el profesor, así como los
estudiantes entre sí en grupos, son elementos importantes en
la impartición de las clases (Ożadowicz, 2020).
La solución parecía estar en combinar la docencia en el aula
con la formación en línea. Tubagus, Muslim, y Suriani (2020)
abogan por una integración óptima entre la enseñanza presencial y el aprendizaje basado en la tecnología. En lo que
respecta a los aspectos tecnológicos, la pandemia sirvió para
poner a prueba la solvencia de herramientas propias de las
universidades que, por norma general, habían quedado relegadas a un segundo o tercer nivel de importancia (Tejedor et
al., 2021a).
En términos parecidos de una educación híbrida, algunos
autores defienden la idea de superar las barreras tradicionales entre la formación presencial y virtual. Pardo y Cobo (2020)
proponen transitar hacia experiencias de aprendizaje expandidas, líquidas y distribuidas en una línea de tiempo narrativa
multimedia a través de la cual los docentes y estudiantes
puedan servirse de modo colaborativo de lo mejor de la presencialidad y la cultura digital.
De cara al curso 2020/2021, muchas universidades adoptaron la enseñanza presencial tradicional con el aprendizaje
digital en línea. Es lo que se conoce como blended learning o
aprendizaje mixto (Garrison y Kanuka, 2004; Bartolomé-Pina,
2008, Castro, 2019), si bien recibe otras denominaciones como
hybrid learning o aprendizaje híbrido (Cochrane et al., 2020),
formación bimodal (Yábar y Barbarà, 1999; Espinoza-Guzmán
y Zermeño, 2017; Steiman y Luna, 2020), B-learning (Bartolomé-Pina, 2004; Núñez-Barriopedro, Monclúz, y Ravina-Ripoll,
2019; Sánchez-Ruiz et al., 2021), o incluso educación flexible
(Salinas, 2000) y enseñanza semipresencial (Leão y Bartolome, 2003). Las instituciones se impusieron la necesidad de
repensar el nuevo escenario educativo ante una situación de
pos pandemia que requería imponer normas sanitarias en particular relacionadas con el contacto interpersonal.
3. La enseñanza bimodal
En el periodo de “nueva normalidad”, cuya duración parecía
difícil de predecir, la obligatoriedad de cumplir las medidas
de prevención y de protección impuestas por las autoridades
sanitarias llevó a las instituciones académicas a diseñar una
serie de pautas de actuación con el objetivo de mantener el
mayor grado posible de presencialidad que fuera compatible
y respetuoso con dichas medidas. Las orientaciones que se
establecieron pretendían preservar, en la medida de lo posible,
el carácter presencial de la docencia de la Universidad, pero
adaptándola a escenarios de distanciamiento social, aforo
limitado y adopción de medidas higiénico-sanitarias.
En un primer momento, las orientaciones para planificar el
curso 2020-2021 continuaban planteando una perspectiva
de formación presencial adaptada, es decir, preferentemente
presencial, pero que se complementara con clases telemáticas y sesiones asíncronas. Se creía que la experiencia obtenida
durante el confinamiento del curso 2019-20 debía ser aprovechada para utilizar formas digitales de interacción en aquellas
actividades que pudieran beneficiarse de dichas metodologías, pero se recomendó que no se impartieran de manera
exclusivamente telemática más de un número concreto de
asignaturas por grupo y cuatrimestre (que variaba en función
de cada universidad), lo que implicaba descartar que pudiera
impartirse un curso entero en modalidad no presencial. Cada
titulación seleccionó aquellas actividades que requirieran
presencialidad para asegurar que el alumnado adquiriera, de
forma adecuada, las competencias recogidas en los correspondientes planes de estudio. Como posibles criterios de
selección, se utilizaron, entre otros, dar prioridad presencial
en modalidades prácticas frente a las de carácter magistral y
expositivo, a los cursos iniciales frente a los cursos superiores
y organizar la docencia por turnos. Se partía de la idea de utilizar la metodología de trabajo habitual de las asignaturas en
su formato presencial, pero parte del alumnado las seguiría a
distancia, a través de la herramienta para videoconferencias
que se pusiera a disposición de la comunidad universitaria,
preferentemente de forma síncrona, lo que le daría la posibilidad de interactuar con el docente y con sus compañeros. En
el caso de las actividades formativas asíncronas la recomendación fue que se mantuviera un nivel de interacción alto con
el alumnado.
Ante esta formación presencial adaptada, se contemplaron
distintas alternativas:
1. Combinar la presencialidad física y la telepresencialidad
con el grupo al completo. Se trataba de que el profesorado y una parte del alumnado compartieran una misma
aula física y el resto participara vía videoconferencia. En
este caso se organizó la alternancia entre ambos grupos
de manera equitativa para el alumnado (p. ej. por semanas:
la mitad del grupo recibía docencia presencial durante una
semana y en la siguiente no acudía al centro y trabajaba de
forma telepresencial).
2. Impartir determinadas modalidades docentes de forma
mayoritariamente a distancia, esto es, determinadas
modalidades docentes de una asignatura se impartieran a
través de videoconferencia sin presencia física del alumnado en el centro.
A la hora de planificar aquellas asignaturas o actividades con
un alto contenido práctico se tuvieron en cuenta una serie de
alternativas, como las de optimizar el tiempo de estancia en
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Terese Mendiguren Galdospin, Koldobika Meso Ayerdi, Jesús Ángel Pérez Dasilva y María Ganzabal Learreta
Figura 1. ¿Qué grado de dificultad o preocupación le generan las siguientes cuestiones? Fuente: Elaboración propia
laboratorios o priorizar aquello que no pudiera ser trabajado
a distancia.
Por lo que respecta a la evaluación, los centros planificaron
ésta de forma presencial, siempre que el cumplimiento de las
medidas establecidas en la normativa vigente lo permitiera.
Ahora bien, dadas las circunstancias excepcionales e imprevisibles en las que todavía continuaba España antes del inicio
del curso 2020-2021, se recomendó que todas las asignaturas
hicieran un planteamiento de evaluación continua además de
una propuesta alternativa de evaluación no presencial.
En lo que respecta a la Universidad del País Vasco, se optó
combinar la presencialidad física y la telepresencialidad para
las clases magistrales con los grupos que superaran los 50
alumnos, tratando de garantizar la presencialidad total en
los subgrupos de las clases prácticas de cada asignatura.
Por tanto, los grupos numerosos (más de 50 alumnos) fueron
organizados para recibir las clases de este modo, que se registró con la denominación de docencia bimodal.
4. Metodología
El estudio de la percepción y la experiencia del profesorado
en torno a la enseñanza bimodal durante el curso 2020/21 se
ha realizado en la Facultad de Ciencias Sociales y de la Comunicación del País Vasco. Se ha optado por una metodología
cualitativa, dividida en dos fases de actuación. En la primera
de ellas, se decidió realizar un Focus Group integrado por
cuatro docentes de los distintos grados de Comunicación2. La
información obtenida gracias al Focus Group fue clave para
la confección de un cuestionario, diseñado ad hoc para este
estudio, que en una segunda fase de actuación fue remitida
al profesorado de la Facultad. A través preguntas cerradas
y semiabiertas, se pretendía conocer más exhaustivamente
el grado de preocupación, cansancio, aprendizaje o crítica
que podía generar la estrategia docente híbrida por parte del
profesorado. Se registró una participación activa de 61 docentes en la encuesta, que tuvo lugar durante la última semana
del mes de abril y la primera del mes de mayo (2021). Para la
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selección de la muestra hubo que hacer un registro del profesorado que imparte docencia bimodal y evitar así un envío
masivo e indiscriminado de la encuesta al personal docente de
la Facultad. Se recurrió a un tipo de muestra no probabilística,
que se suele emplear en los análisis con gran peso cualitativo
y cuando la finalidad del estudio no es generalizar los resultados a toda la población, sino recopilar datos comparativos
y descriptivos. Se trata de determinar la muestra bajo el criterio del equipo investigador tomando como referencia los
objetivos del estudio, lo que Babbie (1999) denomina muestreo
deliberado o crítico. La muestra no probabilística intencional,
que suele ser pequeña y heterogénea, se selecciona procurando que esta sea representativa y depende de la intención
del investigador (Visauta, 1989).
Partíamos de la hipótesis de que el profesorado sentía un nivel
de agotamiento superior al de años anteriores y que había una
general animadversión a la estrategia docente bimodal. Existía además el objetivo de conocer los motivos que contribuían
a ello. Para el análisis de los resultados y su cálculo estadístico
se ha empleado el lenguaje de programación no procedimental que ofrece Microsoft Excel. Así, se ha optado por estudiar
al profesorado de Comunicación, ya que precisamente representa una muestra que hibrida la intención docente con la
intención comunicativa, que es, al fin y al cabo, el objetivo de la
bimodalidad: tratar de garantizar el proceso de aprendizaje del
alumnado, empleando las herramientas necesarias que faciliten la comunicación en los entornos presencial y online.
5. Resultados
5.1. Aspectos relacionados con la tecnología
Ante la pregunta realizada en este estudio sobre el grado de
preocupación que ha supuesto la dependencia de la tecnología
para impartir las clases, algunos docentes sí han reconocido
en este aspecto un quebradero de cabeza, ya que ha sido respondido con un alto grado de preocupación por el 15% de las
personas encuestadas y con algo de preocupación por un 48%
(Figura 1).
Enseñanza bimodal en época de pandemia. Solución provisoria y reto para el futuro en la educación superior
Respecto a la formación recibida para conocer el funcionamiento de la tecnología instalada en las aulas, más de la mitad
de las respuestas consideran que no ha sido escasa, si bien es
cierto que algunas voces apuntan el hecho de que, a la hora de
la verdad, las cosas son más difíciles en la puesta en escena
real que en los cursos de formación. “El problema”, apuntan en
algunos casos concretos, “no es no saber manejarse con tres
pantallas, es utilizarlas a la vez que se expone un Power Point,
porque había que estar pendiente de lo que ven los de clase y
lo que ven los de casa”. Además, existen aulas “en las que el
cañón, el ordenador con la cámara, y el ordenador principal no
están colocados de modo que sea fácil colocarse en un lugar
en el que me vean, escuchen y se pueda manejar todo”. Pero es
algo que apenas ocurre y señalan que “la universidad ha hecho
un esfuerzo en preparar las aulas con la tecnología necesaria
para impartir docencia bimodal” (Figura 2).
Por otro lado, se ha aprendido mucho a lo largo de este curso
porque ahora se sabe que “los micros captan todo el ruido del
aula” y se puede tener en cuenta en los ritmos y las rutinas;
por ejemplo, el docente ahora sabe que no debe empezar a
hablar hasta que haya silencio absoluto en el aula o debe evitar los debates con varias voces al mismo tiempo.
Una gran mayoría reconoce que el alumnado no le ha trasladado su parecer en lo que respecta a la docencia bimodal.
Entre las opiniones recibidas sí se registra una preocupación
por los problemas técnicos (29,5%). La casuística es muy
grande. “Desde el que tiene problemas para conectarse, hasta
el que tiene que compartir ordenador con sus padres, el que
tiene que venir a la universidad para conectarse… o el que está
viendo la clase desde debajo de las mantas con la Tablet”. Pese
a que el 59% de los encuestados consideran que la bimodalidad es una mera solución pasajera ante la situación de la
pandemia (Figura 3), algunas personas opinan que esto ha
llegado para quedarse, quizá no tal y como está introducido
ahora, pero creen que no desaparecerá.
5.2. Aspectos relacionados con el contenido de las materias
El 36% de los profesores y profesoras encuestados afirman
que la adaptación de su asignatura a la bimodalidad le ha
supuesto mucha más carga de trabajo de la habitual. Más de la
Figura 3. ¿Ofrece alguna oportunidad/ventaja esta modalidad
respecto a la presencialidad? Fuente: Elaboración propia
mitad, un 52% reconoce que le ha supuesto algo más de carga
de trabajo y únicamente el 12% no lo considera en absoluto. De
hecho, existe a posteriori un aprendizaje y una experiencia que
va a suponer quizá una segunda readaptación de la estrategia
docente. Mientras un 5% de los docentes afirma que cambiará
su estrategia totalmente para el año siguiente, un 72% reconoce que tendrá que adaptarla en cierta medida. De hecho, la
sensación es que la docencia en la forma bimodal no consiste
únicamente en ser consciente de que se está impartiendo
materia en dos espacios distintos a la vez. Así lo atestiguan
testimonios como “ni el contenido, ni la forma de impartir me
valía. Quizá sí para un aula presencial o para una situación de
confinamiento, pero no para una bimodal”. Únicamente un 10%
reconocía que no tendría que readaptar su estrategia de cara
al siguiente curso. En lo que respecta a la participación del
alumno en el proceso formativo, solamente un 6,5% reconoce
que este tiene un rol activo.
Respecto al ritmo a la hora de impartir la materia, algunos de
los testimonios recogidos tanto en el Focus Group como en las
preguntas semiabiertas de la encuesta, afirman que es inevitable hacer pequeños parones continuos a la hora de mostrar,
entre otras cosas, ejemplos que hay que compartir de modo
presencial y de modo online. “Hay que cambiar la asignatura
para adaptarla a los ritmos de clase, que no son iguales, y para
captar la atención de los que están en casa a la vez que los
que están en clase”. Además, en algunos casos en los que la
práctica también ha sido bimodal, la sustitución de prácticas
grupales por individuales ha supuesto un incremento notable
del tiempo dedicado a las correcciones de ejercicios.
Por otro lado, el 36% de profesorado encuestado asegura que
la formación y el aprendizaje recibidos por parte del alumnado se están viendo muy mermados este año. Un 54% está
bastante o algo de acuerdo con esta afirmación, mientras que
solo un 10% no lo considera en absoluto.
Otro problema detectado entre los testimonios recogidos
es la evasión general del alumnado incluso si está en clase:
“Hay que subir los materiales completos a Egela3 por causa de
Figura 2. El alumnado le ha trasladado su opinión sobre la
bimodal en forma de:... Fuente: Elaboración propia
la bimodalidad, y eso supone la puntilla para su atención. La
palabra hablada les parece redundante”.
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Terese Mendiguren Galdospin, Koldobika Meso Ayerdi, Jesús Ángel Pérez Dasilva y María Ganzabal Learreta
5.3. La atención y el Feed back con el alumnado
Según recoge este estudio, el mayor quebradero de cabeza al
que se ha enfrentado el profesorado es la dificultad de mantener la interrelación y el feed-back con el alumnado presente y
el grupo que se conecta online (Figura 1). No es lo mismo enfocar la estrategia y los esfuerzos en garantizar ese feed-back
cuando toda la audiencia está conectada online, como ocurrió durante el confinamiento riguroso, que cuando se tiene la
mitad del grupo en el aula de forma presencial y la otra mitad
de forma online. El problema no ha estado tanto en jugar con
pantallas, sino en interaccionar realmente con todo el grupo.
“Cuando están en casa, no se sienten parte de la clase. Es muy
difícil interaccionar con la gente que está en el aula y con la
gente que está fuera. Da la sensación de que el mismo mecanismo, las mismas preguntas, las mismas sugerencias, no se
pueden canalizar de la misma manera”.
Existen situaciones, reconocen algunos testimonios de este
estudio, en las que el docente ve inevitable centrarse en los
que están en el aula, pero otros no se lo plantean así: “En el
aula pongo el chat abierto para los que están en casa puedan
interactuar”. El 16% de los encuestados aseguran que el chat
es una buena herramienta para interaccionar y que, en ciertos momentos, es un recurso más empleado que el de alzar la
mano presencialmente en el aula. Sin embargo, el alumnado
dice que cuando está en casa le cuesta mucho conectar con el
grupo en clase. También se ha observado un peligro de absentismo, ya que “los que están en casa se han permitido, según
avanzaba el cuatrimestre, tomarse la clase como opcional”.
El soporte a través del que siguen las clases también influye.
“Mantener la atención en un móvil donde el encuadre abierto
que tienen de la clase hace que te vean muy pequeño. No es
fácil mantener la atención así”.
5.4. Aspectos emocionales
Una gran mayoría, el 84% de las personas participantes en
la encuesta ha reconocido que se encuentra más cansada a
estas alturas del curso, en comparación con años anteriores.
Las casuísticas son variadas, por ejemplo, “en el caso de la
enseñanza cooperativa activa la bimodalidad se convierte en
un handicap emocional y pedagógico. El seguimiento continuo
al alumnado se hace tremendamente difícil y las dinámicas
de trabajo se rompen cuando están en casa. Es un volver a
empezar cada 15 días”. Algunas personas esperaban que esta
situación fuera más sencilla tras pasar por la etapa del confinamiento y haber tenido que adaptar la docencia a un modo
de proceder distinto. Pero reconocen que aquella lógica no
vale de igual modo. Se nota más cansancio, dicen, “no solo por
las circunstancias de la enseñanza bimodal, sino por hablar
con la mascarilla y encima a través de una pantalla”. También encuentran un problema en la pérdida de intimidad que
implica el hecho de que todas las clases puedan ser grabadas
por el alumnado. “Nada les impide grabar lo que se emite por
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BBC. Esto implica que una frase del docente fuera de contexto
puede acabar siendo viral”.
Este tipo de reflexiones ya forman parte de la conversación
habitual entre el profesorado. La enseñanza bimodal es un
tema recurrente entre compañeros para el 64% de las personas encuestadas. Únicamente un 3% opina que no es un tema
habitual de conversación.
En lo que respecta a la asistencia, se temía que hubiera fuga
de presencial a virtual. Esto es, que el alumnado con turno presencial optara por quedarse en casa y conectarse online. Pero
no ha sido así. “Les gusta la semana que les toca venir presencialmente”. Según el informe del Focus Group, lo que más
le gusta al alumnado y cuando más aprende es en los debates
que surgen en clase”.
6. Discusión
La comunidad universitaria ha estado forzada a hacer frente a
una situación de incertidumbre por motivo de la pandemia. La
idea general es que la enseñanza bimodal ofrece la posibilidad
de cursar los estudios de forma semipresencial pero se pierde
la interacción personal, las dinámicas de grupo y los debates
en el aula. Existe una dicotomía entre la ventaja que supone
poder llegar a más alumnado y la calidad en la manera de
llegar a este. Hay más opciones para llegar, pero menos calidad de feedback real, de conexión humana y emocional, cuya
importancia es de sobra reconocida. Entre las ventajas, se ha
fomentado el uso de diferentes plataformas digitales y se ha
reflexionado sobre los materiales que se emplean para impartir docencia y nuevas estrategias de enseñanza. Sin embargo,
el foco del debate hay que ponerlo en el nivel formativo que
encuentra el alumnado en este contexto. Bolonia introdujo
la idea del aprendizaje frente a la idea enseñanza y la del rol
activo del alumnado frente al pasivo. Durante los últimos años
se ha tratado de enfocar los esfuerzos en garantizar la puesta
en marcha de estrategias que garanticen este tipo de aprendizaje. El peligro de la bimodal es enfocar los esfuerzos en el
medio para llegar, más que en el método. Está latente el temor
a volver a las clases magistrales tradicionales a través de una
pantalla y sin la participación real del alumnado.
Por otro lado, no se debe confundir la bimodalidad con la
docencia online, en la que el profesorado puede enfocar sus
esfuerzos de otro modo y conseguir una participación activa.
En la docencia bimodal, existe un público presente ocupando
el mismo espacio que el docente, al que también se debe prestar atención.
La tecnología ofrece la oportunidad de poner en contacto a
las personas que se encuentran a distancia geográfica, pero
ese contacto no garantiza un acercamiento per sé. Para que
llegue, el mensaje debe estar muy focalizado en quién está
detrás de una pantalla o, en su caso, en quien está presente.
Enseñanza bimodal en época de pandemia. Solución provisoria y reto para el futuro en la educación superior
Esta tarea es difícil para el profesorado, que en general ha
sentido frustración y cierta sensación de que no está llegando
a ambos espacios a la vez.
Para que la bimodalidad funcione, toca reflexionar y repensar la forma de impartir las clases. Por ejemplo, fragmentar y
alternar los tiempos de atención al alumnado de los dos espacios. El reto es encontrar una estrategia más eficaz para el
alumnado online y presencial. El objetivo debería ser conseguir
que la docencia bimodal, aparte de ser una solución para hacer
frente a esta situación, se convierta en una opción altamente
eficaz. Actualmente, la sensación general es que de momento
no se están aprovechando las ventajas de la presencialidad, ni
las de la modalidad online.
7. Conclusiones
La universidad se ha adaptado a una circunstancia difícil.
Quizá esta modalidad docente no vino para quedarse, pero lo
cierto es que la estrategia bimodal salvó el curso 2020-21 y
posibilitó cierta presencialidad en el ejercicio de la docencia,
garantizando un entorno seguro en aquellas facultades que
contaban con una elevada tasa de matriculación. Lo cierto es
que se confirma el cansancio y un alto grado de frustración en
el profesorado. De esta experiencia se puede además extraer
un aprendizaje. Se han conocido las necesidades reales y cotidianas que requiere intentar abarcar al mismo tiempo los dos
espacios en los que trabajan los docentes: el presencial y el
virtual. Aunar estos lugares, hibridando dos estrategias distintas en una, no es tarea fácil. Son dos realidades que precisan
necesidades diversas. Se ha demostrado que las estrategias enfocadas únicamente al alumnado que está conectado
no tienen cabida cuando a la vez un grupo asiste de manera
presencial. Existe, por tanto, la necesidad de buscar nuevas
alternativas y estrategias docentes adaptables a la bimodalidad, que conjuguen la capacidad de formar sin olvidar la
emoción y la conexión; encontrar alternativas más creativas e
innovadoras que apelen a la participación; y fomentar más que
nunca el rol activo del alumnado en su proceso de aprendizaje.
La adaptación a la estrategia bimodal requiere un cambio
de paradigma y exigirá esfuerzo y tiempo. Si se entiende la
bimodalidad como una solución temporal y de urgencia ante
una situación extraordinaria y caduca, la experiencia docente
durante el curso 2020-21 puede quedar en una mera reflexión.
Pero lo cierto es que no se puede predecir si en un futuro volverá a ser necesario hacer frente a otra crisis sanitaria que
requiera tener interiorizados modelos educativos y estrategias docentes adaptables.
Financiación
Este trabajo forma parte del proyecto “Noticias, redes y usuarios en el sistema híbrido de medios” (RTI2018-095775-B-C41)”,
financiado por el Plan Nacional del I+D+i, del Ministerio de
Economía y Competitividad, y por el Fondo Europeo de Desarrollo Regional (FEDER) y del Grupo Consolidado IT1112-16 del
Gobierno Vasco.
Notas al final
1.
https://news.un.org/es/story/2020/08/1478302
2.
Un profesor de Comunicación Audiovisual, una profesora de
Publicidad, y dos profesoras de Periodismo, por tratarse del
grado con mayor número de alumnado.
3.
Egela es el nombre de la plataforma que se emplea en la UPV/
EHU para la relación con el alumnado y la compartición de materiales y prácticas, estrategias evaluadoras, foros, etc.
Referencias
Adnan, M. y Anwar, K. (2020). Online Learning amid the COVID-19
Pandemic: Students’ Perspectives. Journal of Pedagogical
Sociology and Psychology, 2(1), 45-51. https://doi.org/10.33902/
JPSP.2020261309
Babbie, E. (1999). Técnicas de la investigación social. Thompson.
Bao, W. (2020). COVID-19 and online teaching in higher education: A
case study of Peking University. Human Behavior and Emerging Technologies, 2(2), 113-115. https://doi.org/10.1002/hbe2.191
Bartolomé-Pina, A. (2004). Blended Learning. Conceptos básicos.
Pixel-Bit, Revista de Medios y Educación, (23), 7-20.
Bartolomé-Pina, A. (2008). Entornos de aprendizaje mixto en educación superior. RIED, 11(1), 15-51. https://doi.org/10.5944/ried.1.11.955
Bartolomé-Pina, A. (2020). Cambios educativos en tiempos de
pandemia. Revista Innovaciones Educativas, (22), 13-16. https://doi.
org/10.22458/ie.v22iespecial.3155
Castro, R. (2019) Blended learning in higher education: Trends and
capabilities. Education Information Technologies, 24, 2523–2546.
https://doi.org/10.1007/s10639-019-09886-3
Chellathurai, G. J. (2020). Future of Education Post Pandemic COVID
-19: Online VS Classroom Learning. Redefining Education, The Researchers’, 6(2), 23-30.
Chipia, J. y Santiago, C. (2020). Educación universitaria: Transición y
disrupción digital, aproximación crítica. GICOS, 5(2), 130-140. http://
portal.amelica.org/ameli/jatsRepo/351/3511475008/index.html
Cochrane, T., Birt, J., Cowie, N., Deneen, C., Goldacre, P., Narayan,
V., Ransom, L., Sinfield, D. y Worthington, T. (2020). A collaborative design model to support hybrid learning environments during
COVID19. Proceedings of the ASCILITE 37th International Conference
on Innovation, Practice and Research in the Use of Educational Technologies in Tertiary Education, Armidale, Australia (pp. 84-89).
Corell, A. y García-Peñalvo, F. J. (2021). COVID-19: La encerrona que
transformó las universidades en virtuales. Gaceta Cultural, (91),
23-26. http://repositorio.grial.eu/handle/grial/2182
De la Calle, C., Miró, S., de Dios, T. y de la Rosa, D. (2021). Adaptación
de una materia universitaria al aprendizaje en línea en tiempos de
COVID-19: una oportunidad de mejora. RED. Revista Educación a
Distancia, 65(21). https://doi.org/10.6018/red.449841
Dodd, R. H., Dadaczynski, K., Okan, O., McCaffery, K. J. y Pickles, K.
(2021). Psychological wellbeing and academic experience of Univer89
Terese Mendiguren Galdospin, Koldobika Meso Ayerdi, Jesús Ángel Pérez Dasilva y María Ganzabal Learreta
sity students in Australia during COVID-19. International Journal of
Environmental Research and Public Health, 18(3), 866. https://doi.
org/10.3390/ijerph18030866
Lee, K., Fanguy, M., Lu, X. S. y Bligh, B. (2021). Student learning during
COVID-19: It was not as bad as we feared. Distance Education, 42(1),
164-172. https://doi.org/10.1080/01587919.2020.1869529
Drane, C. F., Vernon, L. y O’Shea, S. (2020). Vulnerable learners in
the age of COVID-19: A scoping review. The Australian Educational
Researcher, 48, 585-604. https://doi.org/10.1007/s13384-02000409-5
Mishra, L., Gupta, T. y Shree, A. (2020). Online teaching-learning in
higher education during lockdown period of COVID-19 pandemic.
International Journal of Educational Research Open, 1, 100012. https://
doi.org/10.1016/j.ijedro.2020.100012
Ebner, M., Schön, S., Braun, C., Grigoriadis, Y., Haas, M., Leitner, P. y
Taraghi, B. (2020). COVID-19 Epidemic as E-Learning Boost? Chronological Development and Effects at an Austrian University against
the Background of the Concept of “E-Learning Readiness”. Future
Internet, 12(6), 94. https://doi.org/10.3390/fi12060094
Motala, S. y Menon, K. (2020). In search of the ‘new normal’: Reflections on teaching and learning during Covid-19 in a South African
university. Southern African Review of Education, 26(1), 80-99.
https://hdl.handle.net/10520/ejc-sare-v26-n1-a6
Espinoza-Guzmán, J. y Zermeño, M. G. G. (2017). Maturity model
for e-learning classroom, bimodal and virtual courses in higher
education: A preliminary study. International Journal of Web-Based
Learning and Teaching Technologies (IJWLTT), 12(1), 19-31. https://doi.
org/10.4018/IJWLTT.2017010102
Esteban-Guitart, M., Iglesias, E., González-Patiño, J. y González-Ceballos, I. (2020). La personalización educativa en tiempos de cambio
e innovación educativa. Un ejemplo ilustrativo. Aula Abierta, 49(4).
395-404. https://doi.org/10.17811/rifie.49.4.2020
Fawaz, M. y Samaha, A. (2021). E-learning: Depression, anxiety,
and stress symptomatology among Lebanese university students
during COVID-19 quarantine. Nursing Forum, 56(1), 52-57. https://doi.
org/10.1111/nuf.12521
Ferri, F., Grifoni, P. y Guzzo, T. (2020). Online Learning and Emergency
Remote Teaching: Opportunities and Challenges in Emergency Situations. Societies, 10(4), 86. https://doi.org/10.3390/soc10040086
Frau-Meigs, D. (2020). Pédagogie à distance: Les enseignements du
e-confinement. The Conversation. http://theconversation.com/pedagogie-a-distance-les-enseignements-du-e-confinement-137327
García-Peñalvo, F. J. (12 de mayo de 2020). El sistema universitario
ante la COVID-19: Corto, medio y largo plazo. Universidad sí, https://
www.universidadsi.es/sistema-universitario-covid-19/
Garrison, D.R. y Kanuka, H. (2004). Blended learning: Uncovering its
transformative potential in higher education. The Internet and Higher
Education, 7(2), 95-105. https://doi.org/10.1016/j.iheduc.2004.02.001
Giannini, S. (2020). COVID-19 y educación superior: de los efectos
inmediatos al día después. Revista Latinoamericana de Educación
Comparada: RELEC, 11(17), 1-57.
Haider, A. S. y Al-Salman, S. (2020). Dataset of Jordanian university
students’ psychological health impacted by using e-learning tools
during COVID-19. Data in brief, 32, 106104. https://doi.org/10.1016/j.
dib.2020.106104
Hodges, Ch., Moore, S., Lockee, B., Trust, T. y Bond, A. (2020). The
Difference Between Emergency Remote Teaching and Online
Learning. Educause Review. https://er.educause.edu/articles/2020/3/the-difference-between-emergency-remote-teaching-and-online-learning
Murphy, M.P.A. (2020). COVID-19 and emergency eLearning: Consequences of the securitization of higher education for post-pandemic
pedagogy. Contemporary Security Policy, 41(3), 492-505. https://doi.
org/10.1080/13523260.2020.1761749
Novikov, P. (2020). Impact of COVID-19 emergency transition to
on-line learning onto the international students’ perceptions of
educational process at Russian university. Journal of Social Studies
Education Research, 11(3), 270-302.
Núñez-Barriopedro, E., Monclúz, I. M. y Ravina-Ripoll, R. (2019). El
impacto de la utilización de la modalidad B-Learning en la educación
superior. Alteridad. Revista de Educación, 14(1), 26-39.
Olaganwatte, Ch. (2020). The Covid-19: Impact on education. Journal
of Asian and African Social Science and Humanities, 5, 13-14.
Osman, M. E. (2020). Global impact of COVID-19 on education
systems: The emergency remote teaching at Sultan Qaboos University. Journal of Education for Teaching, 46(4), 463-471. https://doi.org/
10.1080/02607476.2020.1802583
Ordorika, I. (2020). Pandemia y educación superior. Revista de la
Educación Superior, 194(49), 1-8. https://www.scielo.org.mx/pdf/
resu/v49n194/0185-2760-resu-49-194-1.pdf.
Ożadowicz, A. (2020). Modified Blended Learning in Engineering
Higher Education during the COVID-19 Lockdown—Building Automation Courses Case Study. Educacion Sciences, 10, 292. https://doi.
org/10.3390/educsci10100292
Ozamiz-Etxebarria, N., Santa-María, M. D., Munitis, A. E. y Gorrotxategi, M. P. (2020). Reduction of COVID-19 anxiety levels through relaxation techniques: A study carried out in northern Spain on a sample of
young university students. Frontiers in Psychology, 11, 2038. https://
doi.org/10.3389/fpsyg.2020.02038
Pardo, H. y Cobo, C. (2020). Expandir la universidad más allá de la
enseñanza remota de emergencia. Ideas hacia un modelo híbrido
post-pandemia. Outliers School.
Pérez-López, E., Vázquez-Atochero, A. y Cambero-Rivero, S. (2020).
Educación a distancia en tiempos de COVID-19: Análisis desde la perspectiva de los estudiantes universitarios. RIED. Revista Iberoamericana de Educación a Distancia, 24(1), 331. https://doi.org/10.5944/
ried.24.1.27855
Iglesias, E., González-Patiño, J., Lalueza, J. L. y Esteban-Guitart, M.
(2020). Manifiesto en tiempos de pandemia: Por una educación
crítica, intergeneracional, sostenible y comunitaria. Revista Internacional de Educación para la Justicia Social, 9(3), 181-198.
Pérez-Dasilva, J.A., Meso-Ayerdi, K. y Mendiguren-Galdospin, T.
(2020). Fake news y coronavirus: Detección de los principales actores
y tendencias a través del análisis de las conversaciones en Twitter.
Profesional de la Información, 29(3). https://doi.org/10.3145/epi.2020.
may.08
Leão, M. y Bartolome, A. (2003). Multiambiente de aprendizagem: a
integração da sala de aula com os laboratorios experimentais e de
multimeios. Revista Brasileira de Tecnologia Educacional, 159(160),
75-80.
Rahiem, M. D. H. (2020). The Emergency Remote Learning Experience of University Students in Indonesia amidst the COVID-19 Crisis.
International Journal of Learning, Teaching and Educational Research,
19(6), 1-26. https://doi.org/10.26803/ijlter.19.6.1
90
Hipertext.net, n. 26. 2023 · https://raco.cat/index.php/Hipertext
Enseñanza bimodal en época de pandemia. Solución provisoria y reto para el futuro en la educación superior
Rapanta, C., Botturi, L., Goodyear, P., Guàrdia, L. y Koole, M. (2020).
Online University Teaching During and After the Covid-19 Crisis: Refocusing Teacher Presence and Learning Activity. Postdigital Science
and Education, 2(3), 923-945. https://doi.org/10.1007/s42438-02000155-y
Sahu, P. (2020). Closure of Universities Due to Coronavirus Disease
2019 (COVID-19): Impact on Education and Mental Health of Students
and Academic Staff. Cureus, 12(4). https://doi.org/10.7759/cureus.7541
Salinas, J. (2000). ¿Qué se entiende por una institución de educación
superior flexible?. En J. Cabero-Almenara, M. Cebrián de la Serna,
A. M. Duarte-Hueros, F. Martínez-Sánchez, J. I. Aguaded-Gómez, J.
Barroso-Osuna, J. M. Fernández-Batanero, J. A. Morales-Lozano
(Coor.), Nuevas tecnologías en la formación flexible y a distancia (pp.
451-466). Kronos.
Sánchez-Ruiz, L. M., Moll-López, S., Moraño-Fernández, J. A. y
Llobregat-Gómez, N. (2021) B-Learning and Technology: Enablers for
University Education Resilience. An Experience Case under COVID-19
in Spain. Sustainability, 13, 3532. https://doi.org/10.3390/su13063532
Sobaih, A. E. E., Hasanein, A. M. y Abu Elnasr, A. E. (2020). Responses
to COVID-19 in Higher Education: Social Media Usage for Sustaining
Formal Academic Communication in Developing Countries. Sustainability, 12(16), 6520. https://doi.org/10.3390/su12166520
Steiman, B. y Luna, A. (2020). La educación bimodal como práctica de
enseñanza innovadora. Hologramática, 33(3), 201-213. https://www.
cienciared.com.ar/ra/doc.php?n=2285
Tarabini, A. (2020). ¿Para qué sirve la escuela? Reflexiones sociológicas en tiempos de pandemia global. Revista de Sociología de la
Educación, 13(2), 145-155. https://doi.org/10.7203/RASE.13.2.17135
Tejedor, S., Cervi, L., Tusa, F. y Parola, A. (2020). Educación en tiempos
de pandemia: reflexiones de alumnos y profesores sobre la enseñanza virtual universitaria en España, Italia y Ecuador. Revista Latina,
(78), 1-21. https://doi.org/10.4185/RLCS-2020-1466
Weeden, K. y Cornwell, B. (2020). TheSmall-World Network of
CollegeClasses: Implications forEpidemic Spread on a University
Campus. Sociological Science, 7, 222-241. https://doi.org/10.15195/
v7.a9
Wind, T. R., Rijkeboer, M., Andersson, G. y Riper, H. (2020). The
COVID-19 pandemic: The ‘black swan’ for mental health care and a
turning point for e-health. Internet Interventions, 20, 100317. https://
doi.org/10.1016/j.invent.2020.100317
Xhelili, P., Ibrahimi, E., Rruci, E. y Sheme, K. (2021). Adaptation and
perception of online learning during COVID-19 pandemic by Albanian
university students. International Journal on Studies in Education,
3(2), 103-111. https://doi.org/10.46328/ijonse.49
Xiong, W., Jiang, J. y Mok, K. H. (2020). Hong Kong university
students’ online learning experiences under the Covid-19 pandemic.
Higher Education Policy Institute–Blog. https://www.hepi.
ac.uk/2020/08/03/hong-kong-university-students-online-learning-experiences-under-the-covid-19-pandemic
Yábar, J. M. y Barbarà, P. L. (1999). La Universitat Autònoma de
Barcelona: el camino hacia una universidad bimodal en el marco de
las Tecnologías de la Información y la Comunicación. Educar, (25),
113-118. https://raco.cat/index.php/Educar/article/view/20718
CV
Terese Mendiguren Galdospin. Es Profesora Agregada de
la Universidad del País Vasco UPV/EHU, Departamento de
Periodismo. Es Doctora en Ciencias de la Información por la
Universidad del País Vasco. Como investigadora, ha participado en 20 proyectos de investigación y ha publicado más
de 30 artículos científicos en revistas de prestigio incluidas
en bases de datos como JCR, SCOPUS o Dice-Cindoc. Actual-
Tejedor, S.; Cervi, L.; Escoda, A.; Parola, A.; Tusa, F. (2021a). Higher
Education response in time of coronavirus: perceptions of teachers
and students, and open innovation. Journal of Open Innovation: Technology, Market, and Complexity, 7(43), 1-15. https://doi.org/10.3390/
joitmc7010043
mente, es miembro del proyecto “Noticias, redes y usuarios
Tejedor, S.; Cervi,L; Tusa, F.; Parola, A. (2021b). Los docentes universitarios frente al cambio a la educación virtual impuesta por el coronavirus. Revista Sociedade e Estado, 36(3). https://doi.org/10.1590/
s0102-6992-202136030004
compagina con la gestión como Vicedecana de Comunicación
Toquero, C.M. (2020). Challenges and Opportunities for Higher Education amid the COVID-19 Pandemic: The Philippine Context. Pedagogical Research, 5(4), em0063. https://doi.org/10.29333/pr/7947
Tubagus, M., Muslim, S. y Suriani, S. (2020). Development of learning
management system-based blended learning model using claroline
in higher education. International Journal of Interactive Mobile Technologies, 14, 186-194. https://doi.org/10.3991/ijim.v14i06.13399
Van Lancker, W. y Parolin, Z. (2020). COVID-19, school closures, and
child poverty: A social crisis in the making. The Lancet Public Health,
5(5), e243-e244. https://doi.org/10.1016/S2468-2667(20)30084-0
Visauta, B. (1989). Técnicas de investigación Social. I: Recogida de
datos. Promociones y Publicaciones Universitarias SA.
Wang, C. y Zhao, H. (2020). The impact of COVID-19 on anxiety in
Chinese university students. Frontiers in psychology, 11, 1168. https://
doi.org/10.3389/fpsyg.2020.01168
en el sistema híbrido de medios” (RTI2018-095775-B-C41)
y del Grupo consolidado de investigación Gureiker (IT111216). Sus líneas de investigación son Periodismo en Internet e
Innovación Educativa y Nuevas Metodologías Docentes, que
de su Facultad.
Koldobika Meso Ayerdi. Es profesor titular y director del
Departamento de Periodismo de la UPV/EHU. Doctor en
Periodismo. Como profesor, ha impartido docencia en Grado
y Posgrado. Ha publicado más de un centenar de trabajos en
forma capítulos, libros y artículos en revistas académicas de
calidad. Ha presentado más de cien ponencias y comunicaciones en congresos nacionales e internacionales. Su labor
investigadora ha sido desarrollada en el marco de su participación continuada en más de una decena de proyectos de
investigación financiados en convocatorias públicas competitivas. También ha dirigido Proyectos de Innovación Educativa y
ha participado en otros como investigador, en el marco de las
actividades del Grupo Especializado en Innovación Educativa
KZBerri. Igualmente, ha participado en diversos contratos de
investigación y transferencia relevantes con entidades públi91
Terese Mendiguren Galdospin, Koldobika Meso Ayerdi, Jesús Ángel Pérez Dasilva y María Ganzabal Learreta
cas (Euskal Irrati Telebista/Radiotelevisión Pública Vasca).
Tiene reconocidos 4 sexenios de investigación.
Jesús Ángel Pérez Dasilva. Es profesor Pleno en el Departamento de Periodismo de la UPV/EHU. Ha sido vicedecano de
la Facultad de Ciencias Sociales y de la Comunicación (20092012), director del Máster de Comunicación Social (2016-2018)
y miembro de la Comisión Universitaria de Evaluación Docente
(Docentiaz, 2016-2022). Forma parte del grupo de investigación Gureiker. Ha sido profesor visitante en la Universidad de
Cambridge (2012) y ha participado en el programa Erasmus+
de movilidad docente en las siguientes universidades: Trieste
(2011), Beira interior (2013), Oporto (2015), Braga (2016),
Wroclaw (2017 y 2019). Ha participado en 27 actividades de
investigación en convocatorias competitivas siendo en 5 de
ellas el investigador principal. Es coautor de 65 artículos científicos (13 Q1 y Q2 en SJR/WOS), 27 capítulos de libros y 100
contribuciones en congresos. Tiene 3 sexenios. Sus líneas de
investigación se centran en el periodismo digital, la innovación educativa y las redes sociales.
María Ganzabal Learreta. Es profesora agregada del Departamento de Periodismo de la Universidad del País Vasco. Es
doctora en Periodismo y máster en Documentación Digital
por la Universidad Pompeu Fabra. Ha trabajado en diversas
agencias de comunicación y en el Departamento de Arte de
la revista americana New Woman. La docencia en materias
de Grado está relacionada con asignaturas como Periodismo
Social y Participativo y Narrativas Transmedia. Asimismo,
imparte docencia en Posgrado, en el Máster de Estudios
Feministas y de Género y en el Máster de Comunicación
Social donde imparte materias relacionadas con el género y
el periodismo. Ha publicado múltiples trabajos en forma capítulos, libros y artículos en revistas académicas de calidad,
También ha presentado ponencias y comunicaciones en congresos nacionales e internacionales. Estas publicaciones se
han desarrollado dentro del marco de investigación del que
forma parte en más de una decena de proyectos de investigación financiados en convocatorias públicas competitivas,
tanto a nivel universitario, autonómico y del Plan Nacional
I+D+I.
PUBLICIDAD
El Observatorio de Cibermedios es una producción
del Grupo de Investigación en Documentación Digital y
Comunicación Interactiva (DigiDoc) del Departamento
de Comunicación de la Universitat Pompeu Fabra.
https://observatoriocibermedios.upf.edu/
92
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El Observatorio de Cibermedios (OCM) forma parte del proyecto del Plan Nacional “Parámetros y estrategias para
incrementar la relevancia de los medios y la comunicación
digital en la sociedad: curación, visualización y visibilidad
(CUVICOM)”. PID2021-123579OB-I00 (MICINN), Ministerio de
Ciencia e Innovación ( España).
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https://openalex.org/W4385786735
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https://journal.uokufa.edu.iq/index.php/kufa_arts/article/download/6161/pdf
|
Chinese
| null |
The exception to linguists and fundamentalists
|
Ādāb al-Kūfaẗ/Ādāb al-kūfaẗ
| 2,017
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cc-by
| 10,662
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ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ ﺍﻟﺪﻛﺘﻮﺭ
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، ذت و واة وا ؛ ا اكُ ا
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ا
ا اّء، و أ اوء ، واع اّغ ، وأمء اء وا
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ل ا . أ إ ا ا أ ت ا ا ام اوأ
ا
. ﻱ
ﺟﺎﻣﻌﺔ ﺍﻟﻜﻮﻓﺔ ـ ﻛﻠﻴﺔ ﺍﻟﻔﻘﻪ ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
.........................................................
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٥٦٣
(
ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
ﺍﳌﻘﺪﻣﺔ
ﺍﻟﺪﻛﺘﻮﺭ
ﻓﺎﺿﻞ ﻛﺎﻣﻞ ﺍﳌﻮﺳﻮﻱ
ﺟﺎﻣﻌﺔ ﺍﻟﻜﻮﻓﺔ ـ ﻛﻠﻴﺔ ﺍﻟﻔﻘﻪ ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
. )
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ٌ ز ُم ؛ا
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، ذت و واة وا ؛ ا اكُ ا
الل وأ و أ ا ا ِدات ا أن او
إذْ ار اوإ
؛ وء اع أو ا ِا ا ط ا َ اُ إ
ل مآن اّ ان ؛ ا ا أ ن ا وإذا
ََْ ِ َاج إ ي ابُ ا أ ا و
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ٍ اع أمِ بِ اَ ا ُ ، و رم ِِم
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.ء اإنا ول اول ا َ ؛ ِ َن أن ا او
ِا َ آراء ت وذ ا و ا وا ءا
ن أر تء ذ ا ان م ان ا . ووا
ا
ا اّء، و أ اوء ، واع اّغ ، وأمء اء وا
، ة ءد وا ا ءَ اّ ء ا ا ان ن
ل ا . أ إ ا ا أ ت ا ا ام اوأ
ا
. )
٦٦٣
( ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
.. ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻷﺻﻮﻟﻴﲔ : ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻷﺻﻮﻟﻴﲔ :
ا ا و واظ ا ء ا إ ا م
)ت ا ، ي ا٨٧٤
، ا لُ )) ام (
:لُ
ل، واذاف ا أط ُ و ب إذا ا و ء إذا ا
)((٧
ا ون اي، وآ ا ا ا ا ( وزاد
)ت ا ا ذ ا٥٠٥
ل :)) ، إذ ا ( ذ
ل ذو َُه أمَو
ل َدُ ر ا أن رةٍ دالٍ ٍ
)(( ولا
٨
) تمّّ ا( و
٣٠٤
) ذ إ (٩
ا ( .وا
ا وأ إ َء اَُ ّ أن إ ا وا اأم
)ت ى ا ،٥٦٤
، ء ا ء: )) ا( أن
ا دوا أن ا ا أن ، إ آ أدِّ ء اجأو إ
ا ن ا ، .... وان ، و : ذ ن ء
، ً ا ودع ز ا : ا
ا ء ، وا ا
)(( اء٠١
اج واوء ادوات ا اج ا وى لا ا( ، و
ن ض امّ ا . ف رأي ا و
أ ء ان فل ال: ))و اذء إا
، أو دا ج
، ٌ ، وذ و م ّم ؛ ؛ ن دا
)(( د ن ب ا َُجْ اّ أم١١
انُ إ .(
ا رؤ ّ وإماْ وا ِء َ ا
ء ِا ّ
. ار ا إظ وظ د و ﺍﻻﺳﺘﺜﻨﺎ
ء
ﺍﺻﻄﻼﺣﺎً ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ
: )ت ء أ٠٨١
د)ت ا( و٥٨٢
(، و
ي )تا
٨٣٥
ا أدوات ودر ا ا ا درم ( ؛
" ُتَ )) : طل ا ا و
، ت ب ان ء ه ة ا ف " ا
ل، دون ل وا اب ا ى ، ء ا ، أوا
(( واا
)٣
)ت ا (٢٩٣
ل (
ء : أن ا:)) و
ه(() أ ه ، أو َ أد َجُ٤
ل ا(و
)ت ٣٤٦
، إذا ا ه لء" ا " ا أن(:)) ا
ف ا ء ،
، وّل او أن اج ا
)((ء ا ، و ءّ ا ، و
( ٥
)ت اّ. ا
٢٧٦
: ))و ف ا ءف ا (
أو وك ر أو ا أو جا
ة (() ا ٦
( م
ي ن ا ا ا اج ، و ا ء ا اّأن
ى أدواتل إ د د اجا ا أن أ، واوا
، و ا إ ا ن دا َجْ أ َ أنُء وا
أن
.ء ا اج إ ، و ء أدوات )
٧٦٣
( ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
... ﺃﺭﻛﺎﻥ
ﲨﻠﺔ ﺍﻻﺳﺘﺜﻨﺎء ء وأداة ا ا ن أر ة ا ء ا
ا ء أداة ا دة ن ا؛أوا
ا ب إ ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
.. ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
.. )
٨٦٣
( ، وإذا ء و أداة ار وإ ا و ا ب ان
اٌ أ إ و ء ا ا ت
ة ا اّ ِّغ ، وء ا ُ ا ؛ و اء إا
ء إ
هّ ء ا اوا إ ، ٌغُ ٌءٌ وا ٌء . ام
. ا ءا ن ا ، ، و
ب أي ا طا ر ان ا اب اَ ا
ط
ن ء ان ظ ن ٌ ط ب ، و ا
. ءَ ا إن ا ق اى ا و :ﻍ
ﱠ
ﺍﻻﺳﺘﺜﻨﺎء ﺍﳌﻔﺮ ا اُْ غءَ ا اُ أن ذ
M
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
[
\
]
^
_
`
a
L
)(. ٢١
و أو ن عا ا أن ن ااو
: ل . ( أو ا )اا
ْْ ٌََ َا إَ َْ
)٣١
( ،
إ
ُم َناّ ، وأ ا اء إ ا .وا
ا ً ؛ إ أذ وأم ء وع ا
. ءاع ارجَ أم َُُْو ا اُْ غءَ ا اُ أن ذ
M
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
[
\
]
^
_
`
a
L
)(. ٢١ ﺃﻧﻮﺍﻉ ﺍﻻﺳﺘﺜﻨﺎء: ء اء ا َّ
وا ا ر
ء ام
ء ا اع ا؛ وا ا ن ا : و
ت وار ا ه ، و ا ن ا : و
ا و وإ و إ
ء و ا ا
ا اّ أم ءال ا ء ا
زا ج وا جُ ولا ا ن واا
٤١ )
وزا وه إذ ن ا (، و
ت ا ه ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
.... )
٩٦٣
( ان ؛ء ال ا ا ا( وا ا )ا
ل أو أو ز وا ا ا
ء ا ا أم( ، و ا ) رة ا، وا
ب)إ
، وإذا ورد ذ ا ء ا )) ز ا ،(
)(( ز ، و٥١
ط ءء اازي: ))ال ا( ، و
ز (() ا ، و ا ٦١
أ ن ل( . إنّ ا
، إدر
ن اّ، أ ًءة ا ا
): الأ
( ٧١ ١راك و ا ، وإذا و ز ، و ا ء إنّ ا
). ًءا
٨١آن ا ا و ا ء أيا ا بوَلَ أ(و
ء ا بر اوأ
: ط ١
راك و ا ، وإذا و ز ، و ا ء إنّ ا
). ًءا
٨١
آن ا ا و ا ء أيا ا بوَلَ أ(و
ء ا بر اوأ
: ط
، ا م أ ا إ ذ م( ، و) ()إ .أ
) إ (ر )إ اي ما
( ٩١
: ا ْب. ان M
Í
Î
Ï
Ð
Ñ
Ò
Ó
Ô
! "
#
$
%
&
'
(
)
L
)٠٢
( . ﺃﻧﻮﺍﻉ ﺍﻻﺳﺘﺜﻨﺎء: ء
د ر ا ء ا ، ا ء إ ا
: ز ، و ا ء ا ، د ر و
M
G
H
I
J
K
L
M
N
O
P
Q
R
L
)١٢
ا (
. و ا
:
M
°
±
²
³
´
µ
¶
L
)٢٢
(، ا
: )) .و ا و ا
إِ ِْ ِِ ُ َ
عَ اَا
((M
Y
Z
[
\
]
^
_
`
a
e
L
)٣٢
ّ( ، وا
: . و ا ، واM
§
¨
©
ª
«
¬
®
¯
°
±
²
³ L
)٤٢
ا ر ، (
):م ال ا ( ٥٢ ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
......................................................... ....... )
٠٧٣
(
أن و
اع ا ل ّ
ي)تل ا٢٥٥
، ء ا ءا اٌ و زه )) : (
ز ا إ ذ و
ر (()وا
٦٢
(. ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
..... أن و
اع ا ل ّ
ي)تل ا٢٥٥
،ء ا ءا اٌ و زه )) : (
ز ا إ ذ و
ر (()وا
٦٢
(.
در أ ن )) : ل ا : إذا ا ا فة او
، ا ط ء واُ درب أ ّول امأي ا ا ، (( إ
ن وَ ا إ درب ا و ء اأي ا او
أرا ا
ا ا إ درن أ ل إن د أن
.ا ٢
) آن ا ا و ا وا ء ا .٧٢
ء ن ا (.)) و
ه (() ا م ن ا ا وارداا
٨٢
( . ٢
) آن ا ا و ا وا ء ا .٧٢
ء ن ا (.)) و ٢
) آن ا ا و ا وا ء ا .٧٢
ء ن ا (.)) و
ه (() ا م ن ا ا وارداا
٨٢
( . ٣
آن ا و . أم
)ا
٩٢
(. ه (() ا م ن ا ا وارداا
٨٢
( . ﺃﻧﻮﺍﻉ ﺍﻻﺳﺘﺜﻨﺎء:
نِ ؛ِ ا ا ِدِهةِ و َءَ اَ اُ ا أَكَ
ٌز ء از، وا ا ا ا ُ ْ أن اا
ُ ّون أم ن اّ . وأٍ ء اي
ُُ ، ٌ ٌءا
ا ونَ ا ن وا از ا ء ا ا
ب ا)) : ل لر ا وب ا ا را
ز و ا ا ول وع ا م َ ان ؛ ا ر
ذ
ول ا ا ا ا أن ورا و ... را ا ا
رٌ( أرادوا ا ا ) : ن ّ ...وأ م ّم
ل ا آد أنُ ن ا ا ا ذر، و إ
)((٠٣
( ،
ُ، لنَ ا ل ال إ م ا أن ا فة او
و م َب ان ٌ ؛ ا ٌ ّبُ إ ا :
ء اّم اّ ، وأ () لا
ا ب ا
ا ) ل وا ا ق ))اّح ا ل ا ،
ر ا و ا ا اذا م( ام زا ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
.. ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
.. )
١٧٣
( ٍ و ب ا إ ا ن َُ ، وإذا أ ل ا
ن ذ
) (( ط ولا
١٣
ا ء نّ ا ا ن د ال ، (
اَ و طا ّ أن ُل ٌ، ب إ ا ن ؛
أو أي أو ط ن ان ا ، ا ب إا
وا ا ق ا ،ب ا ًا أو آ
) عُ ا َاب ا أن ، وا
٢٣
را دة إ :ٌل أ .(
ة ار إ ٌر دل : إ ر ، و ومار إ،
ّن ؛
ء ( أنّ )إ د ، وا اء ا ا ٌَ دا
ر، د ة ّم ة ن ا و () ءت وا
ة . ) ا ًء اة و ٌ و ان٣٣
ء. (
ِارإ
. ِوم و :ﺷﺮﻭﻁ ﺻﺤﺔ ﺍﻻﺳﺘﺜﻨﺎء ط أ ا أنء إ اِّ وط ا وإنّ ا
ء إب ا أ ا أم ا أن، وا
ء ؛ أداة ا ا ن اذا
، ا َ أرب و ا : رأ ًءا
بَ ِا : أ و م وا : إ ُ
بَ اا ا : أ ُ ، و ا ا ا
،ة ق ا ، وطا ا و
ك ءا ءن ا و
: )) ، وا ر ا
َ إَ ااِ أُ
ْا
(()
٤٣
و وّقَ، وا ْ إن ا: ا ( وا
ءً و اء و اد
وء ، وود ا
:ءاّوطنا ١
) ه َْ ، و ءل اب ا. و
( ٥٣
ء ى أدواتِ ا إ اع أي انم ا ل اد اإن
و ن وا
لُ من ا إذام و ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
... ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
.. )
٢٧٣
( . ) أولَُ٦٣
ى ا ا ا َب ا : أ. (
. ا ز إ و
ا ف ل ، و ا و لون ا
، أع امة ا
ءة أراد ا و و اذا ذ اي: إن
رواون إ ة ؟ وه ا ن ء ا ذ
س
ل ، ا ن ، و ء ا ز
). ا ء ا ، ءَ ا ِْ إن ا ا
٧٣
َُْ ( ، إذ
(() إ و أن ا : ))إذا س٨٣
َ ُ م(، و
)ف ذ ٩٣
ّم ؛ د ه ا س ا ق ا(. :ﺷﺮﻭﻁ ﺻﺤﺔ ﺍﻻﺳﺘﺜﻨﺎء ز ا ا: ))وم ا ذ ذ( ، و
(( ) ا ُاوا
٧٤
ك ً ا ء ا ( . و
ذ
:ل ال: ))وأو ا ره ا
در ا ا م ،را إ ا : ، ول ا لا
ل أ ،وا ، را إ ت؛ ا
ءز ا :ا
ا د أن و ، و ا
ن ا أن : وارل ا ... ، وإ
إذا ا ءز ا ّ، وإم ، أورُ
ا ا نرك ا
ل ا أ نر ا وا
:
M
G
H
I
J
K
L
M
N
O
P
Q
R
L
) ( ٨٤
٣. ر ن ا ا ، و ق ن ا أن
: ى اء .و ا ان يا
. أ
ا و ا أن ا ان
ب ط ل م
. ارا
ب
. ا ، و ب ا ط : اق ا أ
(()ا وط ن ء اق ا ))إذا ا ن اوا
٩٤
( . اج إُ ار ا أ ، و م ا؛ ا ن اّوا ٢
.) ا ن ا ْ.أن
٤٤
ي : )) ال ا. (
، ، و ب أّزه أ ، ا ء ا ءا
ة وا ا ، و أوا
ون(() ا ، و
٥٤
( . ن ا ء أنّ ا وأه ا ا
إذا أّ ،را : ا، وارا ٌّ إء أ )) :ل ْ( إذ)أ
، ن ا أ ا ، ل ا إ
، لن ازا
أ ل ّا: إم ، و أ رع اوا و ء ، وا او
(() ٦٤
. ز ا ا: ))وم ا ذ ذ( ، و
(( ) ا ُاوا
٧٤
ك ً ا ء ا ( . :ﺷﺮﻭﻁ ﺻﺤﺔ ﺍﻻﺳﺘﺜﻨﺎء وإط
ة ، َ ا ، و ا ه ذء واع ا ذ
)(( إذا مّ ر : )) واذ ا اد ذ ن ُّّم٠٤
م ، (
)ي ذا
١٤
ذْ إِ( .أ
ن ،ت ا ِ ا ادي ا ُ أن
ف ا ، و ا ، و ءا ْا إن :ل
ريرواه ا با ا ُأت َب أ(.ووط نة ) ا
ا
ا
، ة أ ل (ج )ا
ل ا ر
داود ن ل)) :ل
أة ا اط
، ءَ ا ْ إن ل ، ا ُ رس ً و أو
ة أة وا ا إ ، ءَ ا ان
ُي م ، وا ر ءت
)(( ن أم ا وا ، ءَ ا ل إن ، ه ٍ٢٤
لُ : (. أ
ج ا أة م ا ا ُ ا
ب وٍ و دي أي ْ دون أنا ا
نم ُ ان ر ة ا ا ٍَ أ و
وءَ ا ْلَ : إن ْ أن ؟ ون م إذا ديٍ
م ؛ ا ي اا ا جن م ( ر ) . وا
)ء ا ان
( ٣٤ل ا .
مو
. دُ ان ا ء ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
.. ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
.. )
٣٧٣
( ٢
.) ا ن ا ْ.أن
٤٤
ي : )) ال ا. (
، ، و ب أّزه أ ، ا ء ا ءا
ة وا ا ، و أوا
ون(() ا ، و
٥٤
( . ن ا ء أنّ ا وأه ا ا
إذا أّ ،را : ا، وارا ٌّ إء أ )) :ل ْ( إذ)أ
، ن ا أ ا ، ل ا إ
، لن ازا
أ ل ّا: إم ، و أ رع اوا و ء ، وا او
(() ٦٤
. :ﺷﺮﻭﻁ ﺻﺤﺔ ﺍﻻﺳﺘﺜﻨﺎء و
ذ
:ل ال: ))وأو ا ره ا
در ا ا م ،را إ ا : ، ول ا لا
ل أ ،وا ، را إ ت؛ ا
ءز ا :ا
ا د أن و ، و ا
ن ا أن : وارل ا ... ، وإ
إذا ا ءز ا ّ، وإم ، أورُ
ا ا نرك ا
ل ا أ نر ا وا
:
M
G
H
I
J
K
L
M
N
O
P
Q
R
L
) ( ٨٤ :
M
G
H
I
J
K
L
M
N
O
P
Q
R
L
) ( ٨٤
٣. ر ن ا ا ، و ق ن ا أن
: ى اء .و ا ان يا . أ
ا و ا أن ا ان
ب ط ل م
. ارا ب
. ا ، و ب ا ط : اق ا أ
(()ا وط ن ء اق ا ))إذا ا ن اوا
٩٤
( . اج إُ ار ا أ ، و م ا؛ ا ن اّوا
ا ، زٌ إَ زَ : أ ّل : ))إم ة اذ ا ء ا ا ب
. ا ، و ب ا ط : اق ا أ
(()ا وط ن ء اق ا ))إذا ا ن اوا
٩٤
( .
اج إُ ار ا أ ، و م ا؛ ا ن اّوا
ا ، زٌ إَ زَ : أ ّل : ))إم ة اذ ا ء ا ا ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
.... )
٤٧٣
( (( ) ٍ وا ا وا ّم ؛ ٠٥
ل أن . (
، ار اُ أراد رّّم ة ؛ ا ان ة ، و إمة دم
). :ﺷﺮﻭﻁ ﺻﺤﺔ ﺍﻻﺳﺘﺜﻨﺎء ُُز ر ُاروا
١٥
اف ا)) ا ارنّ ا ( ؛
(()٢٥
أو أم اء ، ء ا )) ان ا (.وا
(()أ
٣٥
ل ،(
، ٍا ار ا ا إ ة إ ٌا
ء ق ا م ء ا ا إ ة إ ّ ل
اق ا اول وء ا ا ا ا ء ا أي اا
. وط و
وذم ار ا إ ة إ ل
م ا ا وا ة ا م
اف ا
م
) ( ٤٥ ﻣﻮﺍﺿﻊ ﰲ ﺍﻻﺳﺘﺜﻨﺎء واع آراءِ ا وا ف ا ء ا ا ه
َم
ء و ا ا ءا
:
:ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﺍﻟﻌﺪﺩ واع آراءِ ا وا ف ا ء ا ا ه
َم
ء و ا ا ءا
:
:ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﺍﻟﻌﺪﺩ :ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﺍﻟﻌﺪﺩ د ، ا ن ان د ا ءِ اِ ن إ اذ
ام ن اّة .ا ون ا ة و ن إ : َل ْ أن
: د اذا ا ءُ اَْ .أ
ل اد ا وا ن ا
وان ا إذاء ا ا ّ : )) إما
(() ةا ( ٥٥ د اذا ا ءُ اَْ .أ
ل اد ا وا ن ا
وان ا إذاء ا ا ّ : )) إما
(() ةا ( ٥٥ ن ) ّد ا ا ء ا .ب
٦٥ :ل ا أ أن ( ، أي
. امة دم
: ل ي ا ا ذ ذ . وإ
، و : وا ، و و و : أل ا ق))
، ت ا ا و : أ ، و و : ل ا ق ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
... )
٥٧٣
( ، ن ذ واذا وا ظ أو
ء ا ق
م ؛ ا ا و و ء ا ، و ا ّم ؛أ
ق (() و ا ( ٧٥
ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﻋﺪﺓ ﲨﻞ
: : ل
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)٨٥
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د َْ ا او ا ( ؟ و ام ) ( أو
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)٩٥
ل ا ،(
د ل ء و ا ا ا ءإنّ ا
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ل ا إ
): ٠٦
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).١٦
(ً ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
... ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
.. )
٦٧٣
( ٢
) ا ء اع ا. ر
٢٦
: .(
M
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٣٦ )ﱠ
(. ، وذ ف ا ا اء و
إ أ ءار ا ج ا ا اع وان ا اذا
ان ء را .ا إا واس واا
س ا ال ا
اا وا اس ا ا واا
)ا
( ٤٦ ٣
ل ا ،ول اء إع ا. ر
ه ، و ا )) :
)(( ا إ ٥٦ ن ول ا د وا ، (
. نم ا ٤
ء ا . :ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﺍﻟﻌﺪﺩ أن
: ا
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)٦٦
دةل ا و ا (ء )ا(
. ل ا دو
ل ا ، ا إ وا ا دق او
دة
ة ا ن ا ؛ وام اء إا
ره ا ة اء إدة ا ، واذا ءن ان وا
ن ن أ و ق ا ُّمء ؛ ا ا ا
ا
ء)٧٦
( واآن ا ا دة ا ا ءد ا و او
؛ وا اُ أنّ ا إ ا ة ا ا
ا و ذ اب ، و ا ي ٌ أ ّم
ه ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
... )
٧٧٣
( ه ا : )) و ن أ ط ا م ، و ام او
ة (( ) ا ض ٨٦
: )) وا ا ا طَ اّ ا( و
ف ، ر ا ن م أ ذ ، ول أ ا ان ، ل
)(( أأ
٩٦
) ا ذ( و
٠٧
( ، وا
دي ) ا
١٧
) من ا ( ، وا
٢٧
ن ) (، وا
٣٧
ن انّ ا ، (
)(( ل ا ا ر ف ، و ه ا ل: ))و٤٧
( . وان أو أ ُ: )) إذا ذ ل ا
ء ، وا
، ن إ ن و : ا ٌم إن
، ه ُِ ؛ ، ص ؛ إذ ا
ُ ) ة ا آ ا وا زل إ اء وث ا
، و ( وِ)أ ، وم (( ذ )إ( إا
، ن إذا ا ذ ، إ َّ و م
وا ، وان أو ا ن إن ء أ او
)(( ا
٥٧
( . :ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﺍﻟﻌﺪﺩ )
٨٧٣
( ة وا : ا ، اد ام ، اء او، وا
(()م ن إا
٦٧
(
ل : ))واذا ن أّوأ
ت وا ءات ا ن
إء ران ا ، ن إ ن و : ا ،وا
ن إ ن ، وا : ا ا ر ا ا إذات ، وا
ل وا وا ان ا ، ن
(ا ا : ) ا
)((تف ا آ
٧٧
. (
) ا ُ ا أنطى و
٨٧
( :
ى و ارادة ا ٌ د ا د ّ : أمأ
: انM
\
]
^
L
((ا ا : )) إ إ
. د ل ، و د إ
، ام ا : ا ، ض وا ّ ا ؛ إن د ّ : أمما
ا وا ، وإ ، إ ، وزرت ا وا
داركء ، وا ا: ا
. ا إ ا ، وار ا
د ُ ء أو ن ا ، واذا داو ن ا : اذاا ة وا : ا ، اد ام ، اء او، وا
(()م ن إا
٦٧
(
ل : ))واذا ن أّوأ
ت وا ءات ا ن
إء ران ا ، ن إ ن و : ا ،وا
ن إ ن ، وا : ا ا ر ا ا إذات ، وا
ل وا وا ان ا ، ن
(ا ا : ) ا
)((تف ا آ
٧٧
. ( )((تف ا آ
٧٧
. (
) ا ُ ا أنطى و
٨٧
( :
ى و ارادة ا ٌ د ا د ّ : أمأ
: انM
\
]
^
L
((ا ا : )) إ إ
. :ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﺍﻟﻌﺪﺩ ه ا : )) و ن أ ط ا م ، و ام او
ة (( ) ا ض ٨٦
: )) وا ا ا طَ اّ ا( و
ف ، ر ا ن م أ ذ ، ول أ ا ان ، ل
)(( أأ
٩٦
) ا ذ( و
٠٧
( ، وا
دي ) ا
١٧
) من ا ( ، وا
٢٧
ن ) (، وا
٣٧
ن انّ ا ، (
)(( ل ا ا ر ف ، و ه ا ل: ))و٤٧
( . وان أو أ ُ: )) إذا ذ ل ا
ء ، وا
، ن إ ن و : ا ٌم إن
، ه ُِ ؛ ، ص ؛ إذ ا
ُ ) ة ا آ ا وا زل إ اء وث ا
، و ( وِ)أ ، وم (( ذ )إ( إا
، ن إذا ا ذ ، إ َّ و م
وا ، وان أو ا ن إن ء أ او
)(( ا
٥٧
( . ل ا ، و ا ا ي وأ وُ ا اّوأ
ل ، ا وذ ا ع ا ، ل ا ل ا إ
إذ ذ ا ا رك ، اح ا دي اء ا و
ال : )) أن
ء ا ا او ، إذا ُ َف ا
:
M
\
]
^
_
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L
ة أنّ ا ا ،
ان ا ( ، أو أنّ ار ) ا (ون) ا
،ل وا ا زع ا ب ن ، ة او اا
وال ا ، ن ا و
ا ، و أو أ
م ة ا ام إن ، واات ا ا ا وم ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
.. ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
. :ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﺍﻟﻌﺪﺩ : ا ا و ا اه ا، وادمل وا
١. ا
ن ا، إذ ا ا وا ا ا ء
دون دا ا و اام
.
٢. ر ا ء ات ا د ٌ ن
. ود اا
٢. ر ا ء ات ا د ٌ ن
. ود اا .٣ء ان ارس ا
ّوه ْ ، ءع ا
ّوه ين ا ا ءب ا ا و اا
ء .ب اا .٣ء ان ارس ا
ّوه ْ ، ءع ا
ّوه ين ا ا ءب ا ا و اا
ء .ب اا ٤. ا ، ا ا واا ا ا ءا
ا ا دون د ا ا وا ّوا
رات وا ا ر ا
. نه ا و ا تظ ٤. ا ، ا ا واا ا ا ءا
ا ا دون د ا ا وا ّوا
رات وا ا ر ا
. نه ا و ا تظ ٥. ا ن ات ودر ا ءن ادرس ا
اد . ا ار ا ق او
.٦ ال وع ا ن ال ااط
ا ا
ء ، ا ، وُ ذ ا ء ا ام ،
وارواوه واا ورس
وااو ٥. ا ن ات ودر ا ءن ادرس ا
اد . ا ار ا ق او .٦ ال وع ا ن ال ااط
ا ا
ء ، ا ، وُ ذ ا ء ا ام ،
و ا رواوه واا و ر س وا او
ق .ا .٦ ال وع ا ن ال ااط
ا ا
ء ، ا ، وُ ذ ا ء ا ام ،
و ا رواوه واا و ر س وا او
ق .ا ٧. ا واط ا أو إ ا اد اد ا ءا
، ا
. ذ ك اده ا ا وا :ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﺍﻟﻌﺪﺩ د ل ، و د إ
، ام ا : ا ، ض وا ّ ا ؛ إن د ّ : أمما
ا وا ، وإ ، إ ، وزرت ا وا
داركء ، وا ا: ا
. ا إ ا ، وار ا
د ُ ء أو ن ا ، واذا داو ن ا : اذاا ) ا ُ ا أنطى و
٨٧
( :
ى و ارادة ا ٌ د ا د ّ : أمأ
: انM
\
]
^
L
((ا ا : )) إ إ
. د ل ، و د إ
، ام ا : ا ، ض وا ّ ا ؛ إن د ّ : أمما
ا وا ، وإ ، إ ، وزرت ا وا
داركء ، وا ا: ا
. ا إ ا ، وار ا
د ُ ء أو ن ا ، واذا داو ن ا : اذاا
. د : اا
ا ؛ إذ ، وإن ا ا : إن اا
اا
. ل وا
ن اذاّ، و ا ءن ا إذا راءه ا ة ن و
ن و و : ا ا ء د ءا
) ا ء ا . ءْ اَ إ
٩٧
( . ﺍﳋﺎﲤــﺔ
ا رب ا
ا وآ ا ا ا ة واوا
ن(.ل و ) ءة وا ا ا ن اذاّ، و ا ءن ا إذا راءه ا ة ن و
ن و و : ا ا ء د ءا
) ا ء ا . ءْ اَ إ
٩٧
( . ا رب ا
ا وآ ا ا ا ة واوا
ن(.ل و ) ءة وا ا ا ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
.... ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
.. )
٩٧٣
( دم وأن ورد ٍ و ز أن ل ا أ ا
: ا ا و ا اه ا، وادمل وا
١. ا
ن ا، إذ ا ا وا ا ا ء
دون دا ا و اام
. Abstract Praise be to Allah, peace and blessings be the best all the people
Mohammed Secretary and Haddat right and the leaders of all creatures
and after: Looks at the knowledge of the language being one of the terms of
the diligent worker with assets of Islamic jurisprudence to ete necessary ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
. ٠٨٣
( to know the holy book of God, which is the first source of Islamic law,
which touches the daily conduct practical for every Muslim such as
prayer, fasting, transactions, etc., it is known that worships rely on
verbal text and acts and statements and approve the infallible up to
taxpayers by speech inaudible or illegible after the era of the infallible;
and if so, must know the Arab speech styles; because the Koran was
revealed. It is the manner of allocation methods and means some of
what covered the speech with being compared with his output falls
within the style allocation among other types, including its relevant
wording of Commons, such as the condition and character and purpose
exception including Mainvsal for the term of Commons such as mental
and auditory, when a speech exception is devoted to the language,
which will be the area of research, God Almighty. Find the three sections was taking exception to the first part, and
idiomatically definition language when linguists and fundamentalists
when it reported the views of some linguists and fundamentalists. The
second section, entitled exception to the Staff stated inter exception
clause exception vacuum, and the types of exception, the terms of the
exception. The third Almiges was titled Placements in exception ensures
the exception of the number and the exception of a few sentences, and
finally was the finale, which contained the most important findings. Alla
ask the Almighty to reconcile ١
ب ن ا (/ ٤١
٥١١
٢
: س ا( ا
٤١٢ /٢
٣
ا ا (/٢
٥٨١
٤
( ا
٨٣
٥
ح ا (/ ١
٤١٤
٦
ح ا (/٢
٢٨١
٧
ن ( ا
/ ١
٧٣١
ز ا اح ا ، و
/ ٣
١٨٢
. م. Abstract ا
٨
( ا
/ ٢
٩٧١ ١
ب ن ا (/ ٤١
٥١١
٢
: س ا( ا
٤١٢ /٢
٣
ا ا (/٢
٥٨١
٤
( ا
٨٣
٥
ح ا (/ ١
٤١٤
٦
ح ا (/٢
٢٨١
٧
ن ( ا
/ ١
٧٣١
ز ا اح ا ، و
/ ٣
١٨٢
. م. ا
٨
( ا
/ ٢
٩٧١ )
١٨٣
( ٩
د ر وا ا : ( ام
٦٢ / ٣
٠١
( ا
١٤٥ /٢
١١
د ر وا( ا
/ ٣
٧٢١
٢١
ان/ ( ال
٤٤١
٣١ ا /ءم( ا
٣
٤١
ا ر: ا( ام
١/
٢٤٥
ل / رج ا :، وام
٣٤
ديء :،وام
ل / ا ل إا
٨٣١
. ٥١
ذا ( ا
. ٥٨ /٢
٦١
ل ( ا
٠٣ /٣
٧١
ل ا( ام
١٣ /٣
. ا ، . ٨١
ر ، ا: ا( ام
٣/
٩٧٢
٩١
ل ا أ ة: ا( ام
٢/
٣٧٦
( ٠٢
/ا
٠٣١٣
١٢
/ ا( ا
٥٢ ٦٢
ء/ ا. و
٢٩ ، ٩٢
،
٧٥١
٢٢
اء/( ا
٧٧
٣٢
/ ا ء( ا
٧٥١
٤٢
د/ (٣٤
٥٢
ص ا م :ح د . م اان ا( د
. ٣٣
٦٢
ل ا ل ا :( ام
١١٢
. ٧٢
ازي ل: ا( ام
١/
٨٠٤
اح ا، و
٣/
٦٨٢
٨٢
. ي ا ا ا ، . د ا يء ا( ا
د داب ، ا ٨٢
٦٠٠٢
.
٩٢
. ر ا : ا( ام
/ ٣
٩٧٢
٠٣
جا ا ، . د ام : ( ام
٢
ص
٤٨٦
ا . اد ،
٧٨٩١
ر .وم
بره ا ر ا ا و/ ١
٣٦٢
. ١٣
( ا
/ ٢
. ٧٨
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/٦ ، ٠٣
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)
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/
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......................................................... ....... )
٣٨٣
( )
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( ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
......................................................... ....... )
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٦٥
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/ ٢
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) و ا ام
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: ااو
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ا ٤
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٧٧
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/ ٢
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/ ٢
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١/
٢١٢
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٢٩
٢٦
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١/
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ح :.وام
اا
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٣١٣
٣٦
ة /( ا
٣٣- ٤٣
٤٦
: ا( ام
١/
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٥٦ ا( ا
) و ا ام
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( . ٠٩ /٦ ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
.. )
٤٨٣
( ﻗﺎﺋﻤﺔ
ﺍﳌﺼﺎﺩﺭ ﻭﺍﳌﺮﺍﺟ
ﻊ ١. .آن اا ٢. وي )ت ل ا ل إج ا جح ا جا
٥٨٦
(
ا
)تا
٦٥٧
)ت ج ا ه(وو
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٤٨٩١
.
٣. )ت أ . ل ا أ ا
٦٥٤
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وت . ةق اا
. ١
٠٨٩١
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٦٥٤
. دار : أ( .م
وت . ةق اا
. ١
٠٨٩١
. ٤. ا ل ا أ ا
ي) ا ١٣٦
زاق ا : .(
ض ودار ا . ا . دار ا١
وت .٣٠٠٢
. ٤. ا ل ا أ ا
ي) ا ١٣٦
زاق ا : .(
ض ودار ا . ا . دار ا١
وت .٣٠٠٢
. ٥. . ي ا أ ا.. د ا يء اا
دداب . ا ٨٢٦٠٠٢
. ٦. )ت ام ا أ ن (و ا) اوا
٠٦٣
(
ض رق: ط
.( ة )د.ت . ا . دار اا ٦. )ت ام ا أ ن (و ا) اوا
٠٦٣
(
ض رق: ط
.( ة )د.ت . ا . دار اا ٧. اج( )ت ا ) ا وفي اي ا ا ا لا
٦١٣
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٦١٣
ن د.ت. . وت . ا . ا ا ، : د. ( ٨. ،من ا ،بن ا َبَف اإر
. ن : ر
. ا، ةا
٨٩٩١
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٥٥٢١
(.
وت ق دار ا :. ١
٠٠٠٢
. ٩. )ت م ا ،ل ا ا ل إد اإر
٥٥٢١
(.
وت ق دار ا :. ١
٠٠٠٢
. . ٠١
)ت ا ر .ال اا
٨٨٣١
وت .رف( دار ا٥٢٤١
. . ١١ اا
) ر ار ا ل ا أ ٤٩٧
. ﻗﺎﺋﻤﺔ
ﺍﳌﺼﺎﺩﺭ ﻭﺍﳌﺮﺍﺟ
ﻊ ا :.(
ف اووزارة ا
٢٩٩١
. . ٢١
ي )ت ا ا ء ا . ل ال ا٢٥٥
: د . . (
ة اث .دار ا ا ز. ١
٢٩٩١
. ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ
..... )
٥٨٣
( . ٤١
)ت ا ا ا .ل ا أ نا
٨٧٤ ا.:د( م
. ء . دار اا
. ٤
٧٩٩١
. . ٥١
ا اق ا ا . ل ا أ ةا
ازي)ت دي اوز
٦٧٤ .د . دار ا : (. ١
٣٨٩١
. . ٥١
ا اق ا ا . ل ا أ ةا
ازي)ت دي اوز
٦٧٤ .د . دار ا : (. ١
٣٨٩١
.
. ٦١
ي )ت ا آن (. أ آي ا ن ا)ي ا٠١٣
. (
وت .اث اء ا . دار ا د ١
وت . .١٠٠٢
.
. ٦١
ي )ت ا آن (. أ آي ا ن ا)ي ا٠١٣
. (
وت .اث اء ا . دار ا د ١
وت . .١٠٠٢
. . ٧١ ا د . ار واا
)ت م ا٣٠٤
ا . :د (
وت . ا . زم أ٨٩٩١
. . ٨١
اح ا . وا ا ا ل، ا ا واا
ج)ت ا
٩٧٨
)ت ا ا ا (١٦٨
وت / ( . دار ا. ١
٦٩٩١
.
ا . ٩١
ي) ا ا. ل ا وع ا ٢٧٧ : د.(
.( )د. تا ا . ٠٢
) ت ذاما أ ظ ، ل ا أ ا
٠١٥
أ .: د (
. ا .ا ٢
وت ..٠٠٠٢
. . ٠٢
) ت ذاما أ ظ ، ل ا أ ا
٠١٥
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. ا .ا ٢
وت ..٠٠٠٢
. . ١٢ ا ا . اا
اج )ت١٨٤
. دري ا: إ. (، ١
١١٤١
. .ان . ا . ر ا . اا . ٢٢
ن )ت ا . مح ا ن ا٦٠٢١
د : (
ة ، ا ا . ا
٢٠٠٢
. . ٣٢
ل ا . ﻗﺎﺋﻤﺔ
ﺍﳌﺼﺎﺩﺭ ﻭﺍﳌﺮﺍﺟ
ﻊ ا روس اا
ول( ا )ا ا
)ت
٦٨٧
. ااث وا . ا ا ا. ( . ٣٢
ل ا . ا روس اا
ول( ا )ا ا
)ت
٦٨٧
. ااث وا . ا ا ا. (
. ٤٢ ار .دار ا : ، م اان اد
١
وت . .٣٩٩١
. . ٤٢ ار .دار ا : ، م اان اد
١
وت . .٣٩٩١
. . ٥٢
: أ. ا ا ا ا .ال ا أ إرا
ه ، دار م اا
٨٤٣١
. . ٥٢
: أ. ا ا ا ا .ال ا أ إرا
ه ، دار م اا
٨٤٣١
.
. ٦٢
)ت ا ا ا. ل ا أظ ا وظ ارو
٠٢٦
: (
وت، . دار ا ا ا ،د
. ٦
٨٩٩١
. )
٦٨٣
( . ٧٢ ر ا . وي ا وي( ااب ا )اا
اد
)ت ار
٨٩٥
ان . . ا . ر ا . ا ا. (
. ٨٢
)ت ا ا ى. أ اا
٨٥٤ در ا : . (
. ن . وت . ا. دار ا
٣ . ٣٠٠٢ . ٩٢
)ت ن ا ا .ء ا ا .٨٤٧
(. ١
٣١٤١
.
ن ، وت . ا . ٩٢
)ت ن ا ا .ء ا ا .٨٤٧
(. ١
٣١٤١
.
ن ، وت . ا . ٠٣
وت ، . ا رق وط در ا :. . ح ا
، ان: دار ا١٠٠٢. . ١ . ٠٣
وت ، . ا رق وط در ا :. . ح ا
، ان: دار ا١٠٠٢. . ١ . ١٣
)ت ل ا، ة ا وة ا ح٢٧٦
ن م : .(
ورين ا ا
. اد . م ا .٨٧٩١
. . ١٣
)ت ل ا، ة ا وة ا ح٢٧٦
ن م : .(
ورين ا ا
. اد . م ا .٨٧٩١
. . ٢٣
دي )ت ا ا ا ا . ﻗﺎﺋﻤﺔ
ﺍﳌﺼﺎﺩﺭ ﻭﺍﳌﺮﺍﺟ
ﻊ ا ا ح٦٨٦
: د (
اب ، دار ا أ
١
وت ،٨٩٩١
. . ٣٣
ر )ت ا وف ا ا أ . اح ا٢٧٩
د(. ا
، ا ى ،و ا ا .٧٨٩١
. . ٤٣
)ت ا ،ح ا٦٤٦
، دار ا ا: أ.(
ا
. ١
٣١٠٢
. د . ٥٣
ر ا ا ح
ي)ت
٦٧٦
. ن . دار ا : (
وت، ٥١
٨٠٠٢
. . ٦٣
)ت ا ا . ل ا ا ةا
٠٦٤
ر:. (
. ريما
١
ان .ا . رة .٧١٤١
. . ٦٣
)ت ا ا . ل ا ا ةا
٠٦٤
ر:. (
. ريما
١
ان .ا . رة .٧١٤١
. . ٧٣
)ت م ا ري ا ح ري ا٢٥٨
ا :،(
ا. دار ا ا اد ز و، ١
٩٨٩١
. . ٨٣
)ت م ا ا . (د)ار وا ا٣٠٤
ا : (
. ز ا ١
ا٨٩٩١ . ٨٣
)ت م ا ا . (د)ار وا ا٣٠٤
ا : (
. ز ا ١
ا٨٩٩١ . ٩٣
دي )ت وز آ ا ا. س اا
٧١٨
. وت . ا ( ٨
،
٥٠٠٢
. . ٩٣
دي )ت وز آ ا ا. س اا
٧١٨
. وت . ا ( ٨
،
٥٠٠٢
. )
٧٨٣
( . ٠٤
)ت ما ر . ل ا د ااط٩٨٤
: . (
وت . ا . دار اا
٧٩٩١
. . ٠٤
)ت ما ر . ل ا د ااط٩٨٤
: . (
وت . ا . دار اا
٧٩٩١
. . ١٤
)ت زا ا . ل اام٠٣٢١
ان . . ط ا ا( . ا
٨٧٣١
. . ٢٤
ر) ت ل ا ،بن ا١١٧
ن وت. در (. دار٥٥٩١
. . ٢٤
ر) ت ل ا ،بن ا١١٧
ن وت. در (. دار٥٥٩١
. . ٣٤
ازي)ت ا ا ،إل ا أ ا
٦٨٤
. ا( ا
. ةا
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|
https://openalex.org/W2118541630
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https://zenodo.org/record/1091/files/Manuscript-Sinoxygen-2012-004.pdf
|
English
| null |
A Versatile Synthesis of Meyers’ Bicyclic Lactams from Furans: Singlet‐Oxygen‐Initiated Reaction Cascade
|
Angewandte Chemie
| 2,012
|
cc-by
| 3,850
|
A versatile new synthesis of Meyers’ bicyclic lactams: singlet oxygen reacts
with furan substrates to initiate a remarkable cascade reaction sequence**
Dimitris Kalaitzakis Tamsyn Montagnon Ioanna Alexopoulou and Georgios Vassilikogiannakis* Dedicated to Professor K. C. Nicolaou range from harnessing microwave energy to accomplish the desired
reaction[6a] or employing Lewis acid catalysis,[6b] to activating the
acid.[6c] A second commonly employed,[7] but stepwise, route to
these bicyclic lactams, also originally emanating from the Meyers’
group,[2a] relies heavily on N-acyliminium chemistry extensively
elucidated by Speckamp.[8] In this case, a succinimide intermediate
is substituted at the C5 position (usually by addition of a Grignard
reagent) and then encouraged to undergo intramolecular cyclisation
under acidic conditions. Several domino reactions affording specific
bicyclic lactams have also been reported recently,[9] as well as, other
stepwise approaches to the scaffold.[10] This unceasing interest in
finding new syntheses for the Meyer’s bicyclic lactams in itself
provides testament to the usefulness of the scaffold and the diversity
of potential applications there are for it. Herein, we introduce a new
and mild method for the synthesis of a wide variety of Meyers’
bicyclic lactams. This entirely novel approach, which uses a singlet
oxygen-mediated cascade reaction sequence, is made particularly
powerful because the one-pot reaction begins from furan substrates
which can be multiply-substituted (A, Scheme 1) at will and with
ease, thus allowing access to highly substituted scaffolds, frequently
with excellent stereoselectivity, as products from the initial reaction
without extra manipulations. In this way, the new methodology also
exhibits a very high degree of step-[11] and atom-economy[12] and
utilises the selective green reagent, singlet oxygen, to achieve these
very rapid increases in molecular complexity with precision and
minimum waste, thereby attaining many of the recently established
criteria for an ideal synthesis.[13] A new and powerful method for the synthesis Meyers’ bicyclic
lactams beginning from furans is reported herein that uses a
remarkable one-pot singlet oxygen-initiated cascade reaction
sequence to deliver the corresponding bicyclic lactams in high yield. Ever since they were pioneered Meyers’ homochiral bicyclic
lactams[1, 2] (B, Scheme 1) have been exceedingly popular and
versatile scaffolds for the enantioselective construction of new
stereogenic centres, including the most challenging type -
quaternary carbon centres. DOI: 10.1002/anie.200((will be filled in by the editorial staff)) DOI: 10.1002/anie.200((will be filled in by the editorial staff)) (Powerful new
method for
synthesizing
bicyclic lactams ) []
The research leading to these results has received funding
from the European Research Council under the European
Union’s Seventh Framework Programme (FP7/2007-
2013)/ERC grant agreement no. 277588. We also thank
Prof. Robert Stockman and Mr. George Procopiou for their
help in taking HRMS. []
Prof. Dr. G. Vassilikogiannakis, Dr. D. Kalaitzakis, Dr. T.
Montagnon, Ms. I. Alexopoulou
Department of Chemistry
University of Crete
Vasilika Vouton, 71003, Iraklion Crete (Greece)
Fax: +30 2810 545166
E-mail: vasil@chemistry.uoc.gr A versatile new synthesis of Meyers’ bicyclic lactams: singlet oxygen reacts
with furan substrates to initiate a remarkable cascade reaction sequence**
Dimitris Kalaitzakis Tamsyn Montagnon Ioanna Alexopoulou and Georgios Vassilikogiannakis* They have been utilised in myriad
different ways to target a wide variety of natural products,[2, 3] non-
natural molecules possessing interesting biological activity,[2, 4] and,
also in a diverse array of other synthetic endeavours.[2, 5] The most
general method for their synthesis, introduced in the seminal work
of Meyers’,[1, 2] wherein a γ-ketoacid is condensed with an
aminoalcohol under dehydrating conditions in refluxing toluene, is
still, by far, the most commonly employed means of accessing the
bicyclic scaffold B. Modifications have been made with the aim of
introducing milder variants[6] to the Meyers’ lactamisation; these Scheme 1. Generalised representation of the transformation
achieved with this new methodology. Scheme 1. Generalised representation of the transformation
achieved with this new methodology. The idea for this new methodology was born from our
experience in the field of furan photooxygenations[14] which has
taught us to regard the furan motif as a readily accessible and easy
to manipulate, 1,4-dienone equivalent (or precursor).[15] Once this
fact was acknowledged, it seemed potentially very interesting to ask
whether the aforementioned intermediate (C, Scheme 2) could be
intercepted by an amino-alcohol, and, from thence, rearrange and
cyclise (under acid catalysis) to afford the Meyer’s bicyclic lactams,
without resorting to the dehydrating conditions, or high
temperatures, which have traditionally been employed. The highly
ambitious concept is summarised, in mechanistic terms, in Scheme 2
(note: only selected steps are shown here). Thus, when a furan is
oxidised, upon exposure to singlet oxygen in MeOH, intermediates
of type C are known to form easily after the in situ reduction (with
Me2S) of a hydroperoxy functionality that remains after the solvent
induced collapse of the initially formed endoperoxide adduct has
occurred.[14,15] We hypothesised that introduction of an 1,2-
aminoalcohol into the flask at this stage should regioselectively
afford the aminal D (via the more stable, more highly substituted, []
Prof. Dr. G. Vassilikogiannakis, Dr. D. Kalaitzakis, Dr. T. Montagnon, Ms. I. Alexopoulou
Department of Chemistry
University of Crete
Vasilika Vouton, 71003, Iraklion Crete (Greece)
Fax: +30 2810 545166
E-mail: vasil@chemistry.uoc.gr
[]
The research leading to these results has received funding
from the European Research Council under the European
Union’s Seventh Framework Programme (FP7/2007-
2013)/ERC grant agreement no. 277588. We also thank
Prof. Robert Stockman and Mr. George Procopiou for their
help in taking HRMS. Supporting information for this article is available on the
WWW under http://www.angewandte.org or from the
author. []
Prof. A versatile new synthesis of Meyers’ bicyclic lactams: singlet oxygen reacts
with furan substrates to initiate a remarkable cascade reaction sequence**
Dimitris Kalaitzakis Tamsyn Montagnon Ioanna Alexopoulou and Georgios Vassilikogiannakis* Dr. G. Vassilikogiannakis, Dr. D. Kalaitzakis, Dr. T. Montagnon, Ms. I. Alexopoulou
Department of Chemistry
University of Crete
Vasilika Vouton, 71003, Iraklion Crete (Greece)
Fax: +30 2810 545166
E-mail: vasil@chemistry.uoc.gr []
Prof. Dr. G. Vassilikogiannakis, Dr. D. Kalaitzakis, Dr. T. Montagnon, Ms. I. Alexopoulou
Department of Chemistry
University of Crete
Vasilika Vouton, 71003, Iraklion Crete (Greece)
Fax: +30 2810 545166
E-mail: vasil@chemistry.uoc.gr []
The research leading to these results has received funding
from the European Research Council under the European
Union’s Seventh Framework Programme (FP7/2007-
2013)/ERC grant agreement no. 277588. We also thank
Prof. Robert Stockman and Mr. George Procopiou for their
help in taking HRMS. Supporting information for this article is available on the
WWW under http://www.angewandte.org or from the
author. Supporting information for this article is available on the
WWW under http://www.angewandte.org or from the
author. 1 Table 1. One-pot synthesis of various bicyclic lactams starting from
furan substrates. Scheme 2. Mechanistic summary for the one-pot cascade reaction
sequence that transforms furans A into bicyclic lactams B. Scheme 2. Mechanistic summary for the one-pot cascade reaction
sequence that transforms furans A into bicyclic lactams B. oxonium cation) that would open to imino enal E, as shown in
Scheme 2, and then close again to afford 2-pyrrolidinone G, via 2H-
pyrrol-2-ol F. It was hoped that addition of an acid at this point,
would affect the desired hydroxy-acyl imminium cyclisation to give
the corresponding bicyclic lactam (H B) as the final product of a
highly ambitious one-pot cascade reaction sequence. Gratifyingly, and, despite the extraordinary complexity of the
designed reaction cascade, we were able to successfully transfer the
concept from a hypothetical “on paper” idea to a working laboratory
protocol, as the results detailed in Table 1 clearly show. Thus, in the
simplest case where 2-methylfuran was oxidised with singlet
oxygen,[16] followed by in situ reduction (Me2S) of the resultant
peroxide, and then by the addition of 2-aminoethanol, and later
catalytic TFA (30 mol%), to the same flask, the desired
corresponding Meyers’ bicyclic lactam was obtained (Entry 1, Table
1). With this proof of principle in hand, we set out to systematically
investigate the effect of varying both the furan and 1,2-aminoalcohol
substitution patterns, and, concomitantly, delineate the precise
stereochemical outcomes of the cascade reaction sequence (Table 1). Keywords: Meyers’ bicyclic lactams · singlet oxygen · cascade
reaction · furan oxidation · 2-pyrrolidinones De Clercq, R. Bouillon, A. Verstuyf, Bioorg. Med. Chem. Lett. 2004, 14, 3885-3888; d) T. D. Aicher, B. Balkan, P. A. Bell, L. J. Brand, S. H. Cheon, R. O. Deems,
J. B. Fell, W. S. Fillers, J. D Fraser, J. Gao, D. C. Knorr, G. G. Kahle,
C. L. Leone, J. Nadelson, R. Simpson, H. C. Smith, J. Med. Chem. 1998, 41, 4556-4566; e) M. D. Ennis, R. L. Hoffman, N. B. Ghazal, D. W. Old, P. A. Mooney, J. Org. Chem. 1996, 61, 5813-5817; f) A. I. Meyers, L. Snyder, J. Org. Chem. 1993, 58, 36-42. l
d
i
l
) S S
i [5] y
,
y
,
g
,
,
For selected representative examples, see: a) S. Sen, V. R. Potti, R. Surakanti, Y. L. N. Murthy, R. Pallepogu, Org. Biomol. Chem. 2011,
9, 358-360; b) J. E. Resek, J. Org. Chem. 2008, 73, 9792-9794; c) D. Enders, O. Niemeier, T. Balensiefer, Angew. Chem. 2006, 118, 1491-
1495; Angew. Chem. Int. Ed. 2006, 45, 1463-1467; d) H. C. Shen, F.-
X. Ding, S. L. Colletti, Org. Lett. 2006, 8, 1447-1450; e) T. E. Nielsen,
S. Le Quement, M. Meldal, Org. Lett. 2005, 7, 3601-3604; f) M. D. Groaning, G. P. Brengel, A. I. Meyers, Tetrahedron 2001, 57, 2635-
2642; g) J. A. Nieman, M. D. Ennis, Org. Lett. 2000, 2, 1395-1397. In the case of Entry 8, the two stereoisomeric final products
could be separated by column chromatography, but subsequently
proved to be highly susceptible to racemisation (even on brief
standing in CDCl3 solution) via opening up of the bicyclic lactam to
the 2-pyrrolidinone (analogous to intermediate G, Scheme 2) and
subsequent reclosure. Finally, and again related to intermediate G;
when 2-benzylfuran was photooxidised and reacted with 2-
aminoethanol, the yield was a mere 30% (Entry 2, Table 1) and the
product recovered was in equilibrium with an open form;[20]
however, when 2-aminoethanol is replaced with either S-serine ethyl
ester (Entry 4, Table 1), or R-phenylglycinol (Entry 6, Table 1), the
product yield improves dramatically to 59 and 58%, respectively,
presumably due to the Thorpe-Ingold angle compression effect
favouring ring closure. 2642; g) J. A. Nieman, M. D. Ennis, Org. Lett. 2000, 2, 1395-13 [6] g
[6]
a) M. Jida, R. Deprez-Poulain, S. Malaquin, P. Roussel, F. Agbossou-
Niedercorn, B. Deprez, G. Laconde, Green Chem. 2010, 12, 961-964;
b) A. N. Cayley, R. J. Cox, C. Keywords: Meyers’ bicyclic lactams · singlet oxygen · cascade
reaction · furan oxidation · 2-pyrrolidinones Keywords: Meyers’ bicyclic lactams · singlet oxygen · cascade
reaction · furan oxidation · 2-pyrrolidinones the
final
bicyclic
lactam
products
with
no
significant
stereoselectivity (dr = 55:45, R1/R3 anti: R1/R3 syn, Entry 7; dr =
55:45, R1/R2 syn: R1/R2 anti, Entry 8), except in the case of
menthofuran (Entry 9) which is unique in its fused bicyclic nature. [1]
For first report, see: A. I. Meyers, M. Harre, R. Garland, J. Am. Chem. Soc. 1984, 106, 1146-1148. [2]
For reviews of the field, see: a) D. Romo, A. I. Meyers, Tetrahedron
1991, 47, 9503-9569; b) M. D. Groaning, A. I. Meyers, Tetrahedron
2000, 56, 9843-9873. The following observations are also worthy of note. It has
been reported in the literature[19] that the product of the reaction
shown in Entry 1, readily opens and reacts intermolecularly to form
the centrosymmetric dimer under aqueous acidic conditions
(Scheme 3). We found this to be true (indeed, it also applied to other
more heavily substituted products, although to a lesser extent), but
we also found that this undesired dimerisation could be avoided by
moderating the amount of TFA employed in the cyclisation step,
and, in the most vulnerable cases by neutralising this TFA with NEt3
prior to concentration of the sample. In general, TFA 20 mol% was
initially added and further additions (up to a maximum of 60 mol%)
were only made when deemed necessary according to tlc analysis of
the reaction mixture. The TFA cyclisation step took between 1 to 9
hours to reach completion. This tendency to dimerise may be the
reason that, somewhat counter intuitively, it would appear that the
bulkier the R1 ring junction substitution is the higher the yields
obtained are (Entries 7 and 9). Likewise, it has been reported in
other methods[10a] that mechanistically converge with ours by going
through intermediates of type G (Scheme 2) that these intermediates
were fleeting and could not be isolated and characterised; however,
though they cannot be described as stable, we have been able to
isolate and take 1H and 13C NMR spectra of some representative
intermediates (see Supporting Info.). ,
,
[3]
For recent representative examples, see: a) M. E. Jung, J. J. Chang,
Org. Lett. 2010, 12, 2962-2965; b) M. Saito, S. Kuwahara, Biosci. Biotechnol. Biochem. 2005, 69, 374-381; c) D. L. J. Clive, J. Wang, J. Org. Chem. 2004, 69, 2773-2784; d) R. C. Hughes, C. A. Dvorak, A. Keywords: Meyers’ bicyclic lactams · singlet oxygen · cascade
reaction · furan oxidation · 2-pyrrolidinones I. Meyers, J. Org. Chem. 2001, 66, 5545-5551; e) M. W. Carson, G. Kim, M. F. Hentemann, D. Trauner, S. J. Danishefsky, Angew. Chem. 2001, 113, 4582-4584; Angew. Chem. Int. Ed. 2001, 40, 4450-4452; f)
D. Trauner, J. B. Schwarz, S. J. Danishefsky, Angew. Chem. 1999,
111, 3756-3758; Angew. Chem. Int. Ed. 1999, 38, 3542-3544. [4] 4]
For some representative examples, see: a) J. D. Hansen, B. J. Newhouse, S. Allen, A. Anderson, T. Eary, J. Schiro, J. Gaudino, E. Laird, A. C. Allen, D. Chantry, C. Eberhardt, L. E. Burgess,
Tetrahedron Lett. 2006, 47, 69-72; b) D. P. Becker, D. L. Flynn, C. I. Villamil, Bioorg. Med. Chem. Lett. 2004, 14, 3073-3075; c) S. Demin,
D. Van Haver, M. Vandewalle, P. J. De Clercq, R. Bouillon, A. Verstuyf, Bioorg. Med. Chem. Lett. 2004, 14, 3885-3888; d) T. D. Aicher, B. Balkan, P. A. Bell, L. J. Brand, S. H. Cheon, R. O. Deems,
J. B. Fell, W. S. Fillers, J. D Fraser, J. Gao, D. C. Knorr, G. G. Kahle,
C. L. Leone, J. Nadelson, R. Simpson, H. C. Smith, J. Med. Chem. 1998, 41, 4556-4566; e) M. D. Ennis, R. L. Hoffman, N. B. Ghazal, D. W. Old, P. A. Mooney, J. Org. Chem. 1996, 61, 5813-5817; f) A. I. Meyers, L. Snyder, J. Org. Chem. 1993, 58, 36-42. 5]
For selected representative examples, see: a) S. Sen, V. R. Potti, R. Surakanti, Y. L. N. Murthy, R. Pallepogu, Org. Biomol. Chem. 2011,
9, 358-360; b) J. E. Resek, J. Org. Chem. 2008, 73, 9792-9794; c) D. Enders, O. Niemeier, T. Balensiefer, Angew. Chem. 2006, 118, 1491-
1495; Angew. Chem. Int. Ed. 2006, 45, 1463-1467; d) H. C. Shen, F.-
X. Ding, S. L. Colletti, Org. Lett. 2006, 8, 1447-1450; e) T. E. Nielsen,
S. Le Quement, M. Meldal, Org. Lett. 2005, 7, 3601-3604; f) M. D. Groaning, G. P. Brengel, A. I. Meyers, Tetrahedron 2001, 57, 2635-
2642; g) J A Nieman M D Ennis Org Lett 2000 2 1395-1397 For some representative examples, see: a) J. D. Hansen, B. J. Newhouse, S. Allen, A. Anderson, T. Eary, J. Schiro, J. Gaudino, E. Laird, A. C. Allen, D. Chantry, C. Eberhardt, L. E. Burgess,
Tetrahedron Lett. 2006, 47, 69-72; b) D. P. Becker, D. L. Flynn, C. I. Villamil, Bioorg. Med. Chem. Lett. 2004, 14, 3073-3075; c) S. Demin,
D. Van Haver, M. Vandewalle, P. J. A versatile new synthesis of Meyers’ bicyclic lactams: singlet oxygen reacts
with furan substrates to initiate a remarkable cascade reaction sequence**
Dimitris Kalaitzakis Tamsyn Montagnon Ioanna Alexopoulou and Georgios Vassilikogiannakis* Commercially available furan starting materials were used
throughout except in the cases of 2-benzylfuran (Entries 2, 4, 6) and
the furan shown in Entry 7, where the requisite substrates were
synthesised using known procedures.[17] In general, the yields are
remarkably high when the large increase in molecular complexity
that has been achieved in this one-pot procedure is taken into
consideration, and given the intricate nature of the cascade reaction
sequence that has been successfully implemented. Regarding the
stereochemical outcomes (confirmed by NOE studies),[18] it was
found that having a defined stereogenic centre adjacent to the amino group (R4 attachment position) on the 1,2-aminoalcohol induces
highly stereoselective formation of the product’s ring junction
(position R1 in the product, Scheme 1), as can be seen from Entries
3 – 10 (Table 1). In contrast, having a defined stereogenic centre
adjacent to the alcohol (R5 attachment position) on the 1,2-amino-
alcohol leads to no significant stereoselectivity being achieved for
the overall reaction, with both ring junction isomers being formed
(dr = 55:45, R1/R5 anti: R1/R5 syn, Entry 11). Similarly, substituents
at the R2 and R3 positions of the furan substrate are incorporated in 2 Scheme 3. Acid catalysed dimerisation of bicyclic lactams. that this method will prove to be a powerful addition to the synthetic
chemist’s armoury. Received: ((will be filled in by the editorial staff))
Published online on ((will be filled in by the editorial staff)) Received: ((will be filled in by the editorial staff))
Published online on ((will be filled in by the editorial staff)) Scheme 3. Acid catalysed dimerisation of bicyclic lactams. Keywords: Meyers’ bicyclic lactams · singlet oxygen · cascade
reaction · furan oxidation · 2-pyrrolidinones Ménard-Moyon, J. P. Schmidt, R. J. K. Taylor, Tetrahedron Lett. 2007, 48, 6556-6560; c) M. Penhoat, S. Leleu, G. Dupas, C. Papamicaël, F. Marsais, V. Levacher,
Tetrahedron Lett. 2005, 46, 8385-8389; d) R. Deprez-Poulain, N. Willand, C. Boutillon, G. Nowogrocki, N. Azaroual, B. Deprez,
Tetrahedron Lett. 2004, 45, 5287-5290. [7]
For a recent example, see: A. A. Bahajaj, M. H. Moore, J. M. Vernon,
Tetrahedron 2004, 60, 1235-1246. In summary, we have introduced a new and versatile method
for the synthesis of Meyers’ bicyclic lactams using a green, step-
and atom-economic singlet-oxygen-initiated cascade reaction
sequence. The mild method’s broad synthetic potential derives from
the fact that it begins from simple furan substrates which can be
readily synthesised and/or substituted to give many possible
variations, as desired; a feature that contrasts with the limitations
(both in terms of their synthesis and subsequent handling and
purification) imposed by the γ-ketoacids precursors used in the
traditional Meyers’ synthesis.[1,2] Furthermore, the cascade initiator,
singlet oxygen is an extremely selective reagent conferring on its
reactions very broad functional group tolerance and a complete lack
of need for protecting groups.[14] For all these reasons it is believed [8]
W. N. Speckamp, H. Hiemstra, Tetrahedron 1985, 41, 4367-4416. [9]
a) S. Comesse, A. Martel, A. Daïch, Org. Lett. 2011, 13, 4004-4007;
b) R. Medimagh, S. Marque, D. Prim, J. Marrot, S. Chatti, Org. Lett. 2009, 11, 1817-1820. [10] a) E. Ascic, J. F. Jensen, T. E. Nielsen, Angew. Chem. 2011, 123,
5294-5297; Angew. Chem. Int. Ed. 2011, 50, 5188-5191; b) I. Kanizsai, Z. Szakonyi, R. Sillanpää, M. D’hooghe, N. De Kimpe, F. Fülöp, Tetrahedron Asymmetry 2006, 17, 2857-2863. [11] P. A. Wender, B. L. Miller Nature 2009, 460, 197-201. [12] B. M. Trost, Science 1991, 254, 1471-1477. [13] a) I. S. Young, P. S. Baran, Nature Chem. 2009, 1, 193-205; b) T. Gaich, P. S. Baran, J. Org. Chem. 2010, 75, 4657-4673; c) T. Newhouse, P. S. Baran, R. W. Hoffmann, Chem. Soc. Rev. 2009, 38, 3 3010-3021; (d) For the first introduction to the "ideal synthesis"
concept, see: Hendrickson, J. B. J. Am. Chem. Soc. 1975, 97, 5784. 3010-3021; (d) For the first introduction to the "ideal synthesis"
concept, see: Hendrickson, J. B. J. Am. Chem. Soc. 1975, 97, 5784. [16] The furan substrate in MeOH at 0 ºC with rose bengal (10-4 M), added
as photosensitiser, was exposed to irradiation from a xenon Variac
Eimac Cermax 300W visible spectrum light for a period of 8 mins
whilst oxygen was gently bubbled through the reaction mixture.
[17]
F
h
i
f b
lf
S
i
I f
i
f Keywords: Meyers’ bicyclic lactams · singlet oxygen · cascade
reaction · furan oxidation · 2-pyrrolidinones Entry for the Table of Contents (Please choose one layout) Entry for the Table of Contents (Please choose one layout) Layout 2:
(Powerful new method for
synthesizing bicyclic lactams )
Dimitris Kalaitzakis, Tamsyn
Montagnon, Ioanna Alexopoulou and
Georgios Vassilikogiannakis*
__________ Page – Page
A versatile new synthesis of Meyers’
bicyclic lactams: singlet oxygen reacts
with furan substrates to initiate a
remarkable cascade reaction sequence
A new and powerful method for the synthesis Meyers’ bicyclic lactams beginning
from furans is reported that uses a remarkable one-pot singlet oxygen-initiated
cascade reaction sequence to deliver the corresponding bicyclic lactams in high
yield. Layout 2: Keywords: Meyers’ bicyclic lactams · singlet oxygen · cascade
reaction · furan oxidation · 2-pyrrolidinones [16] The furan substrate in MeOH at 0 ºC with rose bengal (10-4 M), added
as photosensitiser, was exposed to irradiation from a xenon Variac
Eimac Cermax 300W visible spectrum light for a period of 8 mins
whilst oxygen was gently bubbled through the reaction mixture. 1
h
i
f b
lf
S
i
f
i
f [14] For selected examples see: a) D. Noutsias, I. Alexopouou, T. Montagnon, G. Vassilikogiannakis, Green Chem. 2012, 14, 601-604;
b) A. Kouridaki, T. Montagnon, M. Tofi, G. Vassilikogiannakis, Org. Lett. 2012, 14, 2374-2377; c) G. Vassilikogiannakis, I. Alexopoulou,
M. Tofi, T. Montagnon, Chem. Commun. 2011, 47, 259-261; d) T. Montagnon,
D. Noutsias,
I. Alexopoulou,
M. Tofi,
G. Vassilikogiannakis, Org. Biomol. Chem. 2011, 9, 2031-2039. e) M. Tofi, K. Koltsida, G. Vassilikogiannakis, Org. Lett. 2009, 11, 313-
316; f) T. Montagnon, M. Tofi, G. Vassilikogiannakis, Acc. Chem. Res. 2008, 41, 1001-1011; g) T. Georgiou, M. Tofi, T. Montagnon, G. Vassilikogiannakis,
Org. Lett. 2006, 8, 1945-1948;
h)
G. Vassilikogiannakis, M. Stratakis, Angew. Chem. 2003, 115, 5623-
5626; Angew. Chem. Int. Ed. 2003, 42, 5465-5468; [17] For synthesis of benzylfuran, see: Supporting Information for
procedure; For synthesis of the furan substrate shown in Entry 7
(Table 1), see: A. Aponick, C.-Y. Li, J. Malinge, E. F. Marques, Org. Lett. 2009, 11, 4624-4627. [18] Full details of NOE studies are included in the Supporting Information. [19] C. Wedler, H. Schick, D. Scharfenberg-Pfeiffer, G. Reck, Liebigs Ann. Chem. 1992, 29-32. [20] The open form corresponds to intermediate G except that the preferred
2-pyrrolidinone, in this instance, is that which places the double bond
exo from the five-membered ring and in conjugation with the Ph
group, namely, 5-benzylidene-1-(2-hydroxyethyl)pyrrolidin-2-one. The exact structure is given in the supporting information. [15] For pioneering works on furan photooxygenations, see: a) C. S. Foote,
M. T. Wuesthoff, S. Wexler, I. G. Burstain, R. Denny, G. O. Schenck,
K. H. Schulte-Elte, Tetrahedron 1967, 23, 2583-2599; b) K. Gollnick,
A. Griesbeck, Tetrahedron 1985, 41, 2057-2068; c) B. L. Feringa,
Recl. Trav. Chim. Pays-Bas 1987, 106, 469-488. (Powerful new method for
synthesizing bicyclic lactams ) Dimitris Kalaitzakis, Tamsyn
Montagnon, Ioanna Alexopoulou and
Georgios Vassilikogiannakis*
__________ Page – Page Dimitris Kalaitzakis, Tamsyn
Montagnon, Ioanna Alexopoulou and
Georgios Vassilikogiannakis*
__________ Page – Page A versatile new synthesis of Meyers’
bicyclic lactams: singlet oxygen reacts
with furan substrates to initiate a
remarkable cascade reaction sequence A new and powerful method for the synthesis Meyers’ bicyclic lactams beginning
from furans is reported that uses a remarkable one-pot singlet oxygen-initiated
cascade reaction sequence to deliver the corresponding bicyclic lactams in high
yield. 5
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Cognitive function among older adults
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http://www.rsp.fsp.usp.br/ Rev Saude Publica. 2018;52 Suppl 2:4s Rev Saude Publica. 2018;52 Suppl 2:4s Supplement ELSI-Brazil
Original Article Supplement ELSI-Brazil
Original Article Correspondence:
Erico Castro-Costa
Av. Augusto de Lima, 1715
30190-002 Belo Horizonte, MG, Brasil
E-mail: ericocastrocosta@gmail.com How to cite: Castro-Costa E, Lima-
Costa MF, Bof de Andrade F, Souza-
Junior PRB, Ferri CP. Cognitive
function among older adults: ELSI-
Brazil results. Rev Saude Publica.
2018;52 Suppl 2:4s. Cognitive function among older adults:
ELSI-Brazil results Erico Castro-CostaI, Maria Fernanda Lima-CostaI,II, Fabíola Bof de AndradeI,II, Paulo Roberto
Borges de Souza JuniorIII, Cleusa Pinheiro FerriIV I Fundação Oswaldo Cruz. Instituto René Rachou. Núcleo de Estudos em Saúde Pública e Envelhecimento. Belo
Horizonte, MG, Brasil ,
,
II Fundação Oswaldo Cruz. Instituto René Rachou. Programa de Pós-Graduação em Saúde Coletiva. Belo Horizonte,
MG, Brasil ,
III Fundação Oswaldo Cruz. Instituto de Comunicação e Informação Científica e Tecnológica em Saúde. Rio de
Janeiro, RJ, Brasil ,
III Fundação Oswaldo Cruz. Instituto de Comunicação e Informação Científica e Tecnológica em Saúde. Rio de
Janeiro, RJ, Brasil J
,
J,
IV Universidade Federal de São Paulo. Faculdade de Medicina. Departamento de Psicobiologia. São Paulo, SP, Brasil J
,
J,
IV Universidade Federal de São Paulo. Faculdade de Medicina. Departamento de Psicobiologia. São Paulo, SP, Brasil Data Source The ELSI-Brazil is a population-based cohort study, designed to represent the Brazilian
population aged 50 years or older, and with the objective of investigating the dynamics
of aging in the Brazilian population and its determinants. The baseline was established
between the years 2015/2016. More details can be found on the research’s homepagea and
in another publication21. ELSI-Brazil baseline data collection included: 1) a household interview; 2) an individual
interview with the participant; 3) physical measurements; 4) laboratory tests; 3) storage of
blood aliquots for future analysis. a Fundação Oswaldo Cruz.
Brazilian Longitudinal Study of
Aging. Rio de Janeiro; c2015
[cited 2017 Nov 28]. Available
from: http://elsi.cpqrr.fiocruz.br INTRODUCTION Cognitive function is an important determinant of independence and better quality of life
among older adults1. According to recent projections, the elderly population will triple in
Brazil and will increase from 19.6 million in 2010 to 66.5 million in 20502, making it the
sixth largest elderly population in the world3. Aging is a complex phenomenon and its impact on health conditions and functionality
occurs heterogeneously among older adults. These differences are associated with genetic4,
environmental and social determinants5-8, and individual characteristics of the elderly9,10. Previous population-based studies with national samples from several countries have shown
that socio-demographic factors11,12 and differences between rural and urban residence
areas13,14 are related to alterations in cognitive function among older adults, showing
important cultural and geographic variations11,12, 15,16. Brazil is a country of continental proportions, has one of the highest levels of inequality17,
occupies the 75th position in the Human Development Index (HDI)18 and presents differences
for this index among its macroregions19. There has been an increasing number of studies on
the cognitive function of Brazilian older adults20. However, it was observed that the majority
(72%) was done in the Southeast and none of these studies had a design that allowed the
comparison of cognitive function among Brazilian macroregions20. The Brazilian Longitudinal Study of Aging (ELSI-Brazil) used a national sample
representative of individuals aged 50 years or older and allows us to investigate the
cognitive function of older Brazilian adults. Thus, the objective of the present study
was: 1) to investigate the cognitive function of older adults stratified by the Brazilian
macroregions; 2) to compare the association between sociodemographic factors and the
place of residence with cognitive function and the variations between the macroregions;
3) to investigate whether the variations in the sociodemographic structure and the place
of residence observed are totally or partially responsible for the potential differences in
cognitive function among macroregions. ABSTRACT ABSTRACT OBJECTIVE: To investigate macroregional variations in cognitive function in a national sample
representative of the Brazilian population aged 50 years and older. METHODS: Data from the baseline of the Longitudinal Study of Brazilian Elderly (ELSI-Brazil),
collected between 2015 and 2016, were used. Memory was measured by means of a 10-word list
and executive function, by semantic verbal fluency, based on the naming of animals. Gender,
age, education, and rural or urban residence were potentially confounding RESULTS: Among the 9,412 ELSI-Brazil participants, 9,085 were included in the analysis; 53.9%
were women and the average age was 63.0 (0.42) years. After adjusting for potential confounding
variables, average scores for memory and verbal fluency were lower in the Northeast region
and higher in the Midwest and Southeast, respectively. In the South region, higher scores were
found for immediate and combined memory. In all regions, older participants and those with
lower schooling had worse scores for memory and verbal fluency. CONCLUSIONS: There are differences in cognitive function among older adults in the different
macroregions, independent of age, gender, schooling, and rural or urban residence. Correspondence:
Erico Castro-Costa
Av. Augusto de Lima, 1715
30190-002 Belo Horizonte, MG, Brasil
E-mail: ericocastrocosta@gmail.com DESCRIPTORS: Aged. Cognition. Memory. Socioeconomic factors. Received: Dec 18, 2017
Approved: Mar 18, 2018 How to cite: Castro-Costa E, Lima-
Costa MF, Bof de Andrade F, Souza-
Junior PRB, Ferri CP. Cognitive
function among older adults: ELSI-
Brazil results. Rev Saude Publica. 2018;52 Suppl 2:4s. Copyright: 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 that the original author
and source are credited. 1s Cognitive function among older Brazilian adults
Castro-Costa E et al. Cognitive function among older Brazilian adults
Castro-Costa E et al. Ethical Aspects The ELSI-Brazil Project was approved by the Ethics Commission of Fiocruz, Minas Gerais
(CAAE 34649814.3.0000.5091). All participants signed separate informed consent forms for
all research procedures. Cognitive Function The study adopts a conceptual framework common to other large-scale longitudinal studies
of aging in the world, such as the Health and Retirement Study21. This specific concept of the cognitive function module in ELSI-Brazil allows direct comparison
of Brazilian results with results found in other countries, such as: China [the China Health
and Retirement Longitudinal Study (https://g2aging.org/?section=study&studyid=4)], the
United States [the US Health and Retirement Study (http://hrsonline. isr.umich.edu)], England
[the English Longitudinal Study of Ageing (http://www.natcen.ac.uk/elsa)], Mexico [Mexican
Health and Aging Study(mhasweb.org/Resources.aspx)] etc. Cognitive function among older Brazilian adults
Castro-Costa E et al. In the present analysis, we selected two variables that represent three well-established
domains of cognitive function: memory, language and executive function. Memory
was evaluated through the word list learning test, in which 10 words were read to the
participants. Immediate memory was defined as the repetition of the words immediately
after their reading. Late memory (delayed recall) was their repetition five minutes after
presentation. The participant was kept busy filling out the questionnaire during the
interval between the two tasks. Combined memory was obtained by the sum of the scores
for immediate and late memory. Language and executive function were measured by the
semantic verbal fluency test (animal category), in which participants were asked to say the
name of as many animals as they could in a one-minute period. Statistical Analyses Statistical analyzes were conducted in the Stata 14.1 program. Due to the complex design
of the sample with stratification, all analyzes were done using weighting factors for each
stratum, thus obtaining in the final results the contribution of each stratum according to
its actual weight, and not through its participation in the sample. The use of Stata’s svy
command allowed the output of robust standardized error. To compare the characteristics of the participants among the macroregions, the analysis
of variance (ANOVA) was weighted for continuous variables and the Pearson’s chi-squared
test was weighted for the categorical variables. Multivariate linear regressions with a 95% confidence interval were performed to estimate
the relationship between memory (immediate, late and combined) and verbal fluency with
gender, age, schooling and place of residence for each macroregion. Finally, the average of the variables memory (immediate, late and combined) and verbal
fluency was standardized separately and then simultaneously, by gender, age, schooling
and place of residence, using the direct method22 for each macroregion. Other Variables Also considered were the variables gender, age (50-59, 60-69, 70-79, 80 years or older),
schooling (< 4, 4-7, 8-11, > 11) and place of residence (rural or urban). Ethical Aspects RESULTS Of the 9,412 ELSI-Brazil baseline participants, 9,085 (96.5%) presented complete information
for all variables and were included in this analysis. The main characteristics of the
participants by region are presented in Table 1. In the total sample, the average age was 63.0
years (SE = 0.42) and the majority were female (53.9%) and had less than 11 years of schooling
(76.2%). Table 2 shows the average scores and multivariate analyzes of the association
between memory and verbal fluency scores stratified by macroregions. Residents in the
Southeast, South and Midwest regions had the best performances, both in the memory
and in the verbal fluency evaluation. Older adults in the Southern macroregion had the
best performance in the variables immediate memory and combined memory, while the
best performance in late memory and verbal fluency occurred among residents of the
Midwest and Southeast, respectively. Older residents with low schooling had worse memory
performance (immediate, late and combined) and worse verbal fluency in all macroregions. On the other hand, rural participants showed worse performance in immediate and Cognitive function among older Brazilian adults
Castro-Costa E et al. combined memory in the Northeast and Southeast, and in late memory in the Southeast. In the Midwest, rural residents performed better in late and combined memory. Regarding
verbal fluency, participants from the rural area of the South and Midwest macroregion,
and women from the North and Midwest, presented worse performance of this function. Table 1. Characteristics of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), 2015-2016. RESULTS Characteristic
Total
North
Northeast
Southeast
South
Midwest
p
n = 9,085
n = 713
n = 2,416
n = 3,825
n = 1,234
n = 897
Gender: female (%)
53.9
49.9
54.6
54.5
53.6
52.1
0.934
Age – average (SE)
63.0 (0.42)
62.1 (0.67)
63.3 (0.75)
63.0 (0.72)
63.0 (0.92)
62.3 (1.1)
0.945
Education (years) (%)
< 0.0001
> 11
23.8
26.3
18.3
26.3
24.2
23.7
8–10
11.9
15.6
8.9
12.9
12.2
11.3
4–7
31.4
25.4
23.2
34.4
36.9
31.8
< 4
32.8
32.7
49.5
26.4
26.7
33.2
Place of residence: Rural (%)
15.3
17.7
30.3
6.5
21.6
5.6
0.006
Memory - mean (SE)
Immediate
4.3 (0.05)
4.2 (0.11)
3.8 (0.08)
4.5 (0.06)
4.6 (0.07)
4.3 (0.07)
< 0.0001
Late
2.9 (0.06)
2.7 (0.15)
2.4 (0.09)
3.0 (0.07)
3.0 (0.09)
3.1 (0.13)
< 0.0001
Combined
7.3 (0.10)
7.0 (0.24)
6.2 (0.17)
7.5 (0.13)
7.7 (0.16)
7.6 (0.19)
< 0.0001
Verbal fluency - mean (SE)
12.6 (0.29)
11.4 (0.37)
11.4 (0.85)
13.2 (0.41)
12.6 (0.40)
12.9 (0.38)
0.012
All estimates were weighted by the sample parameters and sample weights of the subjects. SE: standard error. For continuous and categorical variables, F tests were used, and the weighted chi-square, corrected for the study design, was used as a
complex sample. All estimates were weighted by the sample parameters and sample weights of the subjects. SE: standard error. For continuous and categorical variables, F tests were used, and the weighted chi-square, corrected for the study design, was used as a
complex sample. Table 2. Average scores and multivariate analyzes of the association between the cognitive function scores an
chooling and place of residence of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI Table 2. Average scores and multivariate analyzes of the association between the cognitive function scores and the variables gender, age,
schooling and place of residence of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), 2015-2016. F: female; M: male
All multivariate regressions were adjusted for gender, age, schooling and place of residence. RESULTS 4s Cognitive function among older Brazilian adults
Castro-Costa E et al. Cognitive function among older Brazilian adults
Castro-Costa E et al. Standardized average memory (immediate, late and combined) and verbal fluency scores are
arranged and compared between the macroregions in Table 3. The direct standardization
of memory and verbal fluency averages by the individual effect of gender, age, schooling
and place of residence and by the simultaneous effect of these four variables did not alter
the pattern of difference between the regions. The values of the averages observed and simultaneously standardized by the four variables
are shown in the Figure. s of the cognitive function of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), Table 3. Standardized average scores of the cognitive function of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil),
2015-2016. RESULTS Cognitive function
Standardized by gender
Standardized by age
Standardized by
schooling
Standardized by place
of residence
Standardized by the 4
variables
Average
Score
95%CI
Average
Score
95%CI
Average
Score
95%CI
Average
Score
95%CI
Average
Score
95%CI
Immediate memory
North
4.24
4.00–4.47
4.03
3.85–4.20
4.11
3.91–4.31
4.23
4.03–4.43
4.08
3.92–4.24
Northeast
3.83
3.65–4.00
3.74
3.60–3.88
3.97
3.87–4.07
3.89
3.71–4.08
3.82
3.72–3.93
Southeast
4.51
4.40–4.63
4.44
4.34–4.55
4.37
4.30–4.44
4.45
4.36–4.54
4.38
4.31–4.45
South
4.60
4.45–4.74
4.50
4.32–4.68
4.46
4.35–4.57
4.62
4.50–4.74
4.50
4.38–4.61
Midwest
4.43
4.29–4.57
4.29
4.18–4.40
4.37
4.33–4.42
4.41
4.29–4.54
4.33
4.25–4.40
Late Memory
North
2.76
2.45–3.07
2.56
2.24–2.87
2.65
2.26–3.05
2.76
2.44–3.09
2.62
2.25–2.99
Northeast
2.42
2.24–2.61
2.34
2.20–2.48
2.54
2.41–2.68
2.48
2.31–2.66
2.42
2.32–2.52
Southeast
3.05
2.91–3.20
2.97
2.86–3.08
2.89
2.80–2.99
2.98
2.86–3.11
2.90
2.81–2.99
South
3.10
2.92–3.28
2.99
2.81–3.17
2.96
2.81–3.11
3.12
2.98–3.27
3.01
2.86–3.16
Midwest
3.15
2.90–3.42
2.99
2.81–3.18
3.10
2.98–3.22
3.18
2.94–3.41
3.09
2.96–3.22
Combined Memory
North
7.01
6.55–7.48
6.60
6.17–7.03
6.78
6.18–7.37
7.01
6.52–7.49
6.72
6.21–7.22
Northeast
6.26
5.92–6.61
6.09
5.83–6.35
6.52
6.30–6.74
6.38
6.04–6.73
6.25
6.06–6.43
Southeast
7.58
7.33–7.83
7.42
7.22–7.62
7.28
7.13–7.43
7.44
7.24–7.65
7.29
7.14–7.44
South
7.70
7.39–8.01
7.50
7.16–7.84
7.43
7.18–7.67
7.75
7.50–7.99
7.52
7.27–7.76
Midwest
7.60
7.22–7.99
7.29
7.02–7.57
7.49
7.35–7.62
7.62
7.28–7.96
7.46
7.28–7.64
Verbal fluency
North
11.44
10.66–12.23
11.23
10.73–11.73
11.30
10.98–11.71
11.53
10.83–12.24
11.30
10.70–11.89
Northeast
11.41
9.69–13.13
11.23
9.79–12.66
11.79
9.88–13.71
11.60
9.66–13.54
11.54
9.53–13.55
Southeast
13.21
12.41–14.01
13.33
12.34–14.32
13.26
12.10–14.41
13.26
12.31–14.21
13.41
11.94–14.89
South
12.62
11.83–13.40
12.48
11.69–13.26
12.39
11.85–12.92
12.74
12.23–13.26
12.41
11.91–12.91
Midwest
12.97
12.22–13.71
12.75
12.07–13.44
12.86
12.32–13.40
12.87
12.14–13.60
12.77
12.11–13.44 Table 3. Standardized average scores of the cognitive function of the 9,085 sample participants. Brazilian Longitu
2015-2016. 16
14
12
10
18
16
14
12
0
North
observed immediate memory
Northeast
Southeast
South
Midwest
standardized late memory
observed verbal fluency
standardized immediate memory
observed combined memory
observed late memory
standardized combined memory
standardized verbal fluency
Figure. Observed and standardized average scores by gender, age, schooling and place of residence of the cognitive function of the 9,085
sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), 2015-2016. 16
14
12
10
18
16
14
12
0
North
observed immediate memory
Northeast
Southeast
South
Midwest
standardized late memory
observed verbal fluency
standardized immediate memory
observed combined memory
observed late memory
standardized combined memory
standardized verbal fluency
Figure. RESULTS Cognitive function
North
Northeast
Southeast
South
Midwest
Average
95%CI
Average
95%CI
Average
95%CI
Average
95%CI
Average
95%CI
Memory
Immediate
4.22
3.99–4.45
3.83
3.65–4.00
4.51
4.40–4.63
4.60
4.45–4.75
4.43
4.30–4.56
Late
2.76
2.45–3.07
2.42
2.23–2.60
3.05
2.90–3.20
3.09
2.92–3.27
3.15
2.89–3.41
Combined
6.99
6.53–7.46
6.26
5.92–6.60
7.58
7.32–7.83
7.70
7.39–8.01
7.60
7.22–7.99
Verbal fluency
11.52
10.78–12.27
11.41
9.73–13.09
13.24
12.42–14.06
12.64
11.84–13.44
13.01
12.25–13.77
β
95%CI
β
95%CI
β
95%CI
β
95%CI
β
95%CI
Immediate Memory
Gender (F versus M)
0.22
-0.15–0.59
0.01
-0.10–0.13
0.07
-0.07–0.21
0.04
-0.17–0.27
0.03
-0.13–0.19
Age (≥ 75 versus < 75)
-1.95
-2.33– -1.57
-1.31
-1.45– -1.18
-1.20
-1.36– -1.03
-1.55
-1.90– -1.19
-1.49
-1.16– -1.36
Education (< 11 versus ≥ 11)
-0.96
-1.28– -0.64
-1.17
-1.36– -0.97
-1.02
-1.13– -0.91
-1.01
-1.19– -0.82
-1.07
-1.49– -0.66
Place of residence (rural
versus urban)
-0.06
-0.30–0.16
-0.26
-0.39– -0.13
-0.51
-0.66– -0.36
-0.04
-0.32–0.24
0.01
-0.07–0.09
Late Memory
Gender (F versus M)
0.09
-0.17–0.35
0.07
-0.07–0.21
0.07
-0.08–0.23
0.26
0.06–0.46
0.00
-0.44–0.44
Age (≥ 75 versus < 75)
-1.76
-2.23– -1.30
-1.13
-1.30– -0.96
-1.52
-1.70– -1.33
-1.54
-1.76– -1.32
-1.45
-1.70– -1.20
Education (< 11 versus ≥ 11)
-1.02
-1.27– -0.76
-1.22
-1.41– -1.03
-1.21
-1.35– -1.06
-1.11
-1.32– -0.89
-1.12
-1.57– -0.67
Place of residence
(rural versus urban)
0.13
-0.25–0.53
-0.24
-0.53–0.05
-0.54
-0.77– -0.30
-0.22
-0.45–0.02
0.39
0.25–0.52
Combined Memory
Gender (F versus M)
0.31
-0.28–0.90
0.08
-0.14–0.31
0.14
-0.12–0.42
0.31
-0.11–0.73
0.03
-0.55–0.61
Age (≥ 75 versus < 7 5)
-3.72
-4.52– -2.92
-2.44
-2.70– -2.19
-2.72
-3.04– -2.40
-3.09
-3.66– -2.53
-2.94
-3.25– -2.62
Education (< 11 versus ≥ 11)
-1.99
-2.51– -1.46
-2.39
-2.73– -2.05
-2.23
-2.47– -1.99
-2.12
-2.46– -1.78
-2.20
-3.06– -1.34
Place of residence (rural
versus urban)
0.06
-0.50–0.63
-0.50
-0.86– -0.15
-1.05
-1.34– -0.76
-0.26
-0.74–0.22
0.40
0.22–0.58
Verbal Fluency
Gender (F versus M)
-1.24
-2.04– -0.44
0.02
-2.07–2.13
-1.44
-3.34–0.45
-0.46
-0.96–0.03
-0.99
-1.72– -0.27
Age (≥ 75 versus < 75)
-1.84
-2.66– -1.03
-2.33
-4.35– -0.31
3.74
-2.23–9.72
-2.04
-2.71– -1.38
-2.09
-2.76– -1.42
Education (< 11 versus ≥ 11)
-1.57
-2.44– -0.70
-1.75
-4.04–0.52
-2.71
-3.53– -1.88
-3.60
-4.24– -2.97
-2.45
-4.15– -0.75
Place of residence
(rural versus urban)
-0.20
-1.18–0.77
-1.07
-3.53–1.39
0.90
-3.97–5.78
-0.73
-1.46– -0.01
-1.10
-1.84– -0.36
F: female; M: male
All multivariate regressions were adjusted for gender, age, schooling and place of residence. DISCUSSION DISCUSSION This is the first national study with a representative sample of the population aged 50 years
or older that compared cognitive function in the five Brazilian macroregions, where the
same tests were applied in the same period. Residents of the Southeast, South and Midwest
regions presented better cognitive function in all the applied tests. However, there were
variations on the ranking, depending on the ability evaluated. In all regions, there was a
worsening of the three types of memory and verbal fluency correlated with the oldest age and
low level of schooling, except for the Southeast macroregion, where the worsening of verbal
fluency was not correlated with older age. Regarding the place of residence, we observed,
inconsistently, that residents of the rural area exhibited lower scores for tests that evaluate
memory and verbal fluency. However, residents of the rural area of the Midwest performed
better in late memory than those in the urban area. The female gender presented the worst
performance in verbal fluency tests in the North and Midwest macroregions. Finally, the
standardized averages of the three types of memory and of verbal fluency by the effect of
gender, age, schooling and place of residence showed that these variables did not explain
all the differences found among the Brazilian macroregions. This study has several advantages such as: 1) the use of a representative sample of Brazilian
adults aged 50 and older; 2) the direct evaluation of cognitive function through validated
and applied tests in the same period, which allows comparisons between the Brazilian
macroregions and with other countries; 3) training and certification of the interviewers
according to the protocols developed for the study; 4) quality assurance and control of data
collection by conducting previous pilot studies in order to identify and correct potential
problems in procedures and interviews. However, the study also has limitations that must be considered when interpreting the
results. The use of a weighted sample may underestimate the averages for cognitive
function due to its design effect. However, the use of specific analyzes for weighting is
likely to overcome this limitation. Although all dimensions of cognitive function were
not evaluated in this study, scores of our measures (10-word list and verbal fluency test)
were normally distributed with no evidence of floor or ceiling effect23, commonly found
in low education populations. RESULTS Observed and standardized average scores by gender, age, schooling and place of residence of the cognitive function of the 9,085
sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), 2015-2016. Figure. Observed and standardized average scores by gender, age, schooling and place of residence of the cognitive function of the 9,085
sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), 2015-2016. Cognitive function among older Brazilian adults
Castro-Costa E et al. Cognitive function among older Brazilian adults
Castro-Costa E et al. Cognitive function among older Brazilian adults
Castro-Costa E et al. DISCUSSION The results of the present study are directly comparable with studies conducted in high-
income countries (Health Retirement Study [HRS], English Longitudinal Study of Ageing
[ELSA], the Irish Longitudinal Study on Ageing [TILDA])11,12 and in middle income countries,
such as (Mexican Health and Aging Study [MHAS] and the Chinese Health and Retirement
Longitudinal Study [CHARLS])15,16, in which cognitive function was assessed using the same
measure for memory (a list of 10 words, with the exception of the MHAS study, which used a
list of eight words) and verbal fluency (animal category semantic fluency test). ELSI-Brazil,
even with a younger sample due to the inclusion of middle-aged adults, presented lower
averages for all abilities evaluated (immediate, late and combined memory) when compared
to the three studies in high-income countries (HRS, ELSA, TILDA)11,12 which used a sample of
elderly individuals aged 60 years or older. Probably, this occurred due to the great difference
in schooling between these samples, since this factor plays an important role in cognitive
function24. In ELSI-Brazil, 76.2% of the sample of Brazilians aged 50 and older have less than
12 years of schooling; in HRS, ELSA and TILDA, rates are 56%, 48% and 44% for those aged
65 years and older, and 39%, 28%, 28% for those aged 57-64 years, respectively11,12. Compared with studies in middle- and low-income countries, where low schooling was
similar between ELSI-Brazil, MHAS and CHARLS15,16. It was observed that in the Mexican
study, in spite of presenting an older population (adults 60 years old or older), performance
in all three skills was better compared to our findings (immediate memory: 4.8 versus
4.3; late memory: 4.4 versus 2.9, and verbal fluency: 15.3 versus 12.6). This was because the
participants in the MHAS sample were healthier than those in the ELSI-Brazil since the
tests for cognitive evaluation were applied only to those who had not had a stroke or did Cognitive function among older Brazilian adults
Castro-Costa E et al. Cognitive function among older Brazilian adults
Castro-Costa E et al. not present depressive symptoms16. In addition, the memory was tested by a list of eight
words, which probably influenced the results obtained with this version, overestimating
the performance of the participants16. Regarding the Chinese study15, combined memory
performance was worse than in ELSI-Brazil (3.3 versus 7.3), probably because the majority
of participants were from the rural area13,14. DISCUSSION Comparisons with other Brazilian studies are limited due to the differences between the age
groups and the outcomes studied. In a study with employees aged 35-74 at six universities
in the Northeast, Southeast and South macroregions, memory was evaluated by a list of 10
words applied at three different occasions (immediate, late, recognition), while semantic verbal
fluency was evaluated by the animal category test25. The averages for late memory ranged from
four to eight in men and five to eight in women, while the average values for verbal fluency
ranged from 12 to 21 among men and 13 to 21 among women according to participants’
schooling25. Like the Longitudinal Study on Adult Health (ELSA-Brazil), the present study
also demonstrated that older participants with low educational level had worse performance
in memory and verbal fluency. However, the scores were lower in all tests when compared to
those found in ELSA-Brazil. This discrepancy probably occurred because ELSA-Brazil was
conducted exclusively in the urban area (six capitals), while ELSI-Brazil was conducted both
with participants living in the urban area and with those living in rural areas. Memory and Verbal Fluency assessments, in the present study, are part of the cognitive
assessment of the Consortium to Establish the Registry for Alzheimer’s Disease (CERAD)26,
translated and validated to Brazilian Portuguese for carrying out in Brazilian elderly
population27. Previous studies have shown that older people with lower levels of schooling
and those coming from rural areas have worse cognitive performance11-16,27; data for the
gender variable are inconsistent11,12,15,16,28. Our findings replicated the associations previously
demonstrated for memory and verbal fluency in other studies with population-based samples11-
16,26,27, with the exception of results for late and combined memory, which showed that
participants from rural areas in the Midwest had the best performance. This is likely to have
happened because the main activity in this macroregion is farming, which possibly favors
better socioeconomic conditions, which in turn may have contributed to this performance. Finally, the results of the present study showed that the performance of adults aged 50 years or
older on cognitive function tests is worse than that found in high-income countries (United States,
England and Ireland) and inconsistent with that of middle-income countries (worse than that
found in Mexico and better than that found in China). We observed that the association between
sociodemographic, settlement factors and cognitive function had consistent variation patterns
in the Brazilian macroregions. DISCUSSION However, as these factors do not fully explain the differences in
cognitive function in adults aged 50 years or older observed between regions, other contextual
and cultural factors not investigated here may play a relevant role in the differences found. REFERENCES 1. Cigolle CT, Langa KM, Kabeto MU, Tian Z, Blaum CS. Geriatric conditions and
disability: the Health and Retirement Study. Ann Intern Med. 2007;147(3):156-64. https://doi.org/10.7326/0003-4819-147-3-200708070-00004 2. Instituto Brasileiro de Geografia e Estatística, Coordenação de Geografia. Brasil: uma visão
geográfica e ambiental no início do século XXI. Rio de Janeiro: IBGE; 2016. 2. Instituto Brasileiro de Geografia e Estatística, Coordenação de Geografia. Brasil: uma visão
geográfica e ambiental no início do século XXI. Rio de Janeiro: IBGE; 2016. 3. World health Organization. World report on aging and health. Geneva: WHO; 2015 [cited 2017
Nov 25]. Availabe from: http://www.who.int/entity/ageing/publications/world-report-2015/en/ 3. World health Organization. World report on aging and health. Geneva: WHO; 2015 [cited 2017
Nov 25]. Availabe from: http://www.who.int/entity/ageing/publications/world-report-2015/en/ 4. Steves CJ, Spector TD, Jackson SH. Aging, genes, environment and epigenetic: what twin studies
tell us now, and in the future. Age Aging. 2012;41(5):581-6. https://doi.org/10.1093/ageing/afs097 5. Naughton C, Drennan J, Treacy MP, Laerty A, Lyons I, Phelan A, et al. Abuse and neglect of
o older people in Ireland: report on the national study of elder abuse and neglect. Dublin:
National Centre for the Protection of Older People; 2010 [cited 2017 Nov 23]. Available from:
http://www.ncpop.ie/userfiles/file/ncpop%20reports/Study%203%20Prevalence.pdf Cognitive function among older Brazilian adults
Castro-Costa E et al. Cognitive function among older Brazilian adults
Castro-Costa E et al. 6. Wu L, Chen H, Hu Y, Xiang H, Yu X, Zhang T, et al. Prevalence and associated factors of elder
mistreatment in a rural community in People’s Republic of China: a cross-sectional study. PLoS
One. 2012;7(3):e33857. https://doi.org/101371/journal.pone.0033857 7. Shankar A, McMunn A, Banks J, Steptoe A. Loneliness, social isolation, and behavioral and biological
health indicators in older adults. Health Psychol. 2011;30(4):377-85. https://doi.org/101037/a0022826 8. Iliffe S, Kharicha K, Harari D, Swift C, Gillmann G, Stuck AE. Health risk appraisal in older
people 2: the implications for clinicians and commissioners of social isolation risk in older
people. Br J Gen Pract. 2007;57(537):277-82. 9. World Health Organization. Closing the gap in a generation: health equity through action
on social determinants of health: final report of the Commission on Social Determinants of
Health. Geneva: WHO; 2008 [cited 2017 Nov 23]. Available from: http://whqlibdoc.who.int/
publications/2008/9789241563703_eng.pdf 10. Dannefer D. Cumulative advantage/disadvantage and the life course: cross-fertilizing
age and social science theory. J Gerontol B Psychol Sci Soc Sci. 2003;58(6):S327-37. https://doi.org/10.1093/geronb/58.6.s327 11. Langa KM, Llewellyn DJ, Lang IA, Weir DR, Wallace RB, Kabeto MU, et al. REFERENCES Cognitive
health among older adults in the United States and in England. BMC Geriatr. 2009;9:23. https://doi.org/10.1186/1471-2318-9-23 12. Savva GM, Maty SC, Setti A, Feeney J. Cognitive and physical health of the older populations
of England, the United States, and Ireland: international comparability of the Irish Longitudinal
Study on Ageing. J Am Geriatr Soc. 2013;61 Suppl 2:s291-8. https://doi.org/10.111/jgs12196 13. Saenz JL, Downer B, Garcia MAG, Wong R. Cognition and context: rural-urban differences in
cognitive aging among older Mexican adults. J Aging Health. 2017 Apr 1::898264317703560. https://doi.org/10.1177/0898264317703560 14. Cassarino M, O’Sullivan V, Kenny RA, Setti A. Environment and cognitive aging: a cross-sectional
study of place residence and cognitive performance in the Irish Longitudinal Study on Aging. Neuropsychology. 2016;30(5):543-57. https://doi.org/10.1037/neu0000253 15. Lei X, Smith JP, Sun X, Zhao Y. Gender differences in cognition in China and reasons for change over
time: evidence from CHARLS. J Econ Aging. 2014:4:46-55. https://doi.org/10.1016/j.jeoa.2013.11.001 16. Mejía-Arango S, Wong R, Michaels-Obregón A. Normative and standardized data for cognitive
measures in the Mexican Health and Aging Study. Salud Publica Mex. 2015;57 Suppl 1:S90-6. 17. World Bank. Gini Index (World Bank estimate) 2011-2015. Washington (DC); 2015. [cited 2017
Nov 23]. Available from: https://data.worldbank.org/indicator/SI.POV.GINI 18. United Nations Development Programme. Human Development Reports. New York: UNDP
[cited 2017 Nov 23]. Available from: http://hdr.undp.org/en 19. Programa das Nações Unidas para o Desenvolvimento. Desenvolvimento humano nas
macrorregiões brasileiras: 2016. Brasília (DF): PNUD, IPEA, FJP; 2016. 20. Martins NIM, Caldas PR, Cabral ED, Lins CCSA, Coriolano MGWS. Instrumentos de avaliação
cognitiva utilizados nos últimos 5 anos em idosos brasileiros. Cienc Saude Coletiva. 2017 out [cited
2017 Nov 23]:0402. Available from: http://www.cienciaesaudecoletiva.com.br/artigos/instrumentos-
de-avaliacao-cognitiva-utilizados-nos-ultimos-5-anos-em-idosos-brasileiros/16454?id=16454 21. Lima-Costa MF, Andrade FB, Souza Jr PRB, Neri AL, Duarte YAO, Castro-Costa E, de Oliveira C. The Brazilian Longitudinal Study of Aging (ELSI-Brazil): objectives and design. Am J Epidemiol. 2018 Jan 31. https://doi.org/10.1093/aje/kwx387 22. National Institute on Aging; World Health Organization. Global Health and Aging. Bethesda
(MD): National Institute of Health; 2011 [cited 2017 Nov 23]. (NIH publication, 11-773). Available from: http://www.who.int/ageing/publications/global_health.pdf 23. Batty GD, Deary IJ, Zaninotto P. Association of cognitive function with cause-specific mortality
in middle and older age: follow-up of participants in the English Longitudinal Study of Ageing. Am J Epidemiol. 2016;183(3):183-90. https://doi.org/10.1093/aje/kwv139 24. Park DC. The basic mechanism accounting for age-related decline in cognitive function. In: Park
DC, Schwarz N, editors. Cognitive aging: a primer. New York: Psychology Press; 2000. p.3-21. 25. Cognitive function among older Brazilian adults
Castro-Costa E et al. 28. Ferreira L, Ferreira Santos-Galduróz R, Ferri CP, Fernandes Galduróz JC. Rate of cognitive decline
in relation to sex after 60 years-of-age: a systematic review. Geriatr Gerontol Int. 2014;14(1):23-
31. https://doi.org/10.1111/ggi.12093 26. Morris JC, Heyman A, Mohs RC, Hughs JP, Van Belle G, Fillenbaum G, et al. The Consortium to
Establish a Registry for Alzheimer’s Disease (CERAD). Part I. Clinical and neuropsychological assessment
of Alzheirmer’s disease. Neurology. 1989;39(9):1159-65. https://doi.org/10.1212/WNL.39.9.1159 27. Bertolucci PHF, Okamoto IH, Toniolo Neto J, Ramos LR, Brucki SMD. Desempenho da
população brasileira na bateria neuropsicológica do Consortium to Establish a Registry for
Alzheimer’s Disease (CERAD). Rev Psiq Clin (São Paulo). 1998;25(2):80-3. Cognitive function among older Brazilian adults
Castro-Costa E et al. REFERENCES Passos VMA, Giatti L, Bensenor I, Tiemeier H, Ikram MA, Figueiredo RC, et al. Education
plays a greater role than age in cognitive test performance among participants of the
Brazilian Longitudinal Study of Adult Health (ELSA-Brasil). BMC Neurol. 2015;15:191. https://doi.org/10.1186/s12883-015-0454-6 8
https://doi.org/10.11606/S1518-8787.2018052000629 8s Funding: The ELSI-Brazil baseline study was supported by the Brazilian Ministry of Health (DECIT/SCTIE –
Department of Science and Technology from the Secretariat of Science, Technology and Strategic Inputs (Grant
404965/2012-1); COSAPI/DAPES/SAS – Healthcare Coordination of Older Adults, Department of Strategic and
Programmatic Actions from the Secretariat of Health Care) (Grants 20836, 22566, and 23700); and the Brazilian
Ministry of Science, Technology, Innovation and Communication. Authors’ Contribution: Data analysis and interpretation: ECC, MFLC, FBA, PRBSJ, CPF. Manuscript writing:
ECC, MFLC, FBA, PRBSJ, CPF. Manuscript critical review: ECC, MFLC, FBA, PRBSJ, CPF. All authors have approved
the final version of the manuscript and assume public responsibility for its content. Conflict of Interest: The authors declare no conflict of interest. 9s
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Recent Trends in Inequality and Poverty in Developing Countries
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Handbook of income distribution
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www.cedlas.econo.unlp.edu.ar *
This
paper
corresponds
to
chapter
10
of
the
Handbook
of
Income
Distribution,
volume
2,
edited
by
A.
Atkinson
and
F.
Bourguignon.
**
Facundo
Alvaredo
is
with
CONICET,
EMod/OMI-‐Oxford
University
and
Paris
School
of
Economics;
Leonardo
Gasparini
is
with
CEDLAS,
Facultad
de
Ciencias
Económicas,
Universidad
Nacional
de
La
Plata
and
CONICET.
This
paper
was
completed
while
Leonardo
Gasparini
was
visiting
professor
at
the
University
of
British
Columbia.
We
are
grateful
to
participants
to
the
conference
“Recent
Advances
in
the
Economics
of
Income
Distribution”
(Paris),
the
AAEP
Meetings
(Rosario),
and
especially
to
Francois
Bourguignon
and
Tony
Atkinson
for
valuable
comments
and
suggestions.
We
are
also
grateful
to
David
Jaume,
Darío
Tortarolo,
Carolina
López,
Julian
Amendolaggine,
Santiago
Garganta,
Florencia
Pinto,
Pablo
Gluzmann,
Leopoldo
Tornarolli,
Javier
Alejo,
Juan
Zoloa,
and
Carolina
García
Domench
(all
at
CEDLAS)
for
outstanding
research
assistance.
We
alone
are
responsible
for
any
errors.
Financial
support
from
the
ESRC-‐DFID
joint
fund
is
gratefully
acknowledged. Centro de Estudios
Distributivos, Laborales y Sociales
Maestría en Economía
Facultad de Ciencias Económicas
Recent Trends in Inequality and Poverty in
Developing Countries
Facundo Alvaredo y Leonardo Gasparini
Documento de Trabajo Nro. 151
Noviembre, 2013
ISSN 1853-0168 This
version:
November
4,
2013 Recent
Trends
in
Inequality
and
Poverty
in
Developing
Countries
* Facundo
Alvaredo
Leonardo
Gasparini** Abstract This
chapter
reviews
the
empirical
evidence
on
the
levels
and
trends
in
income/consumption
inequality
and
poverty
in
developing
countries. It
includes
a
discussion
of
data
sources
and
measurement
issues,
evidence
on
the
levels
of
inequality
and
poverty
across
countries
and
regions,
an
assessment
of
trends
in
these
variables
since
the
early
1980s,
and
a
general
discussion
of
their
determinants. There
has
been
tremendous
progress
in
the
measurement
of
inequality
and
poverty
in
the
developing
world,
although
serious
problems
of
consistency
and
comparability
still
remain. The
available
evidence
suggests
that
on
average
the
levels
of
national
income
inequality
in
the
developing
world
increased
in
the
1980s
and
1990s,
and
declined
in
the
2000s. There
was
a
remarkable
fall
in
income
poverty
since
the
early
1980s,
driven
by
the
exceptional
performance
of
China
over
the
whole
period,
and
the
generalized
improvement
in
living
standards
in
all
the
regions
of
the
developing
world
in
the
2000s. JEL
Codes:
D31,
I32 JEL
Codes:
D31,
I32 Alvaredo-Gasparini Alvaredo-Gasparini 1. Introduction Poverty and inequality are certainly among the main concerns in the developing world. A typical developing country is characterized by high levels of material deprivation, and
large dispersion in individual wellbeing, at least when compared to a typical developed
economy. Fighting poverty and minimizing the unjust inequalities are top priorities in
the developing world. The United Nations, in the famous declaration of the Millennium
Development Goals (MDGs), proposed as target number 1 to halve income poverty in
the developing world from 1990 to 2015. The reduction of inequality does not occupy
the same privileged position in the agenda, but few would not list it as a central social
concern. While chapter 9 of this Handbook deals with poverty and inequality in the developed
world, this chapter documents patterns and changes in the developing countries. There is no need to argue about the relevance of including a separate chapter in the
Handbook: the developing world is home of 85% of total world population, and bears
levels of poverty and inequality far higher than in the rich countries. While in a typical
developing economy the share of people striving to survive with less than 2 dollars a
day is more than 30%, that share is close to zero in the industrialized countries. In fact,
on this basis poverty is an issue exclusively of the developing world. The differences in
income inequality are presumably also large, although the comparisons are hindered
by the fact that national household surveys typically capture income in developed
countries and consumption expenditures in developing ones. High poverty and inequality are pervasive characteristics of the developing world;
however, they are not immutable features of these economies. There is convincing
evidence pointing to a robust decline in the levels of absolute income poverty in the
developing world over the last decades, and substantial progress in the reduction of
deprivation in various non-monetary dimensions - education, health, sanitation, access
to infrastructure. Changes in income inequality have been much less clear, as relative
inequality has risen in some countries and fallen in others. In fact, the evidence
suggests that on average the developing countries are today (2013) somewhat more
unequal than three decades ago. This chapter reviews the empirical evidence on the levels and trends in income
inequality and poverty in developing countries. INDEX 1. INTRODUCTION ............................................................................................................................ 3
2. THE DEVELOPING WORLD: CHARACTERIZATION AND DATA ..................................... 4
3. INEQUALITY: LEVELS ................................................................................................................. 9
4. INEQUALITY: TRENDS .............................................................................................................. 23
5. POVERTY: LEVELS ..................................................................................................................... 56
6. POVERTY: TRENDS .................................................................................................................... 66
7. CONCLUDING REMARKS ......................................................................................................... 90 2 2 Alvaredo-Gasparini Alvaredo-Gasparini 1. Introduction We focus the analysis on the
income/consumption approximations to welfare; in particular the chapter deals mainly
with relative inequality across individuals in household consumption expenditures per
capita, and with absolute poverty defined over that welfare variable, and considering
alternative international lines defined in US dollars adjusted for purchasing power
parity (PPP). This choice is restricted by space limitations and does not imply ignoring
that a general assessment of poverty and inequality should also include other non-
monetary dimensions (e.g. health, education) and other monetary variables (e.g. wealth). Other chapters in the Handbook contribute to fill those gaps. 3 Alvaredo-Gasparini Alvaredo-Gasparini The analysis in this chapter is mostly focused on inequality and poverty within
countries and not within supra-national regions or in the world.1 Although issues of
global inequality are increasingly relevant, inequality is still primarily a national
concern. People are generally worried about inequality mainly in their countries, and
public policies are typically aimed at reducing disparities among individuals within
national boundaries. The analysis in this chapter is mostly focused on inequality and poverty within
countries and not within supra-national regions or in the world.1 Although issues of
global inequality are increasingly relevant, inequality is still primarily a national
concern. People are generally worried about inequality mainly in their countries, and
public policies are typically aimed at reducing disparities among individuals within
national boundaries. The empirical evidence shown in this chapter is drawn from the academic literature,
regional and country papers, and open-access databases, in particular the PovcalNet
project developed in the World Bank. Although most of the evidence is based on
statistics obtained from national household surveys, we also report results from tax
records (the World Top Incomes Database, WTID) and international surveys (the Gallup
World Poll) to illustrate some issues. Even though the main purpose of the chapter is
presenting basic evidence on levels and trends, we also briefly review the main
discussions on determinants of recent changes in inequality and poverty. The rest of the chapter is organized as follows. In section 2 we briefly characterize the
economies in the developing world, and discuss the data sources and some
measurement issues. The following two sections are assigned to the main topic in this
volume – inequality. In section 3 we document the levels of income inequality in the
developing world, while in section 4 we summarize the evidence on trends since the
early 1980s. 1. Introduction The next two sections repeat the sequence for poverty: section 5
compares levels across countries, and section 6 summarizes trends and discusses the
evidence at the regional level.2 Section 7 closes with a summary and some final
remarks. 2 The separate treatment of inequality and poverty is somewhat artificial, as they are just two
characteristics of the same income distribution. However, and despite some possible overlapping and
duplications, we prefer to follow most of the literature and discuss both concepts separately. 2. The developing world: characterization and data In this section we briefly characterize the economies of the developing world, and
review the sources of data to measure and analyze income poverty and inequality. 1 Global inequality is analyzed in chapter 12 of this volume.
2 2.1. Developing countries The division between developed and developing countries is a helpful simplification
that can be done in different arbitrary ways. In this chapter we follow the World Bank’s
main criterion based on gross national income (GNI) per capita: developing countries
are those with per capita GNI below a certain nominal threshold (US$ 12,276 in 2011). These nations are usually classified into six geographical regions: East Asia and Pacific
(EAP), Eastern Europe and Central Asia (ECA), Latin America and the Caribbean (LAC),
Middle East and North Africa (MENA), South Asia (SA) and Sub-Saharan Africa (SSA). 4 Alvaredo-Gasparini The Appendix includes a list of all the developing countries in each region with their
populations.3 The developing countries cover almost 75% of the total land area in the
world and represent 85% of the total population. Table 2.1 summarizes some basic
demographic and economic statistics. Table 2.1
Population, GNI per capita and Human Development Index, 2010
Developing countries by region Table 2.1
Population, GNI per capita and Human Development Index, 2010
Developing countries, by region
Source: population is taken from the United Nations Demographic Yearbook. Gross National Income
(GNI) per capita in international dollars adjusted for purchasing power parity (PPP) and in current US$
(Atlas method) are taken from World Development Indicators. The Human Development Index (HDI) is
from the UNDP Human Development Report. GNI and HDI are unweighted averages across countries. Countries
Population
(millions)
PPP
Atlas
method
HDI
Developing countries
153
5,840
7,023
4,291
0.608
East Asia and Pacific
24
1,961
4,911
2,992
0.619
Eastern Europe and Central Asia
30
478
12,558
7,815
0.751
Latin America and the Caribbean
31
584
9,789
6,433
0.706
Middle East and North Africa
13
331
6,462
3,647
0.636
South Asia
8
1,633
3,429
1,704
0.535
Sub-Saharan Africa
47
853
3,288
1,798
0.450
Developed countries
62
1,055
37,303
38,818
0.857
00
Total
216
6,894
15,682
14,181
0.663
GNI per capita Table 2.1
Population, GNI per capita and Human Development Index, 2010
Developing countries, by region Source: population is taken from the United Nations Demographic Yearbook. Gross National Income
(GNI) per capita in international dollars adjusted for purchasing power parity (PPP) and in current US$
(Atlas method) are taken from World Development Indicators. The Human Development Index (HDI) is
from the UNDP Human Development Report. GNI and HDI are unweighted averages across countries. 3 In this chapter we include emerging economies as part of the developing world, a decision that implies
some overlapping with chapter 9. In the period under analysis some countries graduated from the set of
developing countries; to avoid selection bias we do not drop them from the analysis. 2.1. Developing countries According to these indicators Eastern Europe and Central Asia is the most developed
region in the group: per capita GNI is almost twice the mean for the developing world,
and the Human Development Index (HDI) is significantly higher. Latin American and
the Caribbean ranks second, and Middle East and North Africa third. Although
economic growth in Asia has been remarkable in the last decades, per capita GNI and
other development indicators are on average still below the mean of the developing
world. South Asia is significantly less developed than East Asia and the Pacific. Sub-
Saharan Africa is the poorest and least developed region of the world. The mean of the
national per capita GNIs in that region is less than 50% of the developing world mean,
and less than 10% of the mean of the industrialized economies. 2.2. Data sources National household surveys are the main source of information for distributive
analysis. Since one of the central goals of these surveys is measuring living standards,
they typically include questions to construct a monetary proxy for wellbeing: income 5 Alvaredo-Gasparini and/or expenditures on consumption goods. Although some developing countries
started to implement national household surveys after World War II, it is only recently
that governments engaged in programs of regularly collecting information through
household surveys, often with the help of some international organization. Distributive
statistics for the developing world are rare before the 1970s, and reasonably robust
only from the 1990s on. There has been a remarkable increase in the availability of
national household surveys over the last decades. A chapter like this one, that includes
a broad assessment of income inequality and poverty in developing countries, could
hardly have been written two decades ago, and is a sign of the huge progress made on
data collection. However, as we discuss below, data limitations are still stringent, and
allow only a still blurred picture of inequality and poverty. The databases for international distributive analysis can be classified into two groups:
those that produce statistics with microdata from surveys or administrative records,
and those that collect, organize and report statistics from other sources. The former
group includes the World Bank´s PovcalNet, the Luxembourg Income Study, the World
Income Distribution database, the World Top Incomes Database and some regional
initiatives. The second one includes the seminal work by Deininger and Squire (1996)
and its follow-up - the WIDER´s World Income Inequality Database, the All the Ginis
database, and some other projects. The main source of information for poverty and inequality analysis at a large
international scale in the developing world is the World Bank´s PovcalNet, a
compilation of distributive data built up from national household surveys, generally
fielded by national statistical offices. PovcalNet, used for the World Bank’s World
Development Indicators, includes statistics constructed mostly from household survey
microdata, and in some few countries from grouped tabulations. At the moment of
writing this database includes more than 850 surveys from almost 130 countries,
representing more than 90% of the population of the developing world, spanning the
period 1979-2011. 4 Statistics are derived from the estimation of a general quadratic and a beta Lorenz curves from
grouped data. Shorrocks and Wan (2008) propose an algorithm that reproduces individual data from
grouped statistics with a higher degree of accuracy. 2.2. Data sources The website of PovcalNet provides public access to data to generate
estimates for selected countries and alternative poverty lines from grouped data.4
Martin Ravallion and Shaohua Chen, the developers of PovcalNet, have produced
several papers exploiting the dataset (Ravallion and Chen, 1997; Chen and Ravallion,
2001, 2010, 2012). This project has been increasingly influential in shaping the
assessment of inequality, and in particular poverty, in the developing world by
researchers and policy practitioners. It is, for instance, the source used to monitor the
poverty-reduction goal of the MDGs. This chapter draws heavily on statistics computed
by the PovcalNet project. Some regional initiatives aimed at computing social statistics from household survey
microdata in a standardized way are useful to study distributive issues in specific 6 Alvaredo-Gasparini geographic areas, and as sources of information for world databases. For instance, the
Socioeconomic Database for Latin America and the Caribbean (SEDLAC), jointly
developed by CEDLAS at the Universidad Nacional de La Plata (Argentina) and the
World Bank’s LAC poverty group (LCSPP), includes distributive and labor statistics for
LAC constructed using consistent criteria across countries and years. BADEINSO,
developed by the United Nations´ ECLAC, is also a large and good-quality database on
economic and social variables in LAC. In Eastern and Central Europe the World Bank
ECA database includes statistics for 28 countries since 1990 computed from direct
access to household surveys. The Household Expenditure and Income Data for
Transitional Economies developed by Branko Milanovic in the World Bank is the
predecessor of that database. Milanovic has also built the World Income Distribution
(WYD) database, which includes data for five benchmark years (1988, 1993, 1998, 2002
and 2005) for 146 countries, 75% obtained from direct access to household surveys. The dataset has been used in several studies to compute global inequality (Milanovic,
2002, 2005, 2012). The Luxembourg Income Study (LIS), described in chapter 9 of this
volume, includes distributive information computed from household survey microdata
for developed countries. LIS also reports statistics for several transitional economies in
Eastern Europe and recently has added some developing countries in Latin America
(Brazil, Colombia, Guatemala, Mexico, Peru and Uruguay). The growth in the availability of distributive statistics stimulated efforts to gather and
organize them. Deininger and Squire (1996) put together a large dataset of quintile
shares and Gini coefficients for most countries since World War II taken from different
studies and national reports. pp
7 SWIID should also be reviewed critically: in many cases it requires a case-by-case analysis, which is
simply a sign that much effort is still needed in putting together comparable statistics. As it is based on
secondary datasets, external problems are inadvertently incorporated. 5 WIID was initially compiled over 1997-1999 for the UNU/WIDER-UNDP project "Rising Income
Inequality and Poverty Reduction: Are They Compatible?" directed by Giovanni Andrea Cornia.
Tabatabai (1996) at International Labour Organization also made an independent effort to put together
distributive statistics for many countries in the world. 6 Analyzing the Deininger and Squire (DS) dataset, Atkinson and Brandolini (2001) conclude that “users
could be seriously misled if they simply download the accept series (i.e., the “high quality” subset)”.
Although WIID implies a significant improvement from the original DS dataset, a similar word of caution
applies. distributive statistics for many countries in the world.
6 Analyzing the Deininger and Squire (DS) dataset, Atkinson and Brandolini (2001) conclude that “users
could be seriously misled if they simply download the accept series (i.e., the “high quality” subset)”.
Although WIID implies a significant improvement from the original DS dataset, a similar word of caution
applies.
7 SWIID should also be reviewed critically: in many cases it requires a case-by-case analysis, which is
simply a sign that much effort is still needed in putting together comparable statistics As it is based on 5 WIID was initially compiled over 1997-1999 for the UNU/WIDER-UNDP project "Rising Income
Inequality and Poverty Reduction: Are They Compatible?" directed by Giovanni Andrea Cornia.
Tabatabai (1996) at International Labour Organization also made an independent effort to put together
distributive statistics for many countries in the world.
6 Analyzing the Deininger and Squire (DS) dataset, Atkinson and Brandolini (2001) conclude that “users
could be seriously misled if they simply download the accept series (i.e., the “high quality” subset)”.
Although WIID implies a significant improvement from the original DS dataset, a similar word of caution
applies.
7 SWIID should also be reviewed critically: in many cases it requires a case-by-case analysis, which is
simply a sign that much effort is still needed in putting together comparable statistics. As it is based on
secondary datasets, external problems are inadvertently incorporated. 2.2. Data sources This panel database, which greatly promoted the
empirical study of the links between inequality and other economic variables, was
updated and extended by the UNU/WIDER-UNDP World Income Inequality Database
(WIID) (WIDER, 2008).5 The WIID database includes Gini coefficients, quintile and
decile shares, and the income shares of the top 5% and bottom 5%. The information is
drawn from very different sources, which raises comparability concerns.6 To provide
guidance in the use of the database, ratings are given to the observations, based on
the survey quality, the coverage, and the quality of the information provided by the
original source. The SWIID database is an effort to identify reasonably comparable
information in WIID (Solt, 2009).7 7 Alvaredo-Gasparini The All the Ginis database, assembled also by Branko Milanovic, is a compilation and
adaptation of Gini coefficients retrieved from five datasets: LIS, SEDLAC, WYD, the
World Bank ECA database, and WIID. Besides gathering all the information in a single
file, the All the Ginis database is useful as it provides information on the welfare
concept and recipient unit to which the reported Gini refers, facilitating the
comparisons. The Chartbook of Economic Inequality, assembled by Atkinson and Morelli (2012),
presents a summary of evidence about changes in economic inequality
(income/consumption, earnings and wealth) in the period from 1911 to 2010 for 25
countries. The information drawn from household surveys for the seven countries in
the developing world included in the database (Argentina, Brazil, India, Indonesia,
Malaysia, Mauritius and South Africa) starts in the 1950s. All the datasets mentioned above are based on data from national household surveys.8
Even when they are the best available source of information for distributive analysis,
household surveys are plagued with problems for international comparative studies,
because, among other reasons, the questionnaires and the procedures to compute
income/consumption variables differ among countries, and frequently also within a
country over time. Some surveys inquire about income and others about consumption,
some capture net income and some gross income, in some cases variables are
reported on a weekly basis and in others on a monthly basis, items as the imputed rent
for owner occupied housing are included in some surveys and ignored in others.9 Even
in those projects that made explicit efforts to reduce these differences, comparability
issues persist, as problems rooted in differences in questionnaires are difficult to be
completely overcome. These problems are well recognized in the literature. 8 The exception is the Chartbook of Economic Inequality, that uses a range of sources.
9 In addition, the typical problems of under-reporting and selective compliance are negligible in some
cases and endemic in others. See Deaton (2003, 2005) and Korinek et al. (2006). 8 The exception is the Chartbook of Economic Inequality, that uses a range of sources.
9 2.2. Data sources For
instance, Chen and Ravallion (2012) state that “…there are problems that we cannot
deal with. For example, it is known that differences in survey methods (such as
questionnaire design) can create non-negligible differences in the estimates obtained
for consumption or income”. In a survey of global income inequality, Anand and Segal
(2008) share those concerns. There are some alternatives to reduce the comparability problems, although they all
come at a price. Gallup conducts a survey in nearly all nations in the world with almost
exactly the same questionnaire. The Gallup World Poll is particularly rich in self-
reported measures of quality of life, opinions, and perceptions, but it also includes
basic questions on demographics, education, and employment, and a question on
household income. In principle, the Gallup World Poll allows a distributive analysis in
nearly all the countries in the world based on the same income question. The
downside is that measurement errors may be very large when reported income is 8 Alvaredo-Gasparini based only on one question and with sample sizes of just around 1000 observations
per country.10 The Estimated Household Income Inequality (EHII) data set produced by the University
of Texas Inequality Project (UTIP) is based on UTIP-UNIDO, a global data set that
calculates industrial pay-inequality measures for 156 countries from 1963 to 2003,
using the between-groups component of a Theil index, measured across industrial
categories in the manufacturing sector (Galbraith and Kum, 2005).11 Although in
principle the use of industrial pay information could lend some homogeneity into the
comparisons, the technique employed may not provide an accurate measure of
inequality over the whole income distribution. 10 Gasparini and Gluzmann (2012) compare basic demographic statistics drawn from the Gallup Poll with
those computed from the national household surveys of the LAC countries for year 2006, and conclude
that in most countries statistics from the Gallup Poll, including income poverty and inequality, are
roughly consistent with those from national household surveys.
11 Specifically, EHII consists on estimates of gross household income inequality, computed from an OLS
regression between the Deininger and Squire inequality measures and the UTIP-UNIDO manufacturing
pay inequality measures, controlling for the source of information in the inequality data
(income/expenditure, gross/net, and household/per capita measures) and for the share of
manufacturing employment in total employment.
12 The drawbacks of computing inequality in the distribution of consumption or income per capita to
measure distributive justice have been widely acknowledged. Among other limitations, it is a one-
dimensional approach, it is focused on results not opportunities, it ignores the value of publicly provided
goods such as education and health services, and it adopts a simple adjustment for demographics
ignoring intra-household inequality, economies of scale and differences in needs (Ferreira and Ravallion,
2009). However, extending inequality measurement to alleviate these limitations in a way that keeps
international comparisons feasible has been proved difficult. 12 The drawbacks of computing inequality in the distribution of consumption or income per capita to
measure distributive justice have been widely acknowledged. Among other limitations, it is a one-
dimensional approach, it is focused on results not opportunities, it ignores the value of publicly provided
goods such as education and health services, and it adopts a simple adjustment for demographics
ignoring intra-household inequality, economies of scale and differences in needs (Ferreira and Ravallion,
2009). However, extending inequality measurement to alleviate these limitations in a way that keeps
international comparisons feasible has been proved difficult. 10 Gasparini and Gluzmann (2012) compare basic demographic statistics drawn from the Gallup Poll with
those computed from the national household surveys of the LAC countries for year 2006, and conclude
that in most countries statistics from the Gallup Poll, including income poverty and inequality, are
roughly consistent with those from national household surveys. Specifically, EHII consists on estimates of gross household income inequality, computed from an OLS
gression between the Deininger and Squire inequality measures and the UTIP-UNIDO manufacturing
y inequality measures, controlling for the source of information in the inequality data
come/expenditure, gross/net, and household/per capita measures) and for the share of
anufacturing employment in total employment. 15 We decided to apply the same coefficient to all LAC countries after failing to find significant
regularities between the ratio consumption/income Ginis and other observable variables for the seven
countries in the sample. WDR (2006) reports consumption and income Ginis in four Latin American
countries; the mean ratio of the Ginis is 0.81. The value is somewhat lower (0.77) for the eight non-LA
countries in the sample. 3. Inequality: levels In this section we present results regarding the level of inequality in the developing
countries, deferring to the next section the discussion of the trends. In most of the
section we measure inequality computed over the distribution of household
consumption per capita, using data from PovcalNet.12 Consumption is usually regarded
as a better measure of current welfare than income on both theoretical and practical
grounds, especially in developing countries (Deaton and Zaidi, 2002). As it is usual in
this literature, we frequently refer to income inequality, despite the fact that statistics
are constructed over the distribution of consumption expenditures. As discussed above, this chapter is mainly focused on within-country inequality, so
welfare disparities are measured among individuals living within the national
boundaries. Although globalization is increasingly raising global inequality concerns,
inequality remains mainly a national matter. This view also leads us to mostly
document unweighted statistics of inequality measures across countries, a practice
that is consistent with the typical cross-country approach in the development
literature. Weighting by population would imply an assessment of inequality in a
region or in the world strongly affected by some highly-populated countries, such as 9 Alvaredo-Gasparini China, India and Indonesia in Asia, or Brazil and Mexico in Latin America, and almost
ignoring the situation in other less-populated nations. Having said that, since the
decision of taking each political entity as a unit in the analysis is certainly debatable;
we show some results using both unweighted and population-weighted statistics.13 13 See some arguments on this debate in Bourguignon et al. (2004). g
g g
(
)
14 The countries are Argentina, Costa Rica, Ecuador, Mexico, Nicaragua, Panama and Peru. 13 See some arguments on this debate in Bourguignon et al. (2004). 3.1. Inequality in the developing countries We start by comparing inequality levels across developing countries based on the Gini
coefficient for the distribution of household consumption per capita for year 2010,
computed in PovcalNet mostly from household survey microdata. Other inequality
measures are highly correlated with the Gini coefficient. For instance, in PovcalNet and
WIID datasets the Pearson and Spearman correlations of the Gini and several extreme
inequality measures (e.g. the 90/10 and 80/20 income-share ratios) exceed 0.9. PovcalNet includes information for the distribution of per capita consumption
expenditures, except in almost all Latin American and a few Caribbean countries, for
which income inequality statistics are reported. In the analysis that follows we adjust
the income Gini coefficients in that region to reflect the gap between income and
consumption inequality estimates. Specifically, we selected seven Latin American
countries with household surveys that include reasonably good consumption and
income data in several years:14 on average the ratio of the consumption/income Ginis
is 0.861 (standard deviation of 0.046). We apply that coefficient to the 22 Latin
American and Caribbean countries with income data to approximate their
consumption Ginis.15 In most cases the observations correspond to year 2010, or adjacent years. However,
some countries are lacking a recent household survey (or it was dropped due to quality
concerns). In fact, in 24 countries the survey used to estimate inequality in 2010 was
carried out between 2000 and 2005, while in 6 cases (5 of them in the Caribbean) the
observation corresponds to the 1990s. With that caveat in mind, the PovcalNet dataset
has relatively recent distributive information for 82% of the countries in the
developing world, representing 97% of its total population (see Table A.1 in the
Appendix). The country coverage across regions is heterogeneous. In East Asia and
Pacific PovcalNet includes 12 out of the 24 developing countries, which nonetheless
represent 96% of the total population of the area. The coverage in Eastern Europe and
Central Asia is almost complete, lacking information only for the small Kosovo. In LAC
the coverage is complete in continental Latin America, but weak in the Caribbean. Anyway, countries with information represent 98% of the total population in LAC (the 10 Alvaredo-Gasparini main missing country in terms of population is Cuba). The dataset in Middle East and
North Africa does not contain information only for Lebanon and Libya, which represent
3% of the MENA population. 16 PovcalNet reports Ginis above 63.1 for Comoros and Seychelles, two small island countries in the
Indian Ocean. However, the results are not well established. For instance, the reported Gini in
Seychelles is 42.7 in 2000 and 65.8 in 2007, a highly implausible change in just seven years. 17 The harmonic mean is similar in SSA than in LAC. 3.1. Inequality in the developing countries In South Asia the only country missing is Afghanistan,
while in Sub-Saharan Africa there is information for 42 out of the 47 countries,
representing 95% of the population. Figure 3.1 displays the range of Gini coefficients for 122 countries around year 2010,
ranking from the least unequal (Ukraine, 25.6) to the most unequal economy (South
Africa, 63.1).16 The mean value is 39.8, while the median is 39.2. More than half of the
observations are in the range [35, 45]. Only seven Eastern Europe countries have Ginis
below 30, and five Sub-Saharan African countries have Ginis higher than 55. The
population-weighted mean is less than one point lower than the simple mean (39.1), a
result affected by the relatively low level of inequality in populous India and Indonesia
(China has a Gini somewhat higher than the world mean). Figure 3.1 shows the
position of some of the most populated countries: Brazil has high inequality levels,
China and Russia intermediate values, and India and Indonesia relatively low levels in
the context of the developing world. Figure 3.1
Gini coefficients for the distribution of household consumption per capita
Developing countries, 2010
Source: own calculations based on PovcalNet (2013). Note: countries sorted by their Gini coefficients. 20
25
30
35
40
45
50
55
60
65
0
10
20
30
40
50
60
70
80
90
100
110
120
130
Gini coefficient
Brazil
China
Indonesia
India
Russia Gini coefficients for the distribution of household consumption per capita
Developing countries, 2010 Source: own calculations based on PovcalNet (2013). Note: countries sorted by their Gini coefficients. The variability of Gini coefficients across countries is large compared to the changes
within countries over time, at least for the period for which we have more robust
information (since the early 1980s). Li, Squire and Zou (1998) find in the Deininger and 11 Alvaredo-Gasparini Squire dataset that 90% of the total variance in the Gini coefficient is explained by
variation across countries, while only a small percentage is accounted by variation over
time. From this observation Li et al. (1998) conclude that inequality should be mainly
determined by factors which differ substantially across countries, but tend to be
relatively stable within countries over time. We find a similar result in a panel of
developing countries from 1981 to 2010 (PovcalNet data): 88.5% of the variance in
that panel is accounted by variation across countries. 3.1. Inequality in the developing countries Squire dataset that 90% of the total variance in the Gini coefficient is explained by
variation across countries, while only a small percentage is accounted by variation over
time. From this observation Li et al. (1998) conclude that inequality should be mainly
determined by factors which differ substantially across countries, but tend to be
relatively stable within countries over time. We find a similar result in a panel of
developing countries from 1981 to 2010 (PovcalNet data): 88.5% of the variance in
that panel is accounted by variation across countries. The inequality rankings are relatively stable over time. The Spearman-rank correlation
coefficient for the Ginis in 1981 and 2010 is 0.68, while it rises to 0.74 for 1990 and
2010, both significant at 1%. The last decades witnessed enormous economic, social
and political changes in the developing world, but, although the income distributions
have been affected with various intensities, the world inequality ranking has not
changed much, a fact that suggests the existence of some underlying factors that are
stronger determinants of the level of inequality. In Figure 3.2 developing countries are grouped in regions. Sub-Saharan Africa is the
geographic area that includes countries with the highest inequality levels, but it is also
the region with the highest dispersion, possibly in part due to measurement errors
(Table 3.1). Although eight out of the ten highest Gini coefficients belong to Sub-
Saharan African countries, and the arithmetic mean of the Gini coefficient is the
highest in the world, the median is lower than in Latin America.17 Figure 3.2
Gini coefficients for the distribution of household consumption per capita
Developing countries, 2010
Source: own calculations based on PovcalNet (2013). Note: each bar represents a country in a given geographic region of the developing world. 20
25
30
35
40
45
50
55
60
65
Gini coefficient
East Asia &
Pacific
Eastern Europe &
Central Asia
Latin America &
the Caribbean
Middle East &
North Africa
South Asia
Sub-Saharan Africa Gini coefficients for the distribution of household consumption per capita
Developing countries, 2010 Source: own calculations based on PovcalNet (2013). (
)
Note: each bar represents a country in a given geographic region of the developing world. Note: each bar represents a country in a given geographic region of the developing world. 3.1. Inequality in the developing countries 12 Alvaredo-Gasparini Table 3.1
Gini coefficients for the distribution of household consumption per capita
Developing countries, 2010
Source: own calculations based on PovcalNet (2013). Note: unweighted statistics. Mean
Median
Coef. Var. Min. Max. East Asia and Pacific
38.1
36.7
0.101
31.9
43.5
Eastern Europe and Central Asia
33.6
33.7
0.144
25.6
43.6
Latin America and the Caribbean
43.8
44.8
0.104
34.7
52.8
Middle East and North Africa
36.0
36.1
0.091
30.8
40.9
South Asia
35.0
36.3
0.081
30.0
38.1
Sub-Saharan Africa
44.4
42.1
0.175
33.3
63.1
Developing countries
39.8
39.2
0.181
25.6
63.1 Table 3.1
Gini coefficients for the distribution of household consumption per capita
Developing countries, 2010 Latin America and the Caribbean has been typically pointed out as the most unequal
region in the world. Deininger and Squire (1996), for instance, state that their dataset
confirm the “familiar fact that inequality in Latin America is considerably higher than in
the rest of the world”.18 This type of assessment however is usually made combining
income Ginis for LAC with consumption Ginis for other regions, and/or ignoring Sub-
Saharan
Africa. With
the
adjustment
mentioned
above
to
take
the
consumption/income gap into consideration (factor 0.861), we find that the mean Gini
for LAC is 43.8, slightly lower than in SSA (44.4), but the median is higher (44.8 in LAC
and 42.1 in SSA). To reach the result of a higher mean Gini in LAC than in SSA we would
need an adjustment parameter higher than 0.92; such value is larger than what we
estimated in all LA countries in the sample, except Mexico. The rest of the regions in the developing world have Ginis mostly below 40. The
arithmetic mean is 38.1 in East Asia and Pacific, 36.0 in Middle East and North Africa,
and 35.0 in South Asia. Inequality is likely to be higher in MENA, since several oil-
producing countries are excluded for being high-income economies (and also for lack
of information).19 Eastern Europe and Central Asia is the region with the lowest
inequality levels, with a mean Gini coefficient of 33.6. Interestingly, the dispersion
measured by the coefficient of variation is higher than in the rest of the regions,
except SSA. Almost all very highly unequal countries (Gini coefficients above 50) are in Sub-Saharan
Africa (Table 3.2). This region, however, has a similar share of countries in the high and
middle categories. 19 Bahrain, Kuwait, Oman, Qatar, Saudi Arabia, United Arab Emirates are in that group. Malta and Isra
are also ignored for being developed and Lebanon and Libya are excluded for lack of information. 18 See also Lopez Calva and Lustig (2010) and Chen and Ravallion (2012).
19 3.1. Inequality in the developing countries In contrast, in LAC most countries have high levels of inequality,
while in EAP, MENA and SA most countries are in the middle-inequality group. Only
ECA has economies with low inequality (Gini coefficients below 30). 13 13 Alvaredo-Gasparini Alvaredo-Gasparini Table 3.2
Classification of countries by level of inequality and by region
Developing countries, 2010
Source: own calculations based on PovcalNet (2013). Note: countries are classified according to the value of the Gini coefficient
for the distribution of household consumption per capita. Very high
High
Middle
Low
Total
[50-70]
[40-50)
[30-40)
[20-30)
East Asia and Pacific
0
3
8
0
11
Eastern Europe and Central Asia
0
5
16
7
28
Latin America and the Caribbean
2
17
6
0
25
Middle East and North Africa
0
1
10
0
11
South Asia
0
0
7
0
7
Sub-Saharan Africa
10
14
16
0
40
Total
12
40
63
7
122
Inequality Table 3.2
Classification of countries by level of inequality and by region
Developing countries, 2010
Source: own calculations based on PovcalNet (2013). Note: countries are classified according to the value of the Gini coefficient
for the distribution of household consumption per capita. Very high
High
Middle
Low
Total
[50-70]
[40-50)
[30-40)
[20-30)
East Asia and Pacific
0
3
8
0
11
Eastern Europe and Central Asia
0
5
16
7
28
Latin America and the Caribbean
2
17
6
0
25
Middle East and North Africa
0
1
10
0
11
South Asia
0
0
7
0
7
Sub-Saharan Africa
10
14
16
0
40
Total
12
40
63
7
122
Inequality Table 3.2
Classification of countries by level of inequality and by region
Developing countries, 2010 Note: countries are classified according to the value of the Gini coefficient
for the distribution of household consumption per capita. The All the Ginis dataset (ATG) includes Gini coefficients from LIS, SEDLAC, WYD, the
World Bank ECA database and WIID. We selected consumption Ginis from ATG for year
2005 or close, and applied a similar adjustment as described above for those countries
in LAC with only income Ginis. The basic results are similar to the ones obtained with
PovcalNet data. The linear correlation coefficient for the Gini between both data
sources is 0.763, while the Spearman rank correlation is 0.771, both significant at 1%. The Gini coefficients in ATG go from 23.1 (Czech Republic) to 62.9 (Comoros). 20 The OECD high-income countries rank as the least unequal in the world with a mean income Gini of
32.8. 3.1. Inequality in the developing countries The
mean and median coincide in 40.1. Again, more than half of the observations are in the
range [35, 45]. Only several Eastern European countries have Ginis below 30, while
only four Sub-Saharan African countries have Ginis higher than 55. The evidence on inequality levels in the developing world drawn from WIID is similar. For instance, based on a sample of income Ginis for around 2005, Gasparini et al. (2013) find that the mean Gini for the six Sub-Saharan African countries in the dataset
is 56.5, followed by Latin America (52.9), Asia (44.7) and Eastern Europe and Central
Asia (34.7).20 We find that the linear correlation coefficient for year 2005 for the Gini
coefficient in PovcalNet and WIID is 0.871, and the Spearman coefficient is 0.820. The Luxembourg Income Study database (see chapter 9 of this volume) covers 36
countries, including 6 in Latin America, which occupy the top places in all the income
inequality rankings. The LA Ginis go from 50.6 in Colombia to 43.9 in Uruguay; the
most unequal non-LA country is Russia with a value of 40.8, while the rest of the
countries in LIS go from 37 (USA) to 22.8 (Denmark). The mean Gini for the Eastern
European countries in LIS is slightly higher than the mean for the high-income
economies. Data from the World Development Indicators also suggest that inequality
in the developing world is significantly higher than in the OECD high-income countries. The mean income Gini for the latter group is 32.2, which is lower than in any other
region in the world. 14 14 Alvaredo-Gasparini The EHII database confirms the high inequality levels of Sub-Saharan Africa and Latin
America, but perhaps surprisingly, it records similar levels in South Asia and Middle
East and North Africa.21 The mean Gini for the period 1998-2002 is 47.3 in SSA, 46.4 in
LAC, 46.4 in MENA and 47.2 in SA. According to this dataset inequality is relatively
lower in East Asia and Pacific (44.9) and Eastern Europe and Central Asia (44.0). The
estimated level of the Gini coefficient is substantially lower in the developed
economies; the mean is equal to 36.5.22 The Pearson (Spearman) correlation
coefficient between EHII and PovcalNet Ginis is 0.642 (0.603), lower than the resulting
value when comparing PovcalNet with WIID or ATG, but still significant at 1%. 22 This mean excludes the oil-rich Arab countries. When including these countries in the sample th
Gi i j
t 39 21 See also Galbraith and Kum (2005). 22 This mean excludes the oil-rich Arab countries. When including these countries in the sample the
mean Gini jumps to 39. 21 See also Galbraith and Kum (2005).
22 This mean excludes the oil-rich Arab countries. When including these countries in the sample the 3.1. Inequality in the developing countries Most international databases do not provide confidence intervals for the point
estimates of the distributive measures, making impossible the assessment of the
statistical significance of the differences among countries. However, given that the
indicators are calculated from large national household surveys, the confidence
intervals are typically relatively narrow. SEDLAC provides the confidence intervals for
all the Gini coefficients in Latin America: for instance, the 95% confidence interval for
the income Gini was [43.9, 44.7] in Argentina 2010, [53.5, 54.0] in Brazil 2009; and
[47.0, 47.9] in Mexico 2010. Differences in the point estimates of more than 1 Gini
point are always statistically significant (Figure 3.3). Figure 3.3
Gini coefficient and confidence intervals (95%)
Distribution of household income per capita
Latin American countries, 2010 Figure 3.3
Gini coefficient and confidence intervals (95%)
Distribution of household income per capita
Latin American countries, 2010
Source: own calculations based on SEDLAC (CEDLAS and the World Bank). 43
48
53
58
Argentina
Brazil
Chile
Colombia
Costa Rica
Dominican R. Ecuador
Honduras
Mexico
Panama
Paraguay
Peru
Uruguay
Gini coefficient Source: own calculations based on SEDLAC (CEDLAS and the World Bank). 15 15 Alvaredo-Gasparini 24 Take the distribution of the shares of deciles 7 to 9 in total consumption across countries. While the
percentile 10 of that distribution is 34.7, the percentile 90 is just 38.3. The corresponding intervals for
the distribution of the share accruing to the poorest 60% [25.4, 38.2] and the richest 10% [24.3, 39.3]
are much wider. 23 Again, figures for Latin American (and a few Caribbean) countries are estimated based on the
comparison of income and consumption microdata of seven countries in that region. 23 Again, figures for Latin American (and a few Caribbean) countries are estimated based on the
comparison of income and consumption microdata of seven countries in that region.
24 Take the distribution of the shares of deciles 7 to 9 in total consumption across countries. While the
percentile 10 of that distribution is 34.7, the percentile 90 is just 38.3. The corresponding intervals for
the distribution of the share accruing to the poorest 60% [25.4, 38.2] and the richest 10% [24.3, 39.3]
are much wider. 23 Again, figures for Latin American (and a few Caribbean) countries are estimated based on the
comparison of income and consumption microdata of seven countries in that region.
24 Take the distribution of the shares of deciles 7 to 9 in total consumption across countries. While the
percentile 10 of that distribution is 34.7, the percentile 90 is just 38.3. The corresponding intervals for
the distribution of the share accruing to the poorest 60% [25 4 38 2] and the richest 10% [24 3 39 3] 3.2. Inequality beyond the Gini coefficient The international databases usually allow a closer look at the distributions in the world
beyond a single parameter, such as the Gini coefficient. Table 3.3 reports some basic
statistics of the decile shares in 120 countries around 2010.23 On average (unweighted)
the poorest 10% of the population in a country accrues 2.6% of total consumption
reported in the survey: that share climbs to 31.5% for the top 10%. In a typical
developing country the aggregate consumption of the poorest 60% of the population is
similar than the consumption of the top 10%. It is interesting to notice that the coefficient of variation of the decile consumption
shares across countries is decreasing up to the top decile, when it strongly rises:
countries in the world seem substantially different in the consumption share of the
poor and the rich, but not in the share of the middle strata, in particular the upper-
middle strata. This fact is naturally related to the typical shape of the real-world
income distributions. For instance, under log-normality, the derivative of the decile
shares with respect to the standard deviation of log incomes is U-shaped. Table 3.4 Table 3.4
Correlation coefficients across countries of decile consumption shares
Developing countries, 2010
Source: own calculations based on PovcalNet (2013). *=significant at 1%. d1
d2
d3
d4
d5
d6
d7
d8
d9
d10
d1
1
d2
0.9355*
1
d3
0.8930*
0.9883*
1
d4
0.8421*
0.9624*
0.9910*
1
d5
0.8042*
0.9273*
0.9647*
0.9787*
1
d6
0.7336*
0.8739*
0.9291*
0.9623*
0.9847*
1
d7
0.6310*
0.7734*
0.8436*
0.8950*
0.9378*
0.9736*
1
d8
0.3127*
0.4711*
0.5624*
0.6446*
0.7253*
0.8085*
0.8982*
1
d9
-0.5793*
-0.4905*
-0.4112*
-0.3258*
-0.2389*
-0.1232
0.0527
0.4390*
1
d10
-0.7844*
-0.9032*
-0.9452*
-0.9689*
-0.9844*
-0.9891*
-0.9650*
-0.7962*
0.118
1 Table 3.4
Correlation coefficients across countries of decile consumption shares
Developing countries, 2010
Source: own calculations based on PovcalNet (2013). *=significant at 1%. d1
d2
d3
d4
d5
d6
d7
d8
d9
d10
d1
1
d2
0.9355*
1
d3
0.8930*
0.9883*
1
d4
0.8421*
0.9624*
0.9910*
1
d5
0.8042*
0.9273*
0.9647*
0.9787*
1
d6
0.7336*
0.8739*
0.9291*
0.9623*
0.9847*
1
d7
0.6310*
0.7734*
0.8436*
0.8950*
0.9378*
0.9736*
1
d8
0.3127*
0.4711*
0.5624*
0.6446*
0.7253*
0.8085*
0.8982*
1
d9
-0.5793*
-0.4905*
-0.4112*
-0.3258*
-0.2389*
-0.1232
0.0527
0.4390*
1
d10
-0.7844*
-0.9032*
-0.9452*
-0.9689*
-0.9844*
-0.9891*
-0.9650*
-0.7962*
0.118
1 Correlation coefficients across countries of decile consumption shares
Developing countries, 2010 Correlation coefficients across countries of decile consumption shares Source: own calculations based on PovcalNet (2013). *=significant at 1%. Table 3.3 Table 3.3
Deciles shares, distribution of household consumption per ca
Developing countries, 2010
Source: own calculations based on PovcalNet (2013). Note: unweighted statistics. Deciles
Mean
Std. Dev. Coef.Var. Min. Max. 1
2.6
0.81
0.31
1.0
4.4
2
3.8
0.86
0.23
1.5
5.8
3
4.8
0.90
0.19
2.0
6.8
4
5.8
0.92
0.16
2.6
7.8
5
6.8
0.92
0.13
3.5
8.8
6
8.1
0.87
0.11
4.7
9.9
7
9.6
0.80
0.08
6.6
11.0
8
11.7
0.65
0.06
9.0
12.7
9
15.3
0.84
0.05
12.7
17.6
10
31.5
6.12
0.19
19.5
51.7 Deciles shares, distribution of household consumption per capita
Developing countries, 2010 The aggregate consumption share of deciles 5 to 9 is on average around 50%, and it is
very stable across countries. Palma (2011) has labeled this phenomenon the
homogeneous middle. Variability across countries is actually smaller in the upper-
middle deciles (deciles 7 to 9).24 The proportion of total consumption accruing to that
group is quite similar in all geographic regions of the world: it ranges from 35.9% in
SSA to 37.3% in ECA. The main difference across regions lies in the share of the bottom 16 Alvaredo-Gasparini 60% compared to those in the upper 10%. For example, while the share of deciles 7 to
9 in total consumption is almost the same in ECA (37.3%) and LAC (37.1%), the share of
the bottom 60% is more than 7 points higher in the former (36.4% and 29.1%). The correlation coefficients for the decile shares in total consumption provide
information about the structure of the distributions across countries (Table 3.4). In a
cross-country perspective, gains are highly positively correlated in the first 8 deciles;
on the other hand, for decile 10 correlations are all negative and large, except with
decile 9, for which the correlation is non-significant. Gains in the participation of the
richest 10% are tightly linked to losses in the share of the poorest 80% of the
population. The table suggests that when we move up in the ladder of countries
according to the share of the bottom deciles, we expect to see gains in the lowest
strata obtained mostly against the share of the upper 10% of the population (and not
for instance against the middle strata, and in alliance with the rich). 3.3. Inequality in the Gallup World Poll The Gallup World Poll provides new evidence on the international comparisons of
income inequality, as it includes identical income and household structure questions
applied to national samples in 132 countries. Of course, the reliability of the national
inequality estimates in Gallup is lower than those obtained with household surveys,
since only one income question is used to approximate well-being, and the sample
sizes are considerably smaller. However, Gluzmann (2012) finds that the correlation
coefficient between the Gini coefficients computed with Gallup microdata and those
reported in the World Development Indicators (WDI) that are based on per capita
income is high (0.85). Interestingly, the relationship with the consumption Ginis in WDI
is much weaker; the linear correlation coefficient is 0.21, non-significant at 10%. International surveys with similar questionnaires across countries, such as the Gallup
World Poll, could hardly be a substitute for household surveys as the main source for
distributive analysis at the country level, but they may have a great potential for 17 17 Alvaredo-Gasparini international comparisons of social variables. Future improvements in the quality of
these surveys could turn them into a very valuable source for comparative
international research. Gasparini and Gluzmann (2012) use microdata from the Gallup World Poll 2006 to
compute inequality in each region of the world. According to the unweighted mean of
the national income Gini coefficients, Latin America is the most unequal region in the
world (excluding Africa, which is not in the sample). The cross-country Gini in Latin
America is 49.9, slightly larger than in South Asia (48.9), and Eastern Asia and Pacific
(47.1). Countries in Eastern Europe and Central Asia (41.8), North America (39.2) and
especially Western Europe (34.0) are the least unequal. Alternatively, regional
inequality can be measured by considering each region as a single unit, and computing
inequality among all individuals in that unit, after translating their incomes to a
common currency - a concept usually labeled global inequality (see chapter 12 of this
Handbook). The global Gini in Latin America is 52.5, a value higher than in Western
Europe (40.2), North America (43.8) and Eastern Europe and Central Asia (49.8), but
lower than in South Asia (53.2) and Eastern Asia and Pacific (59.4). The change in the
rankings between the two concepts of inequality is driven by the differences across
regions in the heterogeneity among countries in terms of mean income. 3.3. Inequality in the Gallup World Poll Gasparini and
Gluzmann (2012) report that the between component in a Theil decomposition
accounts for 8% of total regional inequality in Latin America and 32.4% in East Asia and
Pacific. 3.4. Top incomes Until the recent developments in the literature of top incomes from tax records
(Atkinson and Piketty, 2007, 2010; see also chapter 16 in this volume), inequality
research has been mostly based on household surveys, which suffer from several
limitations when focusing on the upper end of the distribution. Household surveys are
all but ideal for studying top shares: the rich are usually missing from surveys, either
for sampling reasons or because they refuse to cooperate with the time-consuming
task of completing or answering to a long form. Because extreme observations are
sometimes regarded as data “contamination”, the rich may be intentionally excluded
or top coded so as to minimize bias problems generated by presumably less-reliable
outliers, or to preserve anonymity. Additionally, survey data present severe under-
reporting at the top: the richest individuals are more reluctant to disclose their
incomes, or have diversified portfolios with income flows that are difficult to value. Székely and Hilgert (1999) look at surveys from eighteen Latin American household
surveys and confirm that the ten highest incomes reported are often not much larger
than the salary of an average manager in the given country at the time of the survey. In general, the profile of the average individual in the top 10% of the distribution is
closer to the prototype of highly educated professionals earning labor incomes, rather
than capital owners. Consequently, the inequality that we are able to measure with 18 Alvaredo-Gasparini household surveys can be severely affected, regarding both levels and dynamics, in
those cases or periods in which an important part of the story takes place at the top. Tax and register data are being increasingly preferred over surveys in studying
distributive issues at the top. In fact, under certain conditions registry data can provide
valuable information to improve survey-based estimates. Typically, incomes reported
to the surveys are checked against the registers, or incomes are directly taken from
administrative sources for the individuals in the sample. Even if the combination of
survey and administrative data can be seen as an improvement, there remains the
issue of the sampling framework for the top of the distribution.25 In any case, statistics
offices in the developing world are not exploiting register data to complement surveys
yet. The use of tax statistics is not without drawbacks. 26 The reasons for which the rich and wealthy may be particularly dissuaded from disclosing
their fortunes and incomes to authorities in the developing world may go beyond tax concerns,
lest the information revealed fall into the wrong hands. Alvaredo and Londoño (2013) report
that in Colombia, until recently plagued by high insecurity, anecdotal evidence suggests that
during the intense political violence of the 1990s leaked personal tax returns were used by
criminal groups to target victims and kidnap for ransom. 25 If high-income individuals are not properly identified in the sample framework, comparing
the incomes reported to the surveys against those in the registers one by one is only a partial
improvement. In the UK, for example, the ONS scales up the surveys’ incomes so that the
surveys’ averages match the average income in tax data. 27 The National Accounts-based measure of household disposable income has been defined as: balance
of households’ primary incomes + social benefits other than social transfers in kind − employers’ actual
social contributions − imputed social contributions − attributed property income of insurance
policyholders − imputed rentals for owner occupied housing − fixed capital consumption – employees’
social security contributions – taxes on income and wealth paid by households. 3.4. Top incomes First, since only a fraction of the
population files a tax return, studies using tax data are restricted to measuring top
shares, which are silent about changes in the lower and middle part of the distribution. Second, tax data are collected as part of an administrative process and do not seek to
address research needs; both income and tax units are defined by the tax laws and
vary considerably across time and countries. Third and most important, estimates are
affected by tax avoidance and tax evasion; the rich, in particular, have a strong
incentive to understate their taxable incomes. These elements, which are common to
all countries, become critical in the developing world, characterized by tax systems
with low enforcement and multiple legal ways to avoid the tax.26 A number of researchers have addressed the differences in the ability of tax and
survey data to represent income inequality, trying to reconcile the evidence using the
two sources (see Alvaredo, 2011; Burkhauser et al., 2012 for the US). Unfortunately,
and despite multiple requests, at the moment of writing only a few developing
countries have made available microdata from the income tax (namely Colombia,
Ecuador and Uruguay). Alvaredo and Londoño (2013), and Alvaredo and Cano
(forthcoming) show that, in contrast to survey-based results, high-income individuals
are, in essence, rentiers and capital owners. This feature differs from the pattern found
in several developed countries in recent decades, where it has been shown that the
large increase in the share of income going to the top groups has been mainly due to
spectacular increases in executive compensation and high salaries, and to a lesser 26 The reasons for which the rich and wealthy may be particularly dissuaded from disclosing
their fortunes and incomes to authorities in the developing world may go beyond tax concerns,
lest the information revealed fall into the wrong hands. Alvaredo and Londoño (2013) report
that in Colombia, until recently plagued by high insecurity, anecdotal evidence suggests that
during the intense political violence of the 1990s leaked personal tax returns were used by
criminal groups to target victims and kidnap for ransom. 19 19 Alvaredo-Gasparini extent to a partial restoration of capital incomes. 3.4. Top incomes While the working rich have joined
capital owners at the top of the income hierarchy in the United States and other
English-speaking countries, Colombia and Ecuador remain more traditional societies
where the top income recipients are still the owners of the capital stock. Results confirm that incomes reported to the tax authorities are considerably higher
than those recorded in the surveys at the top. A natural question, which has received
much attention lately, is the extent to which tax data can complement household
surveys in examining the level of inequality in developing countries. Alvaredo and
Londoño (2013) compare the Colombian household survey with the tax micro-data
over the years 2007-2010. The total household income from the survey is 60-65% of
the NAS measure of disposable income.27 Such gap cannot be seen as an accurate
measure of missing income in household surveys, because both sources are different,
but a partial explanation may well be at the top of the distribution. As a simple
exercise, these authors replace all the incomes above the percentile 99 in the survey
with those from tax data (net of taxes and social security contributions to render both
sources comparable), under the assumption that the top 1% is poorly captured in the
survey. Two elements are worth mentioning. First, the difference between the
adjusted survey’s total income (that is, the survey’s incomes of the bottom 99% plus
the net-of-tax incomes from the tax data above the percentile 99) and the NAS figure
goes down to 75-80%. Second, the Gini coefficient of individual incomes goes up from
55 to 61 in 2010.28 These findings challenge the general skepticism regarding the use of tax data from
developing countries to study inequality. Such estimates should be regarded as a lower
bound, to take into account the effects of evasion and under reporting. Nevertheless,
they show that incomes reported to tax authorities can be a valuable source of
information, under certain conditions that require a case-by-case analysis. 28 These results are still approximations, as defining individual actual incomes from the Colombian tax
records is not always straightforward. 29 The Gini for the developed countries is computed over the distribution of income per capita, and not
consumption per capita, a fact that probably underestimates the slope of the curve. 30 It is also confirmed estimating GDP with the Atlas method, and using the All the Ginis database.
31 31 The Latin American “excess inequality” is documented in Londoño and Székely (2000); Gasparin
Cruces and Tornarolli (2011), and others. 3.5. Inequality and development Is the level of inequality in a country associated to its development stage? In this
section we take advantage of a cross-section of national Gini coefficients for year 2010
to take a look at this issue. Of course, this topic is related to the long-lasting debate
initiated with the seminal contributions by Lewis (1954) and Kuznets (1955), who
argued that the process of industrialization would imply an inverse U pattern for
inequality. However, the empirical test for the Kuznets curve requires time-series or 28 These results are still approximations, as defining individual actual incomes from the Colombian tax
records is not always straightforward. 20 20 Alvaredo-Gasparini panel data, and not just a cross-section, since it is a hypothesis about the dynamics of
an economy over its development process. The causal relationship between
development and inequality is the subject of a large literature that has to face
numerous empirical challenges, and hence it is far from settled (see Anand and
Kanbur, 1993; Fields, 2002; Banerjee and Duflo, 2003; Dominics et al., 2008; and
Voitchovsky, 2009 for assessments). In this section we simply document the empirical
relationship between these two variables across countries in a recent point in time
without exploring the difficult issue of causality. The first panel in Figure 3.4 plots the Gini coefficient for the distribution of
consumption per capita against per capita gross national income (GNI).29 The figure
seems to reveal a decreasing relationship between inequality and development. The
linear correlation coefficient between the Gini coefficient and per capita GNI is -0.56
(statistically significant at the 1% level). An inverse-U shape shows up in the second
panel of Figure 3.4, when per capita GNI is presented in logs. However, the increasing
segment of the curve covers only very poor Sub-Saharan African countries. The
relationship Gini-GNI is decreasing in the range of GNI of most countries in the world. 21 21 Alvaredo-Gasparini Figure 3.4
Inequality and development
Per capita gross national income (GNI) and Gini coefficient, 2010
Source: own calculations based on WDI and PovcalNet (2013). 32 In order to compute changes we discard countries in PovcalNet with less than four observations over
the period 1981-2010, or with observations concentrated in a narrow time-period. The sample we use
for the calculations on trends include 76 countries that represent 88% of the developing world 3.5. Inequality and development Lind and Mehlum test: H0: monotone or U shape; H1: inverse U shape. Lind and Mehlum test: H0: monotone or U shape; H1 4. Inequality: trends In this section we report the recent trends in income inequality in the developing
countries. We start laying out the general patterns, and then deep into the evidence
for each region. Although most of the section deals with relative inequality, we devote
a section to explore patterns for absolute inequality, and a section to document
aggregate welfare changes. We end with a brief summary of the methodologies and
main issues in the debate on inequality determinants in the developing world. 3.5. Inequality and development 10
20
30
40
50
60
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
Gini coefficient
GNI per capita (PPP)
Developed
EAP
ECA
LAC
MENA
SA
SSA
10
20
30
40
50
60
5
6
7
8
9
10
11
Gini coefficient
log GNI per capita (PPP)
Developed
EAP
ECA
LAC
MENA
SA
SSA Inequality and development GNI per capita (PPP) Source: own calculations based on WDI and PovcalNet (2013). The results of the regressions in Table 3.5 and the Lind and Mehlum (2010) test
confirm an inverse U shape for the relationship between the Gini coefficient and log
GNI per capita in a cross-section of countries.30 The result seems also valid, although
becomes considerable weaker, when restricting the sample to developing economies. The turning points implicit in the regressions correspond to around US$ 1800, a value
that is lower than the per capita GNI of most developing countries, except for some
economies in Sub-Saharan Africa. The inclusion of regional dummies reveals that East
Asian, and especially Latin American and Sub-Saharan African countries are particularly
unequal, even when controlling for their levels of economic development.31 22 Alvaredo-Gasparini Table 3.5
Relationship between Gini coefficient and GNI per capita Table 3.5
Relationship between Gini coefficient and GNI per capita
Note: robust cluster standard errors in brackets. * significant at 10%; ** significant at 5%; *** significant at 1%. Omitted category: Eastern Europe and Central Asia. (i)
(ii)
(iii)
(iv)
log GNIpc
24.24
24.44
18.01
26.54
(9.52)**
(4.48)***
(8.23)*
(6.58)**
log GNIpc squared
-1.606
-1.409
-1.202
-1.541
(0.552)**
(0.34)***
(0.53)*
(0.48)**
Developed countries
-1.416
(2.76)
East Asia & Pacific
7.352
7.170
(1.43)***
(1.62)***
Latin America & Caribbean
10.238
10.157
(0.53)***
(0.62)***
Middle East & North Africa
2.334
2.144
(1.28)
(1.48)
South Asia
1.705
1.515
(1.79)
(1.97)
Sub-Saharan Africa
13.749
13.660
(2.33)***
(2.34)***
Constant
-49.34
-72.10
-61.67
-80.27
(38.69)
(13.17)***
(28.64)**
(20.06)**
Observations
146
146
121
121
R-squared
0.31
0.58
0.07
0.45
Lind and Mehlum test for inverse U shape
| t |
2.72
2.31
1.35
2.0
p-value
0.004
0.011
0.089
0.024
All countries
Only developing countries Table 3.5
Relationship between Gini coefficient and GNI per capita Relationship between Gini coefficient and GNI per capita * significant at 10%; ** significant at 5%; *** significant at 1%. Omitted category: Eastern Europe and Central Asia. Lind and Mehlum test: H0: monotone or U shape; H1: inverse U shape. population. In order to build a constant sample in a few cases Gini coefficients are imputed assuming
constant inequality. Income Ginis in LAC are adjusted as explained in the previous section.
33 This result is in part driven by the lack of information on changes in inequality over this period for
several countries in the developing world. See below. 4.1. General changes The available evidence suggests that on average the levels of national income
inequality in the developing world increased in the 1980s and 1990s, and declined in
the 2000s. Using data from PovcalNet, the mean Gini for the distribution of per capita
consumption expenditures increased from 37.2 in 1981 to 39.4 in 2010 (Figure 4.1).32 23 Alvaredo-Gasparini The mean was basically unchanged between 1981 and 1987,33 then increased more
than three points to reach a value of 40.5 in 1999, and from 2002 it started to fall,
although slowly (from 40.6 in 2002 to 39.4 in 2010). The mean was basically unchanged between 1981 and 1987,33 then increased more
than three points to reach a value of 40.5 in 1999, and from 2002 it started to fall,
although slowly (from 40.6 in 2002 to 39.4 in 2010). Figure 4.1
Gini coefficient
Unweighted mean for developing countries, 1981-2010 Figure 4.1
Gini coefficient
Unweighted mean for developing countries, 1981-2010
Source: own calculations based on PovcalNet (2013). Note: the national Gini coefficients are computed over the distribution of household consumption per
capita. 33
35
37
39
41
43
1981
1984
1987
1990
1993
1996
1999
2002
2005
2008
2010 Note: the national Gini coefficients are computed over the distribution of household consumption per
capita. Figure 4.2 adds to the picture the changes at different percentiles of the distribution of
national Ginis. The figure makes clear that on average the changes in the last decades
have not been large, compared to the range over which the Gini varies across
countries. The picture also reveals that the growth in the mean Gini in the late 1980s
and 1990s was mainly due to the substantial increase in the low-inequality countries,
in particular Eastern Europe and Central Asia economies after the fall of communism,
and also some Asian economies in the early stage of economic take-off. Instead, the
fall in the 2000s was widespread, although more intense in those countries above the
median, such as those in Latin America. This observation suggests convergence in the
levels of inequality in the developing economies. In fact, the standard deviation for the
distribution of Gini coefficients substantially fell over time: 11.2 in 1981, 10.1 in 1990,
7.4 in 1999 and 7.2 in 2010. Countries in the developing world are still very different in
terms of income inequality but differences have become considerably smaller over the
last three decades. 4.1. General changes Figure 4.2 adds to the picture the changes at different percentiles of the distribution of
national Ginis. The figure makes clear that on average the changes in the last decades
have not been large, compared to the range over which the Gini varies across
countries. The picture also reveals that the growth in the mean Gini in the late 1980s
and 1990s was mainly due to the substantial increase in the low-inequality countries,
in particular Eastern Europe and Central Asia economies after the fall of communism,
and also some Asian economies in the early stage of economic take-off. Instead, the
fall in the 2000s was widespread, although more intense in those countries above the
median, such as those in Latin America. This observation suggests convergence in the
levels of inequality in the developing economies. In fact, the standard deviation for the
distribution of Gini coefficients substantially fell over time: 11.2 in 1981, 10.1 in 1990,
7.4 in 1999 and 7.2 in 2010. Countries in the developing world are still very different in
terms of income inequality but differences have become considerably smaller over the
last three decades. 24 Alvaredo-Gasparini Figure 4.2
Gini coefficient g
Gini coefficient
Unweighted statistics for developing countries, 1981-2010
Source: own calculations based on PovcalNet (2013). Note: the national Gini coefficients are computed over the distribution of household consumption per
capita. 10
15
20
25
30
35
40
45
50
55
60
1981
1984
1987
1990
1993
1996
1999
2002
2005
2008
2010
percentile 10%
percentile 90%
percentile 25%
percentile 75%
median
mean Gini coefficient Gini coefficient
Unweighted statistics for developing countries, 1981-2010 Unweighted statistics for developing countries, 1981-2010 10
15
20
25
30
35
40
45
50
55
60
1981
1984
1987
1990
1993
1996
1999
2002
2005
2008
2010
percentile 10%
percentile 90%
percentile 25%
percentile 75%
median
mean Source: own calculations based on PovcalNet (2013). Note: the national Gini coefficients are computed over the distribution of household consumption per
capita. Note: the national Gini coefficients are computed over the distribution of household consumption per
capita. 34 We classify countries in groups according to whether the Gini went up or down by more or less than
2.5% in a period. That threshold is certainly arbitrary. A change of 2.5% applied to the mean Gini in the
developing world - which is around 40- represents 1 Gini point. A change of 1 point in the Gini
coefficient is typically statistically significant, given the sample sizes of the national household surveys. 4.1. General changes A closer inspection of the data reveals that the result of a stable mean Gini in most of
the 1980s is driven by the lack of information for several countries, and by a
substantial heterogeneity in the changes of those with information (Table 4.1).34 The
strong rise in the mean Gini in the 1990s is associated to a large proportion of
countries with growing inequality in a framework of much improved information. The
tide seems to have turned in the 2000s, when most of the countries in the sample
experienced a fall in inequality. But even in this decade of widespread social
improvement, the country performances in terms of inequality reduction were quite
heterogeneous. In fact, in 20% of the economies of the developing world the Gini
coefficient increased between 2002 and 2010, while in 15% of the countries the
changes were smaller than 2.5%. 25 Alvaredo-Gasparini Alvaredo-Gasparini Table 4.1
Proportion of countries classified in groups according
to the change in the Gini coefficient
Source: own calculations based on PovcalNet (2013). Note: “Fall” includes countries where the Gini fell more than 2.5% in the period, “Increase” include
countries where the Gini rose more than 2.5%, “No change” includes countries where the Gini changed
less than 2.5%, “No information” includes countries without two independent observations in each
period. 1981-1990
1990-2002
2002-2010
Fall
14.7
22.7
65.3
No change
21.3
16.0
14.7
Increase
34.7
60.0
20.0
No information
29.3
1.3
0.0
Total
100.0
100.0
100.0 Proportion of countries classified in groups according
to the change in the Gini coefficient Source: own calculations based on PovcalNet (2013). 1981-1990
1990-2002
2002-2010
Fall
14.7
22.7
65.3
No change
21.3
16.0
14.7
Increase
34.7
60.0
20.0
No information
29.3
1.3
0.0
Total
100.0
100.0
100.0 (
)
Note: “Fall” includes countries where the Gini fell more than 2.5% in the period, “Increase” include
countries where the Gini rose more than 2.5%, “No change” includes countries where the Gini changed
less than 2.5%, “No information” includes countries without two independent observations in each
period. The bulk of the countries in the sample (64%) experienced a change in the pattern of
inequality around the turn of the century, from non-falling to decreasing inequality,
while only a few experienced a pattern of continuous increasing (12%) or decreasing
(15%) disparities over the two decades. 4.1. General changes The inverse-U shape for the inequality pattern
observed for many economies in the developing world could be consistent with the
Kuznets story of economic growth for countries located close to the curve turning
point. However, we fail to find any significant correlation between the type of the
inequality pattern and different measures of development and growth. The inverse-U
pattern in the period 1981-2010 appears to have been common to a wide range of
economies (more on this below). The growth in the population-weighted mean of the Gini coefficient across developing
countries was stronger than the increase in the unweighted mean (Figure 4.3). While
the latter increased 2.2 points in the period 1981-2010, the former jumped 7.5 points. The gap between the two means shrunk from 5.4 points in the early 1980s to almost
zero in the late 2000s. This pattern is mainly accounted by the dramatic surge in
income inequality in China over the period. Interestingly, the fall in the unweighted
mean Gini in the 2000s does not show up in the weighted mean, mainly because of the
increase in inequality in China (also in India and Indonesia, but to a lesser extent). Although the Gini coefficient for a typical developing country significantly decreased in
the 2000s, the national Gini for a typical person in the developing world did not fall. 26 Alvaredo-Gasparini Figure 4.3
Gini coefficient
Weighted and unweighted means
Developing countries, 1981-2010
Source: own calculations based on PovcalNet (2013). Note: the Gini coefficients are computed over the distribution of household consumption per capita. 30
32
34
36
38
40
42
1981
1984
1987
1990
1993
1996
1999
2002
2005
2008
2010
unweighted
weighted Figure 4.3
Gini coefficient
Weighted and unweighted means
Developing countries, 1981-2010 Figure 4.3
Gini coefficient
Weighted and unweighted means
Developing countries, 1981-2010 (
)
Note: the Gini coefficients are computed over the distribution of household consumption per capita. In the rest of this section we go beyond the Gini coefficient and track changes along
the distribution. In Figure 4.4 each point in a growth-incidence curve (GIC) indicates
the unweighted mean across countries in the annual rate of growth of real
consumption per capita (in PPP US$) for a given decile of the national distributions.35
There is a stark contrast in the GIC corresponding to the 1990s and the 2000s. 35 The GIC depicted in Figure 4.4 is not the world growth-incidence curve, where for instance decile 1
would include the poorest 10% of the world population. 4.1. General changes The first
one is clearly increasing, suggesting growing inequalities, while the second is
decreasing (and flatter), indicating a fall in well-being disparities in the 2000s. On
average, in that decade consumption per capita grew by more than annual 4% in the
three bottom deciles of the national distributions and by 3% in the top decile. 27 27 Alvaredo-Gasparini Figure 4.4
Growth-incidence curves
Annualized growth rate in consumption per capita by decile
Unweighted mean for developing countries
Source: own estimates based on PovcalNet (2013). Note: annual change in consumption per capita (PPP US$). 1990s=1990-2002, 2000s=2002-2010. -3
-2
-1
0
1
2
3
4
5
1
2
3
4
5
6
7
8
9
10
Deciles
1990s
2000s Figure 4.4
Growth-incidence curves
Annualized growth rate in consumption per capita by decile
Unweighted mean for developing countries 9
10
Deciles Deciles Source: own estimates based on PovcalNet (2013). Note: annual change in consumption per capita (PPP US$). 1990s=1990-2002, 2000s=2002-2010 annual change in consumption per capita (PPP US$). 1990s=1990-2002, 2000s=2002-2010. Naturally, the contrast between decades is also evident when looking at income
shares. The results are summarized in Figure 4.5: while the share of the bottom 60%
fell 2 points in the 1990s and increased 0.9 points in the 2000s, the performance of the
top 10% was almost the exact mirror: increased 2.4 points in the 1990s and fell 1 point
in the following decade. The share of the “middle” (deciles 7 to 9) has remained quite
stable over the two last decades (36.9 in 1990, 36.5 in 1999 and 36.6 in 2010). This
stratum seems not only quite homogeneous across countries but also over time
(Palma, 2011). Figure 4.5
Decile shares
Unweighted mean for developing countries, 1990-2010
Source: own estimates based on PovcalNet (2013). Note: the decile shares are computed over the distribution of household consumption per capita. 28
30
32
34
1990
1993
1996
1999
2002
2005
2008
2010
Deciles 1 to 6
Decile 10 Figure 4.5
Decile shares
Unweighted mean for developing countries, 1990-2010 Unweighted mean for developing countries, 1990 2010
S
i
b
d
P
lN
(2013)
28
30
32
34
1990
1993
1996
1999
2002
2005
2008
2010
Deciles 1 to 6
Decile 10 Source: own estimates based on PovcalNet (2013). (
)
Note: the decile shares are computed over the distribution of household consumption per capita. 36 We prefer not to report the regional patterns before 1990 since the number of observations is small in
several regions. The numbers of countries by region in the sample we use to assess inequality trends
are: 8 in EAP, 20 in ECA, 19 in LAC (all in Latin America, none from the Caribbean), 5 in MENA, 4 in SA
and 20 in SSA. 4.1. General changes (
)
Note: the decile shares are computed over the distribution of household consumption per capit 28 28 Alvaredo-Gasparini Alvaredo-Gasparini 4.2. Changes by region Changes in inequality have been heterogeneous across the six geographical regions of
the developing world (Figure 4.6).36 The mean Gini coefficient in Latin America
increased more than two points in the 1990s, and then dropped in the 2000s by a
larger amount. The data reveals almost no change in inequality in Sub-Saharan Africa
over the two last decades and some decline in the five MENA countries included in the
sample. Instead, the Gini coefficient increased more than two points in Asia, and more
than six points in Eastern Europe and Central Asia. Figure 4.6 suggests again some
pattern toward convergence: the gaps in inequality among regions in the developing
world are smaller now than two decades ago. For instance, while the gap in the Gini
coefficient between Latin America and ECA was 18 points in the early 1990s, it shrank
to 11 points in the late 2000s. Figure 4.6
Gini coefficients
Unweighted means by region, 1990-2010 Figure 4.6
Gini coefficients
Unweighted means by region, 1990-2010
Source: own estimates based on PovcalNet (2013). Note: the Gini coefficients are computed over the distribution of household consumption per capita. 25
30
35
40
45
50
1990
1993
1996
1999
2002
2005
2008
Gini coefficient
Latin America
Middle East & North Africa
East Asia & Pacific
South Asia
Eastern Europe & Central Asia
Sub-Saharan Africa Latin America Sub-Saharan Africa Middle East & North Africa Eastern Europe & Central Asia Source: own estimates based on PovcalNet (2013). Note: the Gini coefficients are computed over the distribution of household consumption per capita. Note: the Gini coefficients are computed over the distribution of household consumption per ca In all regions the share of countries with falling inequality rose in the 2000s, as
compared to the 1990s. The two most remarkable changes in the pattern occurred in
Latin America, and in Eastern Europe and Central Asia. While the Gini went down in
26% of the LA economies in the 1990s, that share increased to 95% in the 2000s. In
ECA while the growth in inequality was generalized in the 1990s, more than half of the
countries experienced reductions in the 2000s. 36 We prefer not to report the regional patterns before 1990 since the number of observations is small in
several regions. 4.2. Changes by region The numbers of countries by region in the sample we use to assess inequality trends
are: 8 in EAP, 20 in ECA, 19 in LAC (all in Latin America, none from the Caribbean), 5 in MENA, 4 in SA
and 20 in SSA. 29 Alvaredo-Gasparini Using data from PovcalNet, Chen and Ravallion (2012) report changes in the within
component of the global mean-log deviation between 1981 and 2008. This within
component is a population-weighted measure of the national inequalities. They find
substantial increases in East Asia and Pacific (from 0.125 to 0.256) and Eastern Europe
and Central Asia (from 0.128 to 0.225), smaller increases in South Asia (from 0.156 to
0.181), Latin America and the Caribbean (from 0.541 to 0.561) and sub-Saharan Africa
(from 0.338 to 0.347) and a fall in MENA (from 0.256 to 0.215). Bastagli et al. (2012)
report similar patterns using data from PovcalNet, SEDLAC and LIS. The picture of national inequalities in the developing world is similar when using other
databases. For instance, the unweighted mean Gini in the All the Ginis database
assembled by Milanovic grew from 36.2 in 1990 to 40.7 in 1999 and then dropped to
39.7 by 2005. While in the 1990s inequality rose in 63% of the economies in the ATG
database, that share dropped to 35% in the 2000s. The recorded increase in the 1990s
was generalized across regions, but especially intense in Eastern Europe and Central
Asia (9 Gini points), while the fall in the 2000s was larger in MENA and Latin America. Cornia and Kiiski (2001), Cornia (2011) and Dhongde and Miao (2013) document
similar results using WIID data. We find that the linear (rank) correlation coefficient for
the change in the Gini coefficient between 1990 and 2005 recorded in PovcalNet and
WIID is 0.776 (0.868), significant at 1%. The corresponding values for the comparison
between PovcalNet and ATG are 0.721 and 0.765. The evidence drawn from the EHII database is also roughly consistent with the
patterns discussed above. The mean Gini for the developing world remained almost
unchanged in the 1980s, increased in the 1990s from 42.5 in 1990 to 47.0 in 1999, and
dropped to 46.5 in 2002 (the latest available date).37 While in 62% of the countries
inequality increased in the early 1990s, that share dropped to 55% between 1993 and
1999, and to 49% between 1999 and 2002. 37 These estimates are computed dropping countries with few observations in the period. 4.2. Changes by region The regional patterns are roughly
consistent with those described above. The main difference is that EHII reveals a
dramatic increase in inequality in the Middle East and North Africa (7 Gini points) that
is not present in the evidence drawn from household surveys. In the rest of this section we briefly review the literature on inequality changes in each
geographic region of the developing world, while we take a closer look to the story of
some particular cases: Brazil, China, India, Indonesia and South Africa. East Asia and Pacific The inequality patterns in East Asia and Pacific can be traced based on information
from only 8 out of the 24 countries in the region, which nonetheless represent 96% of
its total population. This set includes Cambodia, China, Indonesia, Lao PDR, Malaysia,
Philippines, Thailand and Vietnam. There is scattered evidence for Fiji, Micronesia,
Mongolia and Timor-Leste, while information is either lacking or too scarce for 30 30 Alvaredo-Gasparini Alvaredo-Gasparini American Samoa, Kiribati, Korea, Dem. Rep., Marshall Islands, Myanmar, Palau, Papua
New Guinea, Samoa, Solomon Islands, Tonga, Tuvalu and Vanuatu. American Samoa, Kiribati, Korea, Dem. Rep., Marshall Islands, Myanmar, Palau, Papua
New Guinea, Samoa, Solomon Islands, Tonga, Tuvalu and Vanuatu. The slightly increasing pattern showed in Figure 4.6 for the unweighted mean of the
consumption Gini in EAP hides important differences across countries (ADB, 2012;
Chusseau and Hellier, 2012; Ravallion and Chen, 2007; Sharma et al., 2011; Solt, 2009;
Zin, 2005). Consumption inequality increased in most economies in the region during
the 1990s, with the exception of Thailand and Malaysia. The increase was particularly
strong in China, where the consumption Gini climbed around 7 points in that decade. The performance in the 2000s was more heterogeneous: inequality continued
increasing in China, Lao PDR and Indonesia, and also went up in Malaysia, while there
is evidence pointing to a fall in consumption inequality in Cambodia, Philippines,
Thailand and Vietnam. Overall, considering the two decades, EAP combines countries with systematic
increases in inequality (China, Indonesia, Lao PDR), several cases in which inequality
had a cyclical pattern, ending in 2010 at similar levels than in 1990 (Cambodia,
Malaysia, Philippines and Vietnam), and only one successful story of consistent
reduction in consumption inequality: Thailand, for which the estimated reduction in
the Gini coefficient exceeded five points; from 45.3 to 39.4 over the last two decades. Universal social policies, including basic education and health, have been stressed by
many authors as significant drivers of that fall (Jomo and Baudot 2007). Probably the most striking phenomenon regarding inequality in EAP was the strong
rise that took place in the two most populous countries of the region, China and
Indonesia: the Gini coefficient went up around five points in Indonesia and more than
ten points in China over the last two decades. Such dynamics happened in a context of
high growth and falling poverty, most notably in China. Sharma et al. East Asia and Pacific (2011) summarize
the main factors behind these changes: (i) the realignment of activity away from
agriculture and towards industry and services; (ii) the skill premium rose due to the
unmatched growing demand for skills, and even the emigration of skilled workers; (iii)
inequalities in educational attainment in secondary and tertiary schooling have
become an increasingly important source of unequal incomes; (iv) a lack of
infrastructure linking urban areas with rural areas and other barriers to labor mobility
have further accentuated inequality; (v) in China most of income inequality (40 per
cent) is now accounted for by rural-urban differences in income levels, and the
concentration of growth in urban areas is creating a rural-urban divide in employment
and earning possibilities, exacerbated by the much more rapid development that
occurred in coastal areas. The interested reader is referred to the boxes on China and
Indonesia. China China China
Since 1978, when China started the pro-market reforms, GDP has increased at an
average rate close to 10 per cent, and household per capita income has grown more Since 1978, when China started the pro-market reforms, GDP has increased at an
average rate close to 10 per cent, and household per capita income has grown more 31 31 Alvaredo-Gasparini than 7 per cent per year. Such remarkable economic transformation has been
accompanied by important changes in inequality and poverty. China is a successful
story of reduction in absolute poverty (World Bank, 2009; Ravallion and Chen, 2007,
2008; Minoiu and Reddy, 2008). Chen and Ravallion (2008) find that between 1981 and
2005 the number of poor people (with a poverty line of $1.25) went down from 839 to
204 million in terms of consumption, and from 816 to 135 million in terms of income. However, it is argued that it will be harder for China to maintain its past rate of
progress against absolute poverty without addressing the problem of rising inequality. The increase in income inequality in China over the last three decades has been widely
documented. Ravallion and Chen (2007) and World Bank (2009) show that income
inequality rose from the mid-1980s through 1994, dipping a bit in the late 1990s, and
then edging upward thereafter. The Gini coefficient rose from 25 in 1981 to 39 in 1999. Li et al. (2013), among others, document an additional increase in the 2000s, a pattern
explained by the widening of the rural-urban income gap, and the increase in income
from property and assets, driven by the development of urban residential real-estate
markets, the expansion of stock and capital markets, the growth of private enterprises,
and other property rights. In marked contrast to most developing countries, relative inequality is higher in China’s
rural areas than in urban areas. However, there has been convergence over time with
a steeper increase in inequality in urban areas Absolute inequality has increased
appreciably, both between and within both urban and rural areas. Indonesia During the thirty years before the Asian crisis of 1997-1998, which coincided with the
New Order under Suharto’s dictatorship, Indonesia GDP grew at an average rate of 7%
per year, while per capita GDP increased by 3.5 times. The process was not smooth
and went through different phases that implied immense structural change. Agriculture was the main economic sector in the 1970s and benefited from the
introduction of high yielding variety seeds and other technical progress; the oil
commodity boom (1974-1981) generated a windfall income that allowed for the
subsidization of rice production and the improvement of the country’s infrastructure,
with positive effects on life expectancy at birth and school enrollment rates. The
period of deregulation and external oriented policies after 1985 set the basis for the
development of the manufacturing sector. Despite problems with the data, scholars agree in that there was a systematic drop in
poverty rates between 1976 and 1997. At the same time, overall consumption
inequality in Indonesia did not change markedly with development until the late
1980s. On the contrary, the household per capita consumption Gini displayed a falling
trend between 1964 and 1987 (from 35 to 32). Subsequently, although urban
inequality increased, this was partly offset by declines in rural inequality. Cameron
(2002) argues that Indonesia can be considered to be ‘lucky’ in the sense that its 32 Alvaredo-Gasparini industrial center happens to be close to rural Java where many of the country’s
poorest families make their home. These households benefited from the off-farm
employment opportunities that industrialization offered. Alatas and Bourguignon
(2000) found that the returns to land size decreased between 1980 and 1996;
opportunities for off-farm earnings for rural households also contributed to falling
rural inequality. Inequality (but not poverty) started to rise seven years before the crisis. Alatas and
Bourguignon (2000) decompose such increase between components associated with
changes in the structure of earnings, changes in occupational choice and changes in
the socio-demographic structure of the population. They find as main explanations the
migration from rural to urban areas and the increase in non-farm self-employed work. Indonesia was severely hit by a financial crisis: in 1997-1998 GDP dropped by 15%. This
turned into a sharp decrease in inequality and an increase in poverty. Skoufias and
Suryahadi (2000) find that this pattern seems to have arisen from a decrease in
regional inequality. Indonesia Urban areas (which tend to be wealthier than neighboring rural
areas) were hit harder, and the urban middle class, who lost their formal sector jobs,
was harshly affected. As the crisis reduced the per capita expenditure of households,
the percentage reduction was probably less among the poorer population than among
the less poor population. Since 2001, and along with the process of decentralization of
powers to local authorities, a general pattern of rising consumption inequality has
been observed: the Gini increased by 5 points from 33 in 2002 to 38 in 2010. Miranti et
al. (2013) suggest that the recent increases in inequality may be linked to the higher
share of workers employed in the informal sector (70 per cent), hence not covered by
minimum wage legislation or employment protections. 38 Much information also existed about pre-1990 earnings and household incomes in (former)
Czechoslovakia, Hungary and Poland (Atkinson and Micklewright, 1992)
39 Milanovic and Ersado (2010); Ivaschenko (2002); Ferreira (1999); Milanovic (1998); Cornia (1996);
Cornia and Reddy (2001); Mitra and Yemtsov (2006). 39 Milanovic and Ersado (2010); Ivaschenko (2002); Ferreira (1999); Milanovic (1998); Cornia (199
Cornia and Reddy (2001); Mitra and Yemtsov (2006). 38 Much information also existed about pre-1990 earnings and household incomes in (forme
Czechoslovakia, Hungary and Poland (Atkinson and Micklewright, 1992)
39 Eastern Europe and Central Asia The transition from central planning to market system in the countries of Eastern
Europe (EE) and those belonging to the former Soviet Union (FSU) had profound
socioeconomic impacts, which could be better documented (compared to the situation
in previous decades) due to the improvements in the production and release of
information by the new administrations (Milanovic, 1998).38 The fall of the communist
regimes was followed by a substantial increase in inequality in almost all countries.39
According to PovcalNet data the mean Gini for the distribution of per capita
consumption expenditures grew from 26.4 in 1990 to 31.9 in 1996. The increase in the
first half of the 1990s was particularly strong in those countries belonging to the FSU
and in Southeast Europe, and somewhat milder in those economies that joined the
European Union. Such developments have been linked to the process of privatization, 33 Alvaredo-Gasparini which implied an increase in earnings dispersion in comparison to the more
compressed wage structure of the state-owned firms. One key characteristic of the
planned economies was the imposition of wage “grids” that forced a wage
compression; the fast transition from wage setting under the wage grids toward a less
regulated labor market provoked a rise in the returns to education, and hence a surge
in inequality.4041 The economic liberalization also triggered changes in the sectorial
structure of the economy; in particular the ensuing de-industrialization during the
transition is linked to an increase in inequality (Ferreira, 1999, Milanovic, 1999,
Ivaschenko, 2002; Birdsall and Nellis 2003). Milanovic and Ersado (2010) highlight the
role played by the inception or increase of tariffs for utilities, while Standing and
Vaughan-Whitehead (1995) point to the weakening of the minimum wage as key
factors behind the increase in inequality. After the initial surge in the early 1990s, inequality continued growing in the region in
the second half of the 1990s although at slower rates. The patterns were more
heterogeneous in the 2000s; inequality increased in some economies, but went down
in most countries, especially those in the FSU (World Bank, 2005). The mean Gini for
Eastern Europe and Central Asia in the late 2000s was lower than in the late 1990s but
still significantly higher (around 7 Gini points) than before the transition.42 40 See Fleisher, Sabirianova and Wang (2005) for a study of 10 transition economies and Gorodnichenko
and Sabirianova (2005) for the cases of Russia and Ukraine.
41 In Hungary, for instance, the income share accrued by the top 1% almost doubled between 1992 and
2009, from 6-7% to 12%. Half of the increase was due to capital income, while the other half to
increased earnings (Mavridis and Mosberger, forthcoming).
42 The reader is referred to chapter 20 for a survey of cross-country studies on the multiple causes of
inequality in OECD, including many countries in Eastern Europe. 40 See Fleisher, Sabirianova and Wang (2005) for a study of 10 transition economies and Gorodnichenko
and Sabirianova (2005) for the cases of Russia and Ukraine. 41 In Hungary, for instance, the income share accrued by the top 1% almost doubled between 1992 an
2009, from 6-7% to 12%. Half of the increase was due to capital income, while the other half
increased earnings (Mavridis and Mosberger, forthcoming). 42 The reader is referred to chapter 20 for a survey of cross-country studies on the multiple causes
inequality in OECD, including many countries in Eastern Europe. and Sabirianova (2005) for the cases of Russia and Ukraine.
41 In Hungary, for instance, the income share accrued by the top 1% almost doubled between 1992 and
2009, from 6-7% to 12%. Half of the increase was due to capital income, while the other half to
increased earnings (Mavridis and Mosberger, forthcoming).
42 The reader is referred to chapter 20 for a survey of cross-country studies on the multiple causes of
inequality in OECD including many countries in Eastern Europe Latin America and the Caribbean All Latin American countries carry out household surveys that provide information to
obtain estimates of poverty and inequality. All regular surveys in the region include
income questions; still in a few of them consumption expenditures are reported, as
well. The increasing availability of surveys in Latin America allowed the creation of
databases that make efforts to standardize the generation of poverty and inequality
statistics, favoring a close monitoring of the social and labor situation in the region
(SEDLAC by CEDLAS and the World Bank, and BADEINSO by UN´s ECLAC). In contrast,
the situation in the Caribbean is much less favorable, as surveys are isolated and
information is not easy to access. In fact, trends shown in the literature and in this
section are restricted to Latin America, which represents 94% of total LAC population. Latin America experienced two distinct distributive patterns in the last three decades
(IDB, 1999; de Ferranti et al., 2004; Lopez Calva and Lustig, 2010; Gasparini et al.,
2011). During the 1980s, 1990s, and the crises at the turn of the century, income
inequality soared in most countries for which comparable data are available. The mean
Gini for the distribution of household per capita income crawled from 50.1 in 1980 to 34 34 Alvaredo-Gasparini 51.5 in 1986, 51.9 in 1992, 53.0 in 1998 and 53.4 in 2002 (Gasparini et al., 2013). The
frequent macroeconomic crises that hit the region in that period were unequalizing
because the poor were less able to protect themselves from high and runaway
inflation, and adjustments programs frequently hurt the poor and the middle-ranges
disproportionately (Lustig, 1995). The market-oriented reforms that started in Chile in
the 1970s and became widespread in the region in the 1990s were associated with
rising inequality, although this pattern had a notable exception in the case of Brazil
(López Calva and Lustig, 2010). In most countries employment reallocations brought
about by trade liberalization and the skilled-biased technical change associated to the
modernization of the economy implied a sizeable reduction in the demand for
unskilled labor, which led to higher inequality. In some countries adjustments that led
to a contraction in the demand for labor affected unskilled workers disproportionately. All these changes took place in a framework of weak labor institutions and safety nets,
and hence their consequences made a full impact on the social situation (Gasparini and
Lustig, 2011). 43 See Barros et al. (2010), Ferreira et al. (2007), Foguel and Azevedo (2007), Hoffmann (2006) and
Langoni (2005). Latin America and the Caribbean Starting in the late 1990s in a few countries and in the early 2000s for the rest,
inequality began to decline. The mean Gini for the distribution of household per capita
income dropped from 53.4 in 2002 to 50.9 in 2008 (Gasparini et al., 2013). Updated
SEDLAC and BADEINSO statistics suggest that the downward trend continued, and that
at the time of writing (2013) the mean income Gini in the region would be again at
around the value of the early 1980s. The evidence, in fact, indicates that between 2002
and 2012, income inequality went down in all Latin American economies. This
remarkable decline appears to be driven by a large set of factors, including the
improved macroeconomic conditions that fostered employment, the petering out of
the unequalizing effects of the reforms in the 1990s, the expansion of coverage in
basic education, stronger labor institutions, the recovery of some countries from
severe unequalizing crises and a more progressive allocation of government spending,
in particular monetary transfers. The empirical evidence on the driving factors of the
recent fall in inequality is, however, still scarce and fragmentary (López Calva and
Lustig, 2010; Gasparini and Lustig, 2011; Cornia, 2011). Brazil
For decades Brazil was singled out as the most unequal economy in Latin America, and
in some rankings even the most unequal in the world. In the late 1980s the Gini
coefficient for the distribution of household per capita income reached values higher
than 60. But from that point on inequality started to decrease, first slowly in the 1990s,
and then more dramatically in the 2000s.43 By 2011 the Gini reached an
unprecedented low value of 52.7, several points below the level of some other Latin 44 These values are taken from SEDLAC (2013). All sources confirm the strong decreasing pattern in
inequality in Brazil.
45 For evidence and discussion on inequality in the 1990s see Ferreira and Paes de Barros (1999), and
Ferreira et al. (2006). Brazil For decades Brazil was singled out as the most unequal economy in Latin America, and
in some rankings even the most unequal in the world. In the late 1980s the Gini
coefficient for the distribution of household per capita income reached values higher
than 60. But from that point on inequality started to decrease, first slowly in the 1990s,
and then more dramatically in the 2000s.43 By 2011 the Gini reached an
unprecedented low value of 52.7, several points below the level of some other Latin 35 Alvaredo-Gasparini American economies (e.g. Honduras, Colombia, Bolivia).44 Brazil - the fifth most
populous nation in the world - is still a high-inequality country, but it stands out as a
successful case of consistent reduction of income disparities. American economies (e.g. Honduras, Colombia, Bolivia).44 Brazil - the fifth most
populous nation in the world - is still a high-inequality country, but it stands out as a
successful case of consistent reduction of income disparities. Data from the Brazil´s national household survey (PNAD) reveals a drop in the Gini of 2
percentage points in the late 1970s, and no systematic changes for most of the 1980s,
until a deep macroeconomic crisis hit the country, pulling inequality to unprecedented
levels. The Gini went up from 59.2 in 1986 to 62.8 in 1989 and returned to 59.9 in
1993. During the 1990s the Gini moved down very slowly, decreasing by just 1
percentage point between 1993 and 2001. That pace drastically increased in the
2000s: the Gini went down from 58.8 in 2001 to 52.7 in 2011, averaging a fall of 0.6
percentage points a year. During ten years per capita income of the poorest 10% of the
Brazilian population grew at an average annual rate of 7%, almost three times the
national average. In an in-depth study of the determinants of inequality changes in Brazil, Barros et al. (2010) highlight the role played by the sharp fall in earnings inequality and the
substantial increase in public transfers as the two main direct determinants of the
decline in income disparities since the early 2000s.45 They find that half of the
reduction of inequality in labor incomes was associated to the educational progress
that took place over the previous decade, that significantly increased the ratio
between skilled and unskilled workers. Brazil The average years of education for the adult
population grew 22% in the 2000s and the Gini coefficient computed over the
distribution of that variable fell 23%, values well above the mean for Latin America
(Cruces et al., 2013). Using different decomposition techniques, Barros et al. (2010)
and Azevedo et al. (2011) find a sizeable impact of the fall in the returns to education
on earnings inequality. Several authors have also found a reduction in spatial and
sectorial labor market segmentation. Although it is not yet clear which factors are
behind these trends, the evidence seem to point to a relatively faster expansion of
some productive sectors in the Brazilian agricultural ‘hinterland’ as opposed to the
metropolitan/industrial areas (Lustig et al., 2012). The substantial increase in the
minimum wage -68% in real terms between 2002 and 2010 (CEPAL, 2011)- is also
underlined as one important force behind the fall in household income inequality,
given that the minimum wage sets the floor for both unskilled workers earnings and
for social security benefits. The strong expansion of public transfers accounts to a large share of the fall in income
inequality in Brazil (Azevedo et al., 2011; Barros et al., 2010; Bérgolo et al., 2011; Lustig
et al., 2012). The main force was the rapid expansion in the coverage of government
cash transfers targeted to the poor, mainly a transfer to the elderly and disabled 36 36 Alvaredo-Gasparini (Benefício de Prestação Continuada) and Brazil’s signature conditional cash transfer
program Bolsa Família.46 y (
)
(
)
47 See Acar and Dogruel (2012), Adams and Page (2003), Bibi and Nabli (2010), Iqbal (2006), Page (2007),
and Salehi-Isfahani (2010).
48 According to Iqbal (2006) reliable pre-1985 household surveys are only available for Tunisia and Egypt. 47 See Acar and Dogruel (2012), Adams and Page (2003), Bibi and Nabli (2010), Iqbal (2006), Page (2007
and Salehi-Isfahani (2010). 46 See Fiszbein and Schady (2009) and Veras Soares (2007).
47 46 See Fiszbein and Schady (2009) and Veras Soares (2007).
47 (
)
48 According to Iqbal (2006) reliable pre-1985 household surveys are only available for Tunisia and Egyp 49 “In many MENA countries, from the Maghreb to the Arabian peninsula, power is wielded by rather
narrow groups… Seen from this perspective, the most puzzling thing about inequality in the Middle East
is how low it is” (Robinson, 2009). Middle East and North Africa These values place MENA as a region of moderate inequality within the
developing world, a fact that have puzzled some authors, that would predict higher 37 Alvaredo-Gasparini Alvaredo-Gasparini Alvaredo-Gasparini income disparities given the political process and the balance of political power in
those societies.49 income disparities given the political process and the balance of political power in
those societies.49 Middle East and North Africa Data constraints are particularly limiting when analyzing distributive issues in Middle
East and North Africa. The lack of accessible and comparable household surveys makes
it difficult even to identify the extent of poverty and inequality in most MENA
countries. The oil-rich economies (Bahrain, Kuwait, Oman, Qatar, United Arab
Emirates, and Saudi Arabia) enjoy high levels of per capita income, and are usually not
included into the analysis of the developing world. In any case, distributive data is
rarely available for these economies. A second group, by far the largest in terms of
population, consists of middle income countries. Within this group there are no public
accessible information for Lebanon and Libya, just one data point for Djibouti, Iraq,
Syrian Arab Republic, West Bank and Gaza, and only a few for Algeria and Yemen. In
sum, the only MENA countries for which it is possible to track changes in poverty and
inequality over time are Egypt, Iran, Jordan, Morocco and Tunisia, but even in these
cases data is scattered and often of low-quality. MENA has a long way to go to build a
reliable, comparable and sustainable system of household surveys and distributive
statistics. Despite this constraint, several studies shed some light about the trends in inequality
in this region.47 Authors coincide in dividing the last four decades into three periods. The first one, spanning until 1985, was characterized by rapid economic growth. Page
(2007) reports a substantial reduction in income inequality between the mid-1970s
and the early 1990s.48 Data from PovcalNet confirms that fall, although the magnitude
is more modest. “Middle Eastern economies entered their rapid growth period with
income distributions that were becoming more egalitarian, reflecting the political
ideology and policies of post-colonial governments” (Page, 2007). The second period
covers the late 1980s and most of the 1990s, and is characterized by low economic
growth, and meager or no social gains: real per capita incomes increased by less than
1.5% per year, while income distributions were rather stable. The downward pattern in
inequality appeared to have resumed in the 2000s, although at a slow pace. According
to our estimates based on PovcalNet the mean Gini fell from 38.7 in 2002 to 36.8 in
2010. South Asia South Asia has been a region of low inequality for developing world standards, though
rising since the early 1990s. In India, further discussed in a separate box, the
consumption Gini moved from 30.8 in 1993 to 33.9 in 2010. Bangladesh displayed
relatively low inequality throughout the 1980s (Gini equal to 26.1 in 1984), but the
situation worsened since the beginning of the 1990s: the Gini climbed to 32.1 by 2010. Khan (2008) argues that incomes from non-farm sources and the high concentration of
land tenure have all been disequalizing forces, while the positive effects of the more-
evenly distributed farm income were offset partly by its declining share in total
income. Scholars do not always agree about the distributive changes in Pakistan; PovcalNet
helps defining the picture by providing consumption Ginis of 33.2 for 1990, 28.7 for
1996 and 30.0 for 2008. The high economic growth during the 1980s contributed to a
sharp decline in poverty, but accompanied by a mild increase in inequality. The fall in
economic growth during the 1990s resulted in a rise in poverty while inequality
decreased modestly. According with Akmal (2008) in Pakistan there is an institutional
structure that excludes a large proportion of the population from the process of
economic growth as well as governance. Sri Lanka experienced rising inequalities between 1985 (Gini of 32.5) and 2007 (Gini
40.3) –among the highest increase in the region during the period of free market
reforms, integration to the world markets and high growth–, with a reversal of the
trend towards 2010 (Gini of 38.3) and persistent regional disparities due to conflict. Nepal presents similar dynamics. Gosh (2012) notes that rising inequality reflects two
components: first, growing vertical inequality within the modern industrial sector
driven by the returns to skill; and second, increasing disparities between the industrial
fast-growing sector and the traditional agricultural activities. India Chakravarty (1987) argues that even if policymakers in India adopted a development
strategy based on central planning over the forty years following independence, “there
was a tolerance towards income inequality, provided it was not excessive and could be
seen to result in a higher rate of growth than would be possible otherwise.” One of the
explicit goals of the socialist program was to limit the economic power of the elite in
the context of a mixed economy. From the mid-1980s, however, India gradually Chakravarty (1987) argues that even if policymakers in India adopted a development
strategy based on central planning over the forty years following independence, “there
was a tolerance towards income inequality, provided it was not excessive and could be
seen to result in a higher rate of growth than would be possible otherwise.” One of the
explicit goals of the socialist program was to limit the economic power of the elite in
the context of a mixed economy. From the mid-1980s, however, India gradually 38 Alvaredo-Gasparini adopted market-oriented economic reforms. Initially, these were accompanied by an
expansionist fiscal policy involving allocations to rural areas, in order to
counterbalance the negative redistributive effects of the liberalization. The speed of
reforms accelerated during the early 1990s; as in many developing countries
influenced by the Washington consensus, the focus shifted away from state
intervention towards liberalization, privatization and globalization. Most analysis on inequality in India over the last three decades are based on the
observations from the expenditure surveys conducted in 1983, 1987/8, 1993/94,
2004/05 and 2009/10 for urban and rural areas, which have allowed for an analysis pre
and post reforms (see Figure 4.10 below). Inequality has increased significantly in the
post-liberalization years, and this was much more pronounced in the urban areas; on
the contrary, estimates of absolute poverty measures have systematically fallen since
1983. 50 According to Mazundar (2012) the reasons for this pattern of growth are to be found not in the
tertiary sector but in the Indian manufacturing sector, which has been characterized by a dualistic size
distribution where middle-size firms are missing. Sub-Saharan Africa Although recently there have been many improvements, the lack of a consistent body
of household surveys undermine the assessment of income inequality in Sub-Saharan
Africa. Time series data on inequality is severely lacking in most SSA countries,
hindering the inferences about trends in the region. For example, in PovcalNet some
SSA countries are missing (Equatorial Guinea, Eritrea, Mauritius, Somalia, Zimbabwe),
while for thirteen of them there is only one observation in the database for the whole
period 1981-2010. In fact, very few countries have reliable surveys in the 1980s, and it
is not until the mid-1990s when inequality could be really traced with some confidence
in the region. Regional studies typically report a mixed picture, with both increases and decreases in
inequality (e.g. ECA, 2004), a fact that could reflect the heterogeneity in the region, but
also could be caused by noise in the country estimations (Christiansen et al., 2002;
Okojie and Shimeles, 2006). Bigsten and Shimeles (2003) for instance report that for 17
African countries the trend in inequality shows significant variations over short
periods, causing concern about measurement problems. The available evidence seems to support some few broad facts about consumption
inequality in the Sub-Saharan African countries. First, inequality is very high on
average, possibly the highest in the world. This result is in stark contrast with the
presumption of low inequality in SSA, held for a long time based on the predictions of
Kuznets-like models and the absence of reliable data.51 Second, on average inequality
does not seem to have changed much in the 1990s and 2000s. Data from PovcalNet
and other sources suggest a slow downward pattern; but in any case the evidence is
mixed and weak. Third, the heterogeneity among countries in terms of inequality
levels and patterns is large, partly possibly due to various measurement errors. It is
hard to identify a prototype of an inequality pattern in SSA, as in other regions such as
LAC or ECA. The scarce literature on inequality in SSA is consistent with these
observations. Go, Nikitin, Wang and Zou (2007) report that high income inequality
levels in SSA have remained more or less constant over the last four decades. Okojie
and Shimeles (2006) underline the fact that SSA is one of the most unequal regions in
the world, and that disparities have remained persistent over time. India Mazundar (2012) summarizes the main drivers of these changes: (i) the lead in
employment and output growth has been taken not by manufacturing but by the
tertiary sector, which displays higher inequality in pay;50 (ii) much of the labor
reallocated from agriculture is absorbed in the informal sector, where earnings are
only slightly higher than the poverty line; (iii) although numerous social insurance
schemes have been established, their actual impact has been limited and regressive as
they have disproportionately benefited workers in the small formal sector; (iv) the
modest and selective increase in social sector spending is constantly threatened by the
budget deficit; (v) the education polices implemented over the years have been biased
towards the promotion of tertiary education and have neglected basic primary and
lower secondary education. From a different perspective, Banerjee and Piketty (2010) looked at the tax-based
shares of top incomes. Their results suggests that the gradual liberalization of the
Indian economy made it possible for the top 1% to substantially increase their share of
total income, from 4.7% in 1980 to 8.9% in 1999. While in the 1980s the gains were
shared by everyone in the top percentile, in the 1990s it was only those in the top 0.1%
who benefited the most. The authors stress that these results could be linked to the
debate around the Indian growth paradox of the 1990s. According to the household
expenditure survey conducted by the National Sample Survey (NSS), real per capital
growth during the 1990s was fairly limited, in sharp contrast with the fast growth
measured by national accounts. It was suggested that much of such growth could have
gone to the rich, absent from surveys. Banerjee and Piketty (2010) conclude that top
incomes could explain between 20 and 40% of the puzzle, which still leaves the bulk of
the difference unaccounted for. 39 Alvaredo-Gasparini Alvaredo-Gasparini 51 Several studies have sought to explain the unexpected result of high inequality in Sub-Saharan Africa
(Milanovic, 2003; Okojie and Shimeles, 2006; Moradi and Baten, 2005; and van de Walle, 2008). Sub-Saharan Africa In contrast, Sala-i-
Martin and Pinkovskiy (2010) picture a more optimistic scenario, reporting a significant
downward pattern for inequality during the period of growth (1995 to 2006). 52 See, for example, McGrath (1983); McGrath and Whiteford (1994); Klasen (1997 and 2005); Nattrass
and Seekings (1997); Terreblanche (2002); Dollery (2003); van der Berg and Louw (2004); Leibbrandt et
al., (2009); Leibbrandt et al., (2010); and Aron et al., (2009). South Africa Over the last thirty years there have been important studies of overall inequality and
poverty in South Africa, and a heated debate about trends in post-apartheid 40 Alvaredo-Gasparini transition.52 South Africa has long been regarded as having one of the most unequal
societies in the world. Consistent with this view, the country has the highest Gini
coefficient of household consumption per capita (63.1 in 2010). During the early
1970s, the previously constant racial shares of income started to change in favour of
the blacks, at the expense of the whites, in a context of declining per capita incomes
(McGrath, 1983; McGrath and Whiteford, 1994). But while inter-racial inequality fell
throughout the eighties and nineties, inequality within race groups increased (Simkins
1991; Whiteford and Van Seventer 2000). Leibbrandt et al. (2010) provide evidence
from comparable households’ surveys conducted in 1993, 2000 and 2008. These
authors find that since the fall of apartheid inequality continued to increase steadily,
both for the whole population and within each racial group. The high level of overall
income inequality accentuated between 1993 and 2008, incomes becoming
increasingly concentrated in the top decile. The overall Gini coefficient increased by
four percentage points, from 66 to 70. The Gini coefficient for the African and
Asian/Indian population rose more sharply. Van der Berg and Louw (2004) also
conclude that rising black per capita incomes over the past three decades have
narrowed the interracial income gap, although increasing inequality within the black
and Asian/Indian population seems to have prevented any decline in aggregate
inequality. In explaining these changes, scholars agree in that the labor market played a dominant
role, where a rise in the number of blacks employed in skilled jobs (including civil
service and other high-pay government positions) coupled with increasing mean wages
for this group of workers. Leibbrandt et al. (2010) indicate that in the initial post-
apartheid period participation rates increased faster than absorption rates with a
consequent increase in unemployment across all deciles. Since 2000 the aggregate
unemployment rate declined marginally driven by increased absorption of those
individuals in the top deciles. In the lower deciles the early post-apartheid trend
continued to 2008. Indeed, this lack of successful integration into the labor market is
the reason that many of these households find themselves at the bottom of the
income distribution. South Africa State transfers have increased their importance as an income
source but not in a way that has narrowed the distribution of per capita income. They
have, however compensated for the decreasing share of remittance income. Since 1993 poverty has remained virtually constant, or fallen slightly. Leibbrandt et al. (2010) argue that while their impact on poverty incidence remains negligible overall,
government social assistance grants (mainly the child support grant, the disability
grant and the old-age pension) have been crucial in reducing poverty among the
poorest households and the elderly. Two-thirds of income to the bottom quintile now
comes from social assistance, even when many eligible individuals do not receive any 41 41 Alvaredo-Gasparini grant for instrumental reasons. Despite the increase in educational attainment,
younger age cohorts have the highest incidence and shares of poverty and this has not
improved notably over time, suggesting that the labor market has not been playing a
strongly virtuous role as a driver of poverty alleviation over the post-apartheid period. Increasing inequality and stable poverty are consistent with the rising trend in top
income shares recorded between 2002 and 2010 by Alvaredo and Atkinson (2010),
which could be associated to the favorable conditions in the world market for
agricultural commodities, the increase in the value of minerals other than gold, and
the developments in the financial sector. 4.3. Inequality convergence As suggested above, there are signs of inequality convergence among countries in the
developing world. As an example, the mean Gini coefficient for the 20 most unequal
countries in our PovcalNet sample in 1981 fell 11% in the following three decades,
while it increased 58% for the 20 most egalitarian economies. Bénabou (1996) was the first to present empirical evidence for cross-country
convergence in income inequality with data from 1970 to 1990 drawn from the
Deininger and Squire dataset. He found evidence consistent with the predictions of a
neoclassical growth model that yields convergence of the entire income distribution
and not just the first moment. Evidence on inequality convergence was also found in
studies that used improved data: Ravallion (2003) based on PovcalNet, Bleaney and
Nishiyama (2003) based on WIID, and Dhongde and Miao (2013) using both datasets. With variations, a typical inequality convergence study estimates α
β
it
i
i
it
G
G
G
t
e
1
1
-
= (
+
)( -1)+
for t = 2, …, T ; i = 1, …, N, α
β
it
i
i
it
G
G
G
t
e
1
1
-
= (
+
)( -1)+ for t = 2, …, T ; i = 1, …, N, where Git is the Gini coefficient for country i in year t and eit is an heteroscedastic error
term. The parameter β measures the link between the change and the initial Gini, and
therefore β<0 indicates inequality convergence. The intercept adds a notion of the
time required to attain convergence: a higher value for α is associated to a slower
convergence process. Models could be estimated with the Gini coefficient in levels or
logs. In his early study Bénabou (1996) found a β coefficient of -0.039 for a small
sample of around 30 countries. Naturally, estimates of β vary according to the data
used, the period covered, the time horizons considered, and the regression model
applied. Ravallion (2003) estimated a value of -0.028 in the 1990s, Bleany and
Nishiyama (2003) a value of -0.0125 between 1965 and 1990, and Dhongde and Miao
(2013) a value of -0.022 from 1980 to 2005. This literature has also found that the
impact of the initial Gini coefficient on the inequality change diminishes over longer
time horizons, and that the speed of inequality convergence is higher than the speed
of convergence in per capita income. 53 Caselli et al. (1996) and Dhongde and Miao (2013) discuss biases that may arise in an OLS model.
54 See, as an example, the analysis of Atkinson and Lugo (2010) for Tanzania. 4.3. Inequality convergence 42 42 Alvaredo-Gasparini We add to this literature our own estimates, taking advantage of the PovcalNet panel
of 76 countries from 1981 to 2010 used in this section. Table 4.2 shows the OLS and
IVE estimates of α and β for different initial years.53 The parameter β is negative and
significant in all the specifications, suggesting evidence for inequality convergence. The
estimated coefficients are in the range of those estimated in the literature. Table 4.2
Inequality convergence
Models of the change in the Gini coefficient
Note: robust t statistics in parentheses; *significant at 5%; **significant at 1%; the heteroskedasticity-
consistent covariance matrix estimator is used (HC1). IVE estimates use the initial value as the
instrument for the inequality measure in the second survey. The number of observations is 456 in the
first panel, and 281 in the second. Gini Index
OLS
1.098
-0.026
0.49
0.908
-0.023
0.35
(18.90)**
(20.38)**
(11.20)**
(10.97)**
IVE
1.271
-0.031
0.47
0.855
-0.021
0.35
(17.91)**
(17.61)**
(9.83)**
(9.90)**
Difference
0.173
-0.005
-0.053
0.002
Hausman Test
(4.26)**
(4.03)**
(1.69)*
(2.61)**
Log Gini Index
OLS
0.118
-0.032
0.65
0.105
-0.029
0.27
(28.41)**
(28.11)**
(15.53)**
(15.14)**
IVE
0.135
-0.037
0.63
0.104
-0.028
0.27
(25.79)**
(24.75)**
(14.07)**
(13.66)**
Difference
0.017
-0.005
-0.001
0.001
Hausman Test
(5.30)**
(5.01)**
(0.49)
(0.48)
R 2
Initial year 1981
Initial year 1990
R 2
Intercept (α)
Slope (β)
Intercept (α)
Slope (β) Inequality convergence
Models of the change in the Gini coefficient Note: robust t statistics in parentheses; significant at 5%;
significant at 1%; the heteroskedasticity
consistent covariance matrix estimator is used (HC1). IVE estimates use the initial value as the
instrument for the inequality measure in the second survey. The number of observations is 456 in the
first panel, and 281 in the second. While the evidence for inequality convergence in the last decades seems well-
established, the reasons driven that pattern are not clear. As mentioned before,
Bénabou (1996) finds the evidence on convergence consistent within the framework of
a neoclassical growth model. In contrast, the evidence for unconditional inequality
convergence is interpreted by Ravallion (2003) as the result of policy and institutional
convergence since around 1990, when socialist planned economies became more
market-oriented, and non-socialist economies adopted market reforms. 54 See, as an example, the analysis of Atkinson and Lugo (2010) for Tanzania. 4.4. Absolute inequality While relative inequality has been the preferred concept in empirical work in
development economics, absolute views of inequality certainly have some intuitive
appeal (Amiel and Cowell, 1999; Atkinson and Brandolini, 2004). Interestingly, the
trends in the two concepts over the last decades have been different in the developing
world.54 The fact that most countries experienced economic growth, while at the same
time relative inequality did not fall, implied widening absolute income differences. On 43 Alvaredo-Gasparini average, the absolute difference in monthly consumption per capita between the top
and bottom 10% of each country increased over the two decades from US$415 (PPP
adjusted) in 1990, to US$497 in 2002, and US$646 in 2010. In more than 90% of the
countries in the sample that absolute difference was higher in 2010 than in 1990. The contrast between the recent trends in absolute and relative inequality in the
developing countries is illustrated in Figure 4.7. While relative inequality rose in the
late 1980s and early 1990s, absolute inequality declined, driven by a reduction in mean
income. The strong growth in the developing world since mid-1990s is reflected in the
substantial hike in the degree of absolute inequality. Although some equalizing forces
operated in the 2000s that reduced the relative gaps, they were not enough to narrow
the absolute gaps in a context of economic growth. Based on these facts, Ravallion
(2004) argues that the disagreements over whether inequality in the world has gone
up or down may partly be due to differing views about the importance of absolute
versus relative conceptions of inequality. Figure 4.7
Absolute and relative Gini coefficients
Unweighted means, developing countries, 1981-2010
Source: own estimates based on PovcalNet (2013). Note: normalized to 100=mean over the period 1981-2010.The Gini coefficients are computed over the
distribution of household consumption per capita. 80
90
100
110
120
130
140
1981
1984
1987
1990
1993
1996
1999
2002
2005
2008
2010
Relative
Absolute g
Absolute and relative Gini coefficients
Unweighted means, developing countries, 1981-2010 Note: normalized to 100=mean over the period 1981-2010.The Gini coefficients are computed over the
distribution of household consumption per capita. 4.5. Aggregate welfare The typical way of assessing the economic performance of a country is by means of its
per capita income or output. However, this practice is valid only when the evaluator’s
welfare function is utilitarian. Except in this extreme case, measuring aggregate
welfare involves not only knowing the mean but also other elements of the income
distribution, in particular the degree of inequality. Although social welfare functions
are naturally arbitrary, since they depend on the analyst’s value judgments, it is 44 44 Alvaredo-Gasparini common in the literature to work with anonymous, Paretian, symmetric and
quasiconcave functions. For simplicity, here we consider the abbreviated welfare
function proposed by Sen (1976), WS=µ.(1-G), where µ is the mean of the distribution
and G is the Gini coefficient. Figure 4.8 shows the unweighted mean of WS for the
developing countries in the period 1990-2010 computed from household survey data. In general, aggregate welfare has followed changes in per capita consumption. The fall
in mean consumption in the early 1990s (mostly due to the negative performance in
ECA) was reinforced by the increase in inequality, driving welfare down by around
15%. Between 1993 and 2002 mean consumption went up but the change was
counterbalanced by a similar increase in the Gini, keeping welfare roughly constant. The 2000s witnessed a robust increase in mean consumption, along with some fall in
inequality, implying a 40% increase in aggregate welfare between 2002 and 2010. According to these calculations the mean aggregate welfare in the developing
countries was 22% higher in 2010 than in 1990, implying an annual growth rate of
around 1%. Figure 4.8
Aggregate welfare
Sen welfare function, unweighted mean, developing countries, 1990-2010
Source: own estimates based on PovcalNet (2013). Note: normalized to 100=value in 1990. 60
70
80
90
100
110
120
130
140
1990
1993
1996
1999
2002
2005
2008
2010
Mean
Gini
Welfare Figure 4.8
Aggregate welfare
Sen welfare function, unweighted mean, developing countries, 1990-2010
Source: own estimates based on PovcalNet (2013). Note: normalized to 100=value in 1990. In order to calculate welfare it is necessary to have estimates of the mean income and
some inequality measure. Ideally, both parameters should be estimated from the same
source, typically a household survey, as we have done so far. Some authors have taken
a different approach, anchoring the mean to a variable from National Accounts, such
as per capita GDP or aggregate household consumption expenditures. 4.5. Aggregate welfare For several
reasons changes in mean income from household surveys tend to differ significantly
from changes in per capita GDP (Deaton, 2003, 2005; Anand and Segal, 2008). Some of
these differences are natural since per capita income and GDP are different concepts,
but some are rooted in measurement errors both in household surveys and in National
60
70
80
90
100
110
120
130
140
1990
1993
1996
1999
2002
2005
2008
2010
Mean
Gini
Welfare gg
g
Sen welfare function, unweighted mean, developing countries, 1990-2010 In order to calculate welfare it is necessary to have estimates of the mean income and
some inequality measure. Ideally, both parameters should be estimated from the same
source, typically a household survey, as we have done so far. Some authors have taken
a different approach, anchoring the mean to a variable from National Accounts, such
as per capita GDP or aggregate household consumption expenditures. For several
reasons changes in mean income from household surveys tend to differ significantly
from changes in per capita GDP (Deaton, 2003, 2005; Anand and Segal, 2008). Some of
these differences are natural since per capita income and GDP are different concepts,
but some are rooted in measurement errors both in household surveys and in National
Accounts. Some authors pay the price of the potential inconsistency of using two 45 Alvaredo-Gasparini different data sources (i) in order to avoid departing from the typical growth and
development literature that is based on National Accounts data, (ii) as a way to
alleviate the underreporting issue in household surveys, and (iii) to avoid problems
related to the unavailability of surveys for many years in several countries (Ahluwalia
et al., 1979; Bourguignon and Morrison, 2002; Sala-i-Martin, 2006; Bhalla, 2002). In Figure 4.9 we report the results of computing the unweighted average of aggregate
welfare across developing countries using alternative mean income variables. According to these estimates mean welfare in the developing world grew at an annual
1% from 1990 to 2010 using mean consumption per capita from household surveys,
1.6% using per capita GNI from WDI, and 2.1% using per capita GDP from Penn World
Tables (PWT) (Heston et al., 2012).55 These discrepancies are worrying and call for
increasing efforts to understand and reduce the gaps among data sources. 55 In fact, this difference comes from the combination of higher growth recorded in the National
Accounts in the 1990s compared to household surveys, and the opposite result in the 2000s. For
instance, while per capita GDP slightly fell between 2008 and 2010, mean consumption in household
surveys increased at annual 2%. y
56 Some authors have computed global welfare, ignoring the division of the world in countries. The
evidence suggest an increase in aggregate welfare in the developing world in the last decades
(Pinkovskiy and Sala-i-Martin, 2009; Atkinson and Brandolini, 2010; Pinkovskiy, 2013). 55 In fact, this difference comes from the combination of higher growth recorded in the National
Accounts in the 1990s compared to household surveys, and the opposite result in the 2000s. For
instance, while per capita GDP slightly fell between 2008 and 2010, mean consumption in household
surveys increased at annual 2%.
56 Some authors have computed global welfare, ignoring the division of the world in countries. The
evidence suggest an increase in aggregate welfare in the developing world in the last decades
(Pinkovskiy and Sala-i-Martin, 2009; Atkinson and Brandolini, 2010; Pinkovskiy, 2013). 57 The results for China (1986-2003) and Indonesia (1982-2004) in the WTID are based on household
surveys and not on tax records. The fact that top income share estimates are lower than in the most
egalitarian developed countries shows that they are likely to be underestimated. In the case of China,
the rising trend is robust and can be taken as an indicator of the true dynamics of concentration at the
top. Piketty and Qian (2009) show that top income shares increased at a very high rate during the
period, which is consistent with the evidence discussed in section 4. The top decile share rose from
about 17 per cent in 1986 to almost 28 per cent in 2003—that is, by more than 60 per cent. The top 1
per cent income share more than doubled between 1986 and 2001, from slightly more than 2.6 per cent
in 1986 to 5.9 per cent in 2003.
58 4.5. Aggregate welfare Figure 4.9
Aggregate welfare for alternative mean variables
Sen welfare function, unweighted mean, developing countries, 1990-2010
Source: own estimates based on PovcalNet (2013), WDI and PWT. Note: mean anchored to per capita consumption (PovcalNet), GNI per capita (WDI) and GDP per capita
(PWT). Normalized to 100=value in 1990. 60
80
100
120
140
160
1990
1993
1996
1999
2002
2005
2008
2010
consumption (surveys)
GNI (WDI)
GDP (PWT) Aggregate welfare for alternative mean variables
Sen welfare function, unweighted mean, developing countries, 1990-2010 (
),
Note: mean anchored to per capita consumption (PovcalNet), GNI per capita (WDI) and GDP per capi
(PWT). Normalized to 100=value in 1990. Note: mean anchored to per capita consumption (PovcalNet), GNI per capita (WDI) and GDP per capita
(PWT). Normalized to 100=value in 1990. The population-weighted mean of the welfare measure grew at a much higher rate,
due to the positive performance of several large countries.56 The growth rate between
1990 and 2010 was 2.3% using household survey data, 3% anchoring mean income to
per capita GNI from WDI, and 3.3% when using the Penn World Tables. 46 46 Alvaredo-Gasparini Alvaredo-Gasparini 58 We do not review the results for former colonial territories being prepared as part of an on-going
project carried out by Atkinson (British colonies), and Alvaredo, Cogneau and Piketty (French colonies).
This project has assembled data for some forty colonies covering the periods before and after
independence. Unfortunately, the series stop before 1970 in most cases due to unavailability of recent
data. 4.6. Trends from tax records At the moment of writing (2013), the WTID offers estimates of the tax-based shares of
top incomes for a small number of developing countries: Argentina, Colombia, India,
Mauritius and South Africa.57 Malaysia is available from Atkinson (forthcoming), and
will be included in the WTID as soon as the proper documentation is finished. Preliminary results for Ecuador are available from Alvaredo and Cano (forthcoming).58
Ongoing research analyzes the cases of Brazil, Chile, Ecuador and Uruguay. Consequently, evidence in this respect is still fragmentary, not only because this
particular research program is rather recent in what concerns the developing world,
but also because of the unavailability of tax data. The results for the top 1% income share are presented in Figure 4.10, together with
survey-based Gini coefficients. Several elements are worth mentioning. First, both
sources are not directly comparable for the reasons discussed in section 3. The top
share estimates are in general before taxes, while survey Ginis are net of taxes; in
addition the units of analysis usually do not match. Second, there is substantial
heterogeneity in this group, both in levels and dynamics, compared to the evidence
discussed in chapter 16 for developed countries. It should be borne in mind that the
differences in the tax systems across countries imply different income concepts, so
that top share levels should be read with this caveat in mind. Third, Leigh (2007), who
analyzes 13 developed countries, and find that there is a strong and significant
relationship between top income shares and broader inequality measures, concludes
that “panel data on top income shares may be a useful substitute for other measures
of inequality over periods when alternative income distribution measures are of low
quality, or unavailable”. Figure 4.10 seems to agree with the results in Leigh (2007) in
some cases but not in others. For example, the evolutions of the Gini and the top 1%
share in Colombia are particularly diverging, while the dynamics in Mauritius is
remarkably similar. Top shares and broader, synthetic inequality measures can very well display different
trends. The problem arises when the top group plays a major role in the changes in
inequality and survey data fail to capture high incomes. 58 We do not review the results for former colonial territories being prepared as part of an on-going
project carried out by Atkinson (British colonies), and Alvaredo, Cogneau and Piketty (French colonies). 59 Bourguignon et al. (2008) and Ferreira (2010) are excellent references for methodological issues in
recent research on inequality determinants. 4.6. Trends from tax records Note: Gini coefficient for Malaysia 2004 identified as outlier. 4.6. Trends from tax records This project has assembled data for some forty colonies covering the periods before and after
independence. Unfortunately, the series stop before 1970 in most cases due to unavailability of recent
data. 47 47 Alvaredo-Gasparini Figure 4.10 Top 1% income share and Gini coefficients Top 1% income share and Gini coefficients
Argentina, Colombia, Malaysia, Mauritius, India and South Africa
Source: own estimates based on WTID. Note: Gini coefficient for Malaysia 2004 identified as outlier. 4.6. Trends from tax records 25
35
45
55
65
75
0
5
10
15
20
25
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
Gini coefficient
Top 1% income share (%)
Argentina
top 1% income share
Gini-Urban HH per capita income (SEDLAC)
25
35
45
55
65
75
0
5
10
15
20
25
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
Gini coefficient
Top 1% income share (%)
Colombia
top 1% income share
Gini-HH per capita income (Povcalnet)
25
35
45
55
65
75
0
5
10
15
20
25
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
Gini coefficient
Top 1% income share (%)
Malaysia
top 1% income share
Gini-HH per capita income (Povcalnet)
25
35
45
55
65
75
0
5
10
15
20
25
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
Gini coefficient
Top 1% income share (%)
Mauritius
top 1% income share
Gini-HH disposable income (Atkinson and Morelli, 2012)
25
35
45
55
65
75
0
5
10
15
20
25
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
Gini coefficient
Top 1% income share (%)
India
top 1% income share
Gini-Urban HH per capita consumption (Povcalnet)
Gini-Rural HH per capita consumption (Povcalnet)
25
35
45
55
65
75
0
5
10
15
20
25
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
Gini coefficient
Top 1% income share (%)
South Africa
top 1% income share
Gini-HH per capita income (Leibbrandt et al., 2010) Argentina, Colombia, Malaysia, Mauritius, India and South Africa Argentina, Colombia, Malaysia, Mauritius, India and South Africa 35
45
55
65
75
5
10
15
20
25
Gini coefficient
Top 1% income share (%)
Argentina
top 1% income share
Gini-Urban HH per capita income (SEDLAC) 25
35
45
55
65
75
0
5
10
15
20
25
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
Gini coefficient
Top 1% income share (%)
Colombia
top 1% income share
Gini-HH per capita income (Povcalnet) Top 1% income share (%) 25
0
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012 25
0
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012 25
35
45
55
65
75
0
5
10
15
20
25
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
Gini coefficient
Top 1% income share (%)
Mauritius
top 1% income share
Gini-HH disposable income (Atkinson and Morelli, 2012) 25
35
45
55
65
75
0
5
10
15
20
25
Gini coefficient
Top 1% income share (%)
Malaysia
top 1% income share
Gini-HH per capita income (Povcalnet) 25
35
45
55
65
0
5
10
15
20
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
Gini coefficient
Top 1% income share (%)
Gini-HH per capita income (Povcalnet)
25
35
45
55
65
75
0
5
10
15
20
25
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
Gini coefficient
Top 1% income share (%)
India
top 1% income share
Gini-Urban HH per capita consumption (Povcalnet)
Gini-Rural HH per capita consumption (Povcalnet) Top 1% income share (%) 25
0
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012 25
0
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012 South Africa 0
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012 25
0
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012 Source: own estimates based on WTID. 4.7. Exploring inequality changes Explaining changes in the income distribution is a very difficult and challenging task
that lies well beyond the objectives of this chapter. In this section we briefly review
some methodologies to study the determinants of the income distribution changes
and lay out some of the main results regarding developing countries.59 Certainly, there
has been sustained progress in our understanding of the factors that shape the income
distributions, but yet the image that emerges from reviewing the literature is still that
of a patchwork of numerous hypotheses without conclusive empirical support. 48 48 Alvaredo-Gasparini Decompositions are one of the most widely used techniques to characterize income
distribution changes. Typically, an income model is estimated, and a counterfactual
distribution is simulated modifying some elements of the estimated income model
(e.g. parameters or the distribution of observable factors), while keeping the rest fixed. The difference between the actual and the simulated distribution captures the first-
round partial-equilibrium effect of the change under study.60 The method generates
entire counterfactual distributions, and hence can capture the heterogeneity of
impacts throughout the distribution. The decompositions have been typically used to
shed light on the impact of changes in the returns to education, in the demographic,
sectorial, occupational and educational composition of the population, and in labor
and social policies. The decompositions do not allow for the identification of causal
effects, and suffer from the usual problems of equilibrium-inconsistency and path
dependence. Nevertheless, these types of exercises are informative about the relative
strength of several direct determinants that may be driving the distributive changes,
and therefore could be useful in identifying areas in which to focus the research
efforts. Ideally, income distribution changes should be studied in a general equilibrium
framework, since they are the result of complex processes that involve all sorts of
effects and interactions throughout the economy. Computable general equilibrium
(CGE) models have been applied to study changes in the income distributions around
the developing world. These exercises, however, depend critically on parameters and
functions that are difficult to estimate, and rely on many simplifying assumptions. The
more recent macro-micro approach combines a CGE model (the macro component)
with a microsimulation (the micro component). CGE models provide a framework to
assess consistency of policy alternatives, but lack the necessary disaggregation for the
analysis of distributional issues, which is provided by the microsimulations. 60 See Bourguignon et al. (2005); Barros et al. (2006) and Bourguignon et al. (2008), for methodological
proposals; Bourguignon et al. (2005) for applications to Asia and Latin America, and Inchauste et al.
(2012) for a recent application to poverty reduction in Bangladesh, Peru and Thailand. Fortin et al.
(2011) and Essama Nssah (2012) are useful surveys of the economic literature on decompositions.
61 Ferreira et al. (2010) use this approach to estimate the effect of a trade liberalization episode on the
distributions of wages and household incomes in Brazil. 61 Ferreira et al. (2010) use this approach to estimate the effect of a trade liberalization episode on the
distributions of wages and household incomes in Brazil. 4.7. Exploring inequality changes The macro
and micro components of this methodology communicate through aggregate variables
such as employment levels and wage rates that are generated by the CGE model and
used as inputs in the microsimulations (Bourguignon et al., 2008; Bourguignon and
Bussolo, 2012). In a related approach, rather than building a full general equilibrium
model of the economy, researchers rely on a reduced-form relationship between a set
of observed exogenous variables (such as changes in tariff rates) and a set of sector-
level variables (such as industry-skill wage premia), that serve as inputs in the
microsimulations.61 A very different strand of the literature involves the estimation of cross-country
regressions, typically with panel data, where an aggregate measure of overall
inequality, such as the Gini coefficient, is linked to various potential causal factors (e.g. 49 49 Alvaredo-Gasparini Li et al., 1998; Anderson, 2005). Naturally, endogeneity problems are endemic to this
approach, which is useful to characterize the structure of correlations among variables,
but less successful in identifying causal links. Most of the literature takes a less ambitious but probably more productive road, and
focus on the partial-equilibrium impact of specific shocks and policy changes, using
different identification strategies depending on the characteristics of the shock/policy
and the data available. Examples of these methodologies include (i) a typical supply
and demand approach, where the impact of indicators of trade, technology or other
factors on the relative wage between skilled and unskilled workers is estimated,
controlling for relative supply; (ii) the cost function approach, where the impact of
several indicators on the share of skilled wages in total wage bill is estimated, using
flexible cost functions (usually a translog cost production function); and (iii) mandated
wage regressions.62 When experiments with random assignment are available, causal
links are more clearly identified. For instance, the conditional cash transfer program
Progresa in Mexico was initially implemented with a random assignment of treated
and control rural villages, which allowed a rigorous impact evaluation. (
)
63 See also Parker and Skoufias (2001) and Gertler (2004) for impact evaluations studies of the Mexican
conditional cash transfer program.
64 For instance, according to SEDLAC data, on average in Latin America in 2010 the share of labor income
in total household income was 82.3%, the share of transfers (including pensions) 13.9%, and the rest of
the sources just 3.8%.
65 62 See Anderson (2005).
63 See also Parker and Skoufias (2001) and Gertler (2004) for impact evaluations studies of the Mexican
conditional cash transfer program.
64 For instance, according to SEDLAC data, on average in Latin America in 2010 the share of labor income
in total household income was 82.3%, the share of transfers (including pensions) 13.9%, and the rest of
the sources just 3.8%.
65 See Caselli and Michaels (2013). 62 See Anderson (2005). (
)
63 See also Parker and Skoufias (2001) and Gertler (2004) for impact evaluations studies of the Mexica
conditional cash transfer program.
64 65 See Caselli and Michaels (2013). 64 For instance, according to SEDLAC data, on average in Latin America in 2010 the share of labor incom
in total household income was 82.3%, the share of transfers (including pensions) 13.9%, and the rest
the sources just 3.8%. 4.7. Exploring inequality changes Taking
advantage of that design, Todd and Wolpin (2006) estimate a full structural model of
behavior, including education, fertility and labor supply decisions, a model that can be
used to simulate the distributive impact of policies and shocks.63 The bulk of the distributive analysis in developing countries has focused mainly on the
labor market and on public and private transfers, while largely setting aside the role
played by other sources of income, such as capital, land rents and business profits. The
neglect of these other factors is essentially due to the fact that household surveys fail
to capture these income sources properly.64 This shortcoming has, for instance,
severely limited the study of the impact of the natural resources exploitation on
inequality, a relevant topic in several developing countries.65 Most studies narrow the
analysis to particular indicators of the labor market, such as the wage gap between
skilled and unskilled labor (the wage premium) or the returns to education. For
instance, Bourguignon et al. (2005) show that increases in returns to schooling were
large contributors to increasing inequality in East Asia and Latin America in the 1990s. In what follows we review some general debates on the determinants of recent
inequality changes. Growth and development. As shown in Section 3.5 there is a significant negative
relationship between inequality and measures of development, such as GNI per capita,
in a cross-section of countries. From this evidence Ferreira and Ravallion (2009) 50 50 Alvaredo-Gasparini conclude that “high inequality is a feature of underdevelopment”. However, the short
or medium run relationship between inequality and development has proved to be
elusive. There appears to be no evidence in the last decades of a significant correlation
between the growth rate of an economy and the change in the inequality level
(Ravallion and Chen, 1997; Ravallion, 2001; Dollar and Kraay, 2002; Ferreira and
Ravallion, 2009).66 Ravallion (2007), for example, analyzed 290 episodes in 80
countries in 1980-2000 and found a correlation coefficient non-significant at the 10%
level between the changes in the log of the Gini coefficient and changes in the log of
mean income in real terms between successive household surveys. The analysis of
more recent data from PovcalNet leads to the same conclusion. Using 473 spells in the
period 1981-2010 we find a non-significant coefficient of -0.0094. 68 Ravallion (2004) argues that, on average, growth is not associated with increases in relative inequality
but absolute inequality, and it is these higher absolute gaps between "rich" and "poor" that generate
the perception of an unequal growth processes. 66 A related literature finds no support for a Kuznets curve with longitudinal data (Bruno, 1998; Field
2002; Hellier and Lambrecht, 2012).
67 67 The relationship becomes negative, although just slightly significant, when using the change in log re
per capita GDP (Penn World Tables) or per capita GNI (WDI) as measures of growth.
68 pp
g
2002; Hellier and Lambrecht, 2012).
67 The relationship becomes negative, although just slightly significant, when using the change in log real
per capita GDP (Penn World Tables) or per capita GNI (WDI) as measures of growth.
68 Ravallion (2004) argues that, on average, growth is not associated with increases in relative inequality
but absolute inequality, and it is these higher absolute gaps between "rich" and "poor" that generate
the perception of an unequal growth processes. 67 The relationship becomes negative, although just slightly significant, when using the change in log real
per capita GDP (Penn World Tables) or per capita GNI (WDI) as measures of growth.
68 Ravallion (2004) argues that, on average, growth is not associated with increases in relative inequality
b t
b
l t
i
lit
d it i th
hi h
b
l t
b t
" i h"
d "
" th t
t 4.7. Exploring inequality changes A similar result
applies when restricting the sample to observations after 1990 or 2000, or when
considering longer spells.67 The data suggests that among both growing and
contracting economies, inequality increased about as often as it fell. In the last
decades economic growth has been distribution-neutral on average in the developing
countries.68 Globalization. Much of the recent public and academic debate on inequality changes
has been related to the rise in globalization. In the latest decades most developing
countries have experienced increasing openness to international trade, capital markets
flows and foreign direct investment. The theoretical channels linking these changes to
inequality are multiple and complex, which accounts for the lack of conclusive
empirical results (Wood, 1997; Rama, 2003; Winters et al., 2004; Anderson, 2005;
Goldberg and Pavcnik, 2007; Harrison et al., 2011). While studies in cross sections of
developing countries are inconclusive in relation to the impact of globalization upon
inequality, several longitudinal estimates concerning countries taken separately or in
small groups reveal a positive correlation between openness and the relative demand
for skilled labor (Anderson, 2005; Goldberg and Pavnick, 2007; Harrison et al., 2011;
Chusseau and Hellier, 2012). Trade openness may affect the income distribution
through various channels. The traditional Stolper-Samuelson effect predicts a
reduction in the skill premium in unskilled-labor-abundant developing countries, a
prediction that does not appear to be confirmed by the facts (Goldberg and Pavnick,
2007; Feenstra, 2008). While some of the research has pointed then to non-trade
factors - such as skill-biased technological change and labor institutions - to explain
rising wage gaps, in recent years new mechanisms have been explored through which
trade can increase income inequality. These mechanisms include heterogeneous firms 51 Alvaredo-Gasparini and bargaining, trade in tasks, labor frictions and incomplete contracts (Harrison et al.,
2011). In addition, competition among developing countries may increase inequality in
middle-income countries (e.g. Latin America) competing with low-income
economies.69 Also, the growing size of the developing world, as new countries enter
the world markets, may foster inequality by augmenting the world endowment of
unskilled labor. The literature finds that the mechanisms through which globalization
affects income distribution are country, time, and case specific, and therefore the
impacts of trade liberalization need to be examined in conjunction with other
concurrent policy reforms (Goldberg and Pavnick, 2007). 69 The increase in inequality in Latin America has also been explained arguing that it is a region relatively
abundant in natural resources, while in the onset of liberalization Asian countries were relatively
abundant in unskilled labor (Wood, 1997). 70 According to Tinbergen (1975) changes in earnings inequality are the outcome of a “race” between
technological progress raising the demand for skills, and the expansion of education raising the supply of
skills. 71 See Gasparini et al. (2011) for Latin America. 71 See Gasparini et al. (2011) for Latin America. 4.7. Exploring inequality changes In addition, and due to
various limitations, the literature is mostly focused on the static link between
globalization and income distribution that typically operates through changes in
relative prices and wages, rather than on the dynamic, more indirect link from trade to
growth, and then to poverty and inequality. Technology and education. Skill-biased technological change has been a popular
explanation for the rise in inequality in the developed countries. Changes in
technology, such as the use of computers, increase the relative demand for skilled
workers driving the skill premium up. This hypothesis is also plausible in the developing
world, where globalization increased the transfers of more skill-intensive technologies
from the North, and fostered imports of capital goods, typically complementary of
skilled labor. Several studies find that openness-driven technological transfers tend to
increase inequality in emerging countries (Conte and Vivarelli, 2007). The increase in
the wage skill premium may be temporary, as the introduction of new technologies
requires a transitional period during which skilled workers are employed to adapt the
firm to the new technology (Pissarides, 1997; Helpman and Trajtenberg, 1998). The
empirical applications usually show evidence on the short and medium-run effects of
the reforms, failing to capture the long-run impact. The generalized fall in inequality in
the 2000s in the developing world might be in part attributed to the petering out of
the unequalizing initial impact of the liberalizing reforms and technological shocks
experienced by many countries in the 1990s. The increase in education may counteract the effect of skill-biased technological
change in the Tinbergen´s race between education and technology.70 In fact, education
has expanded in the developing world at high rates during the last decades, mitigating
the impact of other factors that tend to increase the wage premium.71 However, the
link between education and income inequality may not be that straightforward. Given 52 52 Alvaredo-Gasparini the convexities in the returns to education, even an equalizing increase in schooling
may generate an unequalizing change in the distribution of earnings. Bourguignon,
Ferreira and Lustig (2005) have labeled this phenomenon “the paradox of progress”, a
situation where an educational expansion is associated to higher inequality.72 Market reforms. Several developing countries have implemented market-oriented
reforms in the last decades, reducing regulations and privatizing firms. 74 For instance, Lustig (2012) finds that in Latin America when indirect taxes are taken into account, the
net income of the poor and the near poor can be lower than it was before taxes and cash transfers.
75 ILO (2010) reports that in the early 2000s the share of the population above the legal retirement age
receiving a pension in developing economies was, on average, around 40 per cent, as compared to 90
per cent in European economies. 73 See Cornia (1996), Milanovic (1998), Ferreira (1999), Cornia and Reddy (2001), and Milanovic an
Ersado (2010).
74 p
p
75 ILO (2010) reports that in the early 2000s the share of the population above the legal retirement age
receiving a pension in developing economies was, on average, around 40 per cent, as compared to 90
per cent in European economies. 74 For instance, Lustig (2012) finds that in Latin America when indirect taxes are taken into account, th
net income of the poor and the near poor can be lower than it was before taxes and cash transfers. 72 Inequality may also increase after an education expansion, given that wage dispersion is larger
higher educational levels (Alejo, 2012).
73 Inequality may also increase after an education expansion, given that wage dispersion is larger at
higher educational levels (Alejo, 2012).
73 See Cornia (1996), Milanovic (1998), Ferreira (1999), Cornia and Reddy (2001), and Milanovic and
Ersado (2010).
74 4.7. Exploring inequality changes The
paradigmatic case includes the former socialist planned economies in ECA, but the
transition from centrally planned to market-oriented economies was also experienced
by several African and Asian countries, including China. The evidence suggests a
significant increase in inequality over the transition period. That surge has been linked
to the process of privatizations, that implied an increase in the earnings dispersion in
comparison to the more compressed wage structure of the state-own firms, and the
institutional and regulatory reforms that have increased competition in product and
factor markets and decreased the bargaining power of labor.73 Other non-socialist
economies also adopted market-friendly reforms; Ravallion (2003) argues that in some
cases (e.g. Brazil) pre-reform controls benefited the rich and kept inequality high, and
then reforms help lowering inequality, while in some others (e.g. India) the controls
(and the reforms) had the opposite effect. Fiscal and social policy. Developing countries are characterized by relatively low levels
of taxation, heavy reliance on regressive revenue instruments, and low coverage and
benefit levels of transfer programs (World Bank, 2006). This structure limits the
redistributive potential of fiscal policy and in some cases even exacerbates the market
income disparities.74 While average tax ratios for advanced economies exceed 30 per
cent of GDP, ratios in developing economies (excluding emerging Europe) generally fall
in the range of 15–20 per cent of GDP (Baltagi et al., 2012). Tax collection is not only
lower but also more regressive than in developed countries. The difficulties in
collecting more progressive taxes are related to the high levels of self-employment and
sizeable informal sectors, which limit the capacity of the tax authorities to verified
taxpayers´ income and assets. On the spending side, in most developing economies
social spending is relatively low, and participation in social insurance schemes is
restricted to high-income workers in the formal sector and to public sector
employees.75 All these factors combine for a low redistributive impact of the fiscal 53 53 Alvaredo-Gasparini policy. For instance, Goñi et al. (2008) and Lustig (2012) find that the tax and transfer
system in Latin America decreased the market Gini by only 2 percentage points, a
meager impact compared to the 20-points impact estimated in 15 European
economies. Since the mid-1990s there have been some encouraging signs of improvement,
especially in terms of increasing coverage and better targeting of social policies. 77 A collapse is identified as a cumulative percentage drop in per capita GDP (from peak to trough) of at
least 9.5% for 1911 to 1950 and of 5% for the post-1950 period. Their results are somewhat different
when they look at banking crisis and consumption collapses, but not more conclusive. 76 In the case of the Gini coefficient, a change is considered significant when it is higher than 0.7
percentage points (that is, 1/3 of 2 Gini points).
77 4.7. Exploring inequality changes The
recent expansion of conditional cash transfer programs (CCTs) implies a promising
approach for enhancing the distributive impact of public spending in developing
economies. CCTs typically transfer income to poor households, conditional on
households making certain investments on their children's human capital – education,
health and nutrition. Such programs have been adopted in many developing
economies, including some Sub-Saharan African countries, although on a smaller scale
(Fiszbein and Shady, 2009; Garcia and Moore, 2012). CCTs became particularly popular
in LAC: by 2010 there were 18 countries in Latin America and the Caribbean applying
CCTs, covering 20% of total LAC population, and spending on average 0.40% of GDP
(Cruces and Gasparini, 2012). Soares et al. (2009) estimate that the CCTs in Brazil and
Mexico reduced the Gini for disposable income by 2.7 percentage points, accounting
for about a fifth of the decrease in that index between the mid-1990s and the mid-
2000s. Macroeconomic crises. The scale of the recent crisis has placed the distributional
impact of macroeconomic shocks back on the agenda. Banking crises, crashes in stock
and real estate markets and GDP collapses are events with potential large effects on
the income distribution. Atkinson and Morelli (2011) were the first in addressing this
issue from an empirical, historical and global perspective. They investigated the effect
of crisis on inequality as well as the impact of inequality on the probability of economic
crises, by analyzing the history of banking, consumption and GDP collapses over a 100-
year period in 25 countries, out of which only 6 are developing economies. These
authors observed the variation in distributional variables taking a 5-year window
before and after the crisis date, and classified each one according to whether
inequality was increasing, constant or decreasing before and afterwards.76 Table 4.3,
panel A, reproduces their results specifically regarding GDP collapses.77 They identified
103 crises, but for only one third there is information on inequality changes. The
shadowed diagonal shows combinations where the trajectory was unchanged; above
the diagonal are cases where the trajectory “bent” downward; below the diagonal are
cases where the trajectory “bent” upward. 4.7. Exploring inequality changes As it is readily apparent, one cannot draw
firm conclusions: (i) the raw totals show that most crisis did not involve changes in 54 54 Alvaredo-Gasparini inequality ex-post; (ii) the number of cases above the diagonal is low and not very
different from the cases below the diagonal, which means that GDP crisis are not
necessarily associated with a specific direction in the change of inequality; (iii) the
inverted V shape (inequality increasing and then decreasing) is not prevalent. Atkinson
and Morelli (2011) conclude that “economic crises differ a great deal in whether or not
they were preceded by rising inequality, and, in any case, where there was such a rise,
causality is not easy to establish.” When banking crisis are analyzed instead of GDP
drops, the cases in which inequality tend to increase following a crisis are in majority. We replicated their methodology for the years 1980-2010 to take into account the set
of developing countries, and the results are shown in panel B of Table 4.3.78 Even if our
list is not exhaustive and could be considerably improved, we identified at least 67
crises episodes. As in general they occurred during the 1980s or early 1990s, it is not
surprising that in most cases inequality changes before the crises remain unknown due
to data unavailability. There is a tendency for inequality to rise after a GDP collapse (10
cases), but again the numbers are too small to draw conclusions, and this could just be
the continuation of a previous tendency. This is not necessarily in contradiction with
Atkinson and Morelli (2011) due to at least two reasons: (i) GDP crisis may well be
more correlated with financial crisis in the developing world, and (ii) such conclusion is
highly influenced by the experience of the transition economies after the fall of the
Berlin Wall. It should also be noted that several of the canonical Latin American
exchange rate crisis of the 1980s and 1990s, with the exception of Argentina and Brazil
(included in Atkinson and Morelli, 2011) do not fall within our classification of a
collapse. In this sense, there is much work to be done about the magnitude of a crisis
and its sensitivity on the two-way relationship with inequality. 79 The case studies are numerous; see for example Forbes (2011) and Lustig (1990). 78 Given the higher volatility of per capita GDP in developing countries, we have kept the threshold of
9.5% drop to identify crisis for 1980-2010. The data come from the World Bank Development Indicators. p
y
Changes in Gini coefficient are taken from PovcalNet. 78 Given the higher volatility of per capita GDP in developing countries, we have kept the threshold of
9.5% drop to identify crisis for 1980-2010. The data come from the World Bank Development Indicators.
Changes in Gini coefficient are taken from PovcalNet.
79 The case studies are numerous; see for example Forbes (2011) and Lustig (1990). 4.7. Exploring inequality changes Ferreira
and Ravallion (2009), for instance, report that in Indonesia a large share of the increase
in inequality was associated with migration from wage employment in agriculture to
urban self-employment. Milanovic (2012) argues about the key role of migration in
global inequality. 4.7. Exploring inequality changes Of course this brief review does not exhaust the multiple factors behind
distributive changes in the developing world; in fact, arguably any shock or policy could
affect the income distribution. For instance, demographic factors, such as the decline
in fertility, the rise in life expectancy and the growing importance of assortative mating
and single-parent households have been identified as relevant sources of inequality
changes. Labor policies are a key target for research, as well. Several studies find that
the weakening of labor institutions such as unions and the declining real value of
minimum wages were responsible for the increase in earnings inequality in several
developing countries, especially in the 1990s, while more ambitious labor policies
contributed to the reduction in inequality in the 2000s. Migration and sector changes
are also determinants of inequality changes, studied at least since the seminal
contributions by Lewis (1954) and Kuznets (1955). Changes in inequality are associated
to the geographic and sectorial pattern of growth (Loayza and Raddatz, 2010). Ferreira
and Ravallion (2009), for instance, report that in Indonesia a large share of the increase
in inequality was associated with migration from wage employment in agriculture to
urban self-employment. Milanovic (2012) argues about the key role of migration in
global inequality. Others. Of course this brief review does not exhaust the multiple factors behind
distributive changes in the developing world; in fact, arguably any shock or policy could
affect the income distribution. For instance, demographic factors, such as the decline
in fertility, the rise in life expectancy and the growing importance of assortative mating
and single-parent households have been identified as relevant sources of inequality
changes. Labor policies are a key target for research, as well. Several studies find that
the weakening of labor institutions such as unions and the declining real value of
minimum wages were responsible for the increase in earnings inequality in several
developing countries, especially in the 1990s, while more ambitious labor policies
contributed to the reduction in inequality in the 2000s. Migration and sector changes
are also determinants of inequality changes, studied at least since the seminal
contributions by Lewis (1954) and Kuznets (1955). Changes in inequality are associated
to the geographic and sectorial pattern of growth (Loayza and Raddatz, 2010). 4.7. Exploring inequality changes In any case, the pattern
in Latin America points to an increase in inequality before the crashes (regressive
inflation tax, rise in unemployment due to openness to trade and loss of
competitiveness from exchange rate mismanagement), then followed by short-term
reductions after stabilization programs.79 55 Alvaredo-Gasparini Table 4.3
Inequality and GDP collapses
Source: own calculations based on PovcalNet (2013) and WDI. Panel A (Atkinson and Morelli, 2011)
Panel B (developing countries 1980-2010) Table 4.3
Inequality and GDP collapses
Source: own calculations based on PovcalNet (2013) and WDI. Others. Of course this brief review does not exhaust the multiple factors behind
distributive changes in the developing world; in fact, arguably any shock or policy could
affect the income distribution. For instance, demographic factors, such as the decline
in fertility, the rise in life expectancy and the growing importance of assortative mating
and single-parent households have been identified as relevant sources of inequality
changes. Labor policies are a key target for research, as well. Several studies find that
the weakening of labor institutions such as unions and the declining real value of
minimum wages were responsible for the increase in earnings inequality in several
developing countries, especially in the 1990s, while more ambitious labor policies
contributed to the reduction in inequality in the 2000s. Migration and sector changes
are also determinants of inequality changes, studied at least since the seminal
contributions by Lewis (1954) and Kuznets (1955). Changes in inequality are associated
to the geographic and sectorial pattern of growth (Loayza and Raddatz, 2010). Ferreira
and Ravallion (2009), for instance, report that in Indonesia a large share of the increase
in inequality was associated with migration from wage employment in agriculture to
urban self-employment. Milanovic (2012) argues about the key role of migration in
global inequality. Panel A (Atkinson and Morelli, 2011)
Panel B (developing countries 1980-2010) Table 4.3
Inequality and GDP collapses
Source: own calculations based on PovcalNet (2013) and WDI. Panel A (Atkinson and Morelli, 2011)
Panel B (developing countries 1980-2010) Inequality and GDP collapses Inequality and GDP collapses Inequality and GDP collapses and WDI. Panel B (developing countries 1980-2010) Panel B (developing countries 1980-2010) Panel B (developing countries 1980-2010) Panel A (Atkinson and Morelli, 2011) Panel A (Atkinson and Morelli, 2011) (
,
) Source: own calculations based on PovcalNet (2013) and WDI. Others. 5. Poverty: levels The vast literature on poverty measurement suggests that there are neither normative
nor objective arguments to set an unambiguous threshold below which everybody is
poor and above which everyone is non-poor (Deaton, 1997). Despite this central
conceptual ambiguity, reducing poverty is a deliberate policy objective for
governments around the world. The international community has embraced this goal
as reflected in the first Millennium Development Goal of halving poverty by 2015. In
this section we focus on measures of poverty in the income/consumption space using
international poverty lines in terms of US dollars adjusted for purchasing power parity
(PPP). This choice implies taking a one-dimensional, monetary, static, absolute view of
poverty, that certainly has many limitations and drawbacks, but it is still the best
available paradigm to summarize deprivations in the world. 56 Alvaredo-Gasparini The $1-a-day per person at PPP is a poverty standard meant to define an international
norm to gauge at the inability to pay for food needs.80 The $1 line, proposed in
Ravallion et al. (1991) and used in World Bank (1990), was chosen as being
representative of the national poverty lines found among low-income countries. The
line was recalculated in 1993 PPP terms at $1.0763 a day (Chen and Ravallion, 2001),
and more recently in 2005 PPP at $1.25 a day (Ravallion et al., 2009). The PPP
adjustment is based on the 2005 round of the International Comparison Program
(World Bank, 2008).81 The $1.25 line is usually deemed too low for middle-income
countries; for that reason it is typical to compute poverty with the $2-a-day standard,
which is close to the median of the official poverty lines chosen by the developing
countries. Although these international lines have been criticized, their simplicity and
the lack of reasonable and easy-to-implement alternatives have made them the
standard for international poverty comparisons.82 While the measurement of poverty
with national lines takes into consideration that societies differ in the criteria used to
identify the poor, the international lines are unavoidable instruments to compare
absolute poverty levels and trends across countries, and provide regional and world
poverty counts. The World Bank is the main institution that regularly produces information on poverty
measurement in the developing world drawn from original microdata from household
surveys.83 In 2013, the World Bank released an update of the developing world's
poverty estimates for 1981-2010. p
y
83 Ahulwalia et al. (1979) was an early attempt to measure poverty in the developing countries. 81 See the discussions on the PPP adjustments in Deaton and Dupriez (2008), Deaton and Heston (201
and Ravallion (2010).
82 82 For the debate on the international measurement of poverty, see Reddy and Pogge (2010), Deaton
(2010) and chapter 12 by Anand and Segal in this volume. Gentilini and Sumner (2012) compute global
poverty using the national poverty lines officially set in each country instead of using international
poverty standards. 80 For simplicity, the sign $ is used to refer to US dollars.
81 80 For simplicity, the sign $ is used to refer to US dollars.
81 80 For simplicity, the sign $ is used to refer to US dollars.
81 See the discussions on the PPP adjustments in Deaton and Dupriez (2008), Deaton and Heston (2010)
and Ravallion (2010).
82 For the debate on the international measurement of poverty, see Reddy and Pogge (2010), Deaton
(2010) and chapter 12 by Anand and Segal in this volume. Gentilini and Sumner (2012) compute global
poverty using the national poverty lines officially set in each country instead of using international
poverty standards.
83 Ahulwalia et al. (1979) was an early attempt to measure poverty in the developing countries. 5. Poverty: levels The new poverty estimates combine the PPP
exchange rates for household consumption from the 2005 International Comparison
Program with data from more than 850 household surveys across 127 developing
countries. In this section we rely heavily on that dataset (PovcalNet). The problem of the choice of the welfare variable discussed for inequality in section 3
applies to the measurement of poverty, as well. While poverty estimates in PovcalNet
refer to consumption deprivation, in most countries in Latin America and a few others
in the rest of the world they are constructed from income data. After computing
consumption and income poverty in 22 household surveys of 7 Latin American
countries using the $2 standard, we find that on average the ratio
consumption/income poverty is 0.97 with only small differences across countries. Given this piece of evidence we decided not to perform an adjustment for income
poverty figures in the analysis that follows. 57 57 Alvaredo-Gasparini Alvaredo-Gasparini 5.1. Income poverty in the developing world Although poverty is a ubiquitous characteristic of the developing economies, its
severity widely varies across countries. Figure 5.1 shows the poverty headcount ratio
in most of the developing countries in the world, using the $2-a-day poverty line. The
figure reveals the enormous differences among developing nations in terms of
monetary deprivation. While there are economies where the proportion of the
population living with less than 2 dollars a day is below 2%, in several countries that
proportion exceeds 80%. The problem of absolute income poverty has a radically
different scale in some countries compared to others, even in the developing world. Figure 5.1
Poverty headcount ratio
Developing countries, 2010
Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
$2-a-day line. 0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
110
120
130
Poverty headcount
China
Indonesia
Brazil
India Source: own calculations based on PovcalNet (2013). Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
$2-a-day line. Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjuste
$2-a-day line. In 2010, 41 per cent of the population in the developing world lived with less than $2 a
day. The unweighted mean headcount ratio was significantly lower: in a typical
developing country 33 per cent of the population was poor according to that criterion. The difference between the weighted and unweighted mean is not determined by
China, as the incidence of poverty in that country is similar to the developing world
mean, but by India (and to a lesser extent Indonesia and Pakistan), where the
deprivation measures are substantially higher. In fact, when ignoring India both the
weighted and unweighted headcount ratios become very close (33.3 and 32.7). The
median poverty rate is also lower than the mean (23.5 for the $2 line). Table 5.1
reports these results for other indices and poverty lines. Interestingly, when using the
$1.25 line the weighted mean is lower than the unweighted mean for the poverty gap 58 Alvaredo-Gasparini and the squared poverty gap, a result driven by the relatively low value of these
indicators in China and Indonesia. 84 Rwanda, Mozambique, Malawi, Nigeria, Tanzania, Zambia, Madagascar, Burundi, Congo Dem. Rep.
and Liberia.
85 The share of India and China in the world poor is 47% with the $1.25 line, 52% with the $2 line and
51% with the $4 line. 85 The share of India and China in the world poor is 47% with the $1.25 line, 52% with the $2 line an
51% with the $4 line. 5.1. Income poverty in the developing world and the squared poverty gap, a result driven by the relatively low value of these
indicators in China and Indonesia. Table 5.1
Poverty measures
Developing countries, 2010
Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita. weighted
mean
std.dev
median
$ 1.25 poverty line
Headcount
21.4
19.6
22.9
9.3
Poverty gap
6.5
7.5
10.7
2.9
Squared poverty gap
3.0
4.0
6.5
1.0
$ 2 poverty line
Headcount
41.0
33.0
30.1
23.5
Poverty gap
15.8
14.5
16.4
7.1
Squared poverty gap
8.1
8.3
10.9
4.0
$ 4 poverty line
Headcount
66.7
54.8
33.6
59.3
Poverty gap
35.9
29.6
24.0
25.4
Squared poverty gap
22.9
19.5
18.5
13.2
unweighted Note: poverty computed over the distribution of consumption/income per capita. The picture of poverty in the developing world is not significantly affected by changing
the poverty indicator or the poverty line. The correlations across countries when using
alternatively the headcount (H), the poverty gap (PG) and the squared poverty gap
(SPG) with a given poverty line are all higher than 0.9. For a fixed indicator the
correlations are higher than 0.95 when changing the poverty line. The correlations are
only slightly lower when changing both the indicator and the line (e.g. 0.85 for SPG
with the $1 line and H with the $2 line). The top ten steps in the poverty ladder, using the headcount ratio with the $2 line, are
all occupied by Sub-Saharan African countries.84 The following ten features also eight
SSA economies, in addition to a Caribbean country (Haiti), and a South Asian nation
(Bangladesh). However, given its size, India is the country with the largest number of
poor people. While around 840 million people in that country live with less than $2 a
day, the number in the second nation in that ranking, China, is less than a half (359). Both countries are home of 52% of the poor in the world, while the following four
countries - Nigeria, Bangladesh, Indonesia and Pakistan – represent 19%. Of course,
these exact figures are valid only for a specific definition of income poverty, but the
main results are robust to changes in indices and poverty lines.85 59 59 Alvaredo-Gasparini Alvaredo-Gasparini Alvaredo-Gasparini Alvaredo-Gasparini As expected, the relationship between mean consumption and poverty is very tight
(Figure 5.2). 5.1. Income poverty in the developing world A simple model of the headcount ratio ($2 line) on log mean consumption
per capita estimated in a cross section of developing countries for 2010 accounts for
more than 70% of the variation in the data. Figure 5.2
Poverty headcount ratio and log consumption per capita
Developing countries, 2010
Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
2$-a-day line. 0
10
20
30
40
50
60
70
80
90
100
3
3.5
4
4.5
5
5.5
6
6.5
7
headcount ratio
ln consumption per capita (PPP US$) Figure 5.2
Poverty headcount ratio and log consumption per capita
Developing countries, 2010 ln consumption per capita (PPP US$) Source: own calculations based on PovcalNet (2013). Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
2$-a-day line. (
)
Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
2$-a-day line. Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjuste
2$-a-day line. The cross-country relationship between poverty and inequality is much looser (Figure
5.3). The correlation coefficient between the headcount ($2 line) and the Gini is 0.17
(barely significant at 5%). The relationship is somewhat tighter with other poverty
indices, but still in all cases the correlation coefficients are lower than 0.3. The
magnitude of the correlations is similar when considering different income shares as
measures of inequality. 60 60 Alvaredo-Gasparini Alvaredo-Gasparini Alvaredo Gasparini
Figure 5.3
Poverty headcount ratio and Gini coefficient
Developing countries, 2010
Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
$2-a-day line. 0
10
20
30
40
50
60
70
80
90
100
20
25
30
35
40
45
50
55
60
65
70
headcount ratio
Gini coefficient Figure 5.3
Poverty headcount ratio and Gini coefficient
Developing countries, 2010
l
l
b
d
l
(
)
0
10
20
30
40
50
60
70
80
90
100
20
25
30
35
40
45
50
55
60
65
70
headcount ratio
Gini coefficient Figure 5.3
Poverty headcount ratio and Gini coefficient
Developing countries, 2010 Source: own calculations based on PovcalNet (2013). (
)
Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
$2-a-day line. 5.1. Income poverty in the developing world (
)
Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
$2-a-day line. Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjuste
$2-a-day line. Table 5.2 shows some simple regressions aimed at characterizing the relationship
between poverty, mean income and inequality in a cross-section of developing
countries. The results of course do not have any causal implication, and then are not
helpful to orient policy, but nonetheless are illustrative of the empirical relationship
among these three variables. An increase (cross-country) of 1% in mean consumption
is associated to a fall of around 2% in the headcount ratio, while a drop of 1% in the
Gini coefficient is associated to a reduction of around 3.5% in poverty measured by the
headcount. The results are similar when measuring deprivation with the squared
poverty gap. Table 5.2
Regressions of poverty measures
Developing countries, 2010
Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
$2-a-day line. ln y=log mean household consumption/income per capita; share d1= share of decile 1 in
the household consumption/income per capita distribution; share d1-d6=cumulative share of deciles 1
to 6. Robust standard deviations are shown under the coefficients. *** significant at 1% level. (i)
(ii)
(iii)
(iv)
(v)
(vi)
(vii)
(viii)
ln y
-2.0
-2.0
-2.1
-2.0
-2.1
-2.0
-2.2
-2.1
(0.114)***
(0.085)***
(0.086)***
(0.086)***
(0.127)***
(0.092)***
(0.084)***
(0.091)***
ln Gini
3.32
3.76
(0.329)***
(0.349)***
Share d1
-0.74
-0.91
(0.073)***
(0.071)***
Share d1-d6
-0.12
-0.13
(0.011)***
(0.012)***
Constant
12.6
0.1
14.8
16.1
11.0
-3.1
13.8
15.1
(0.577)***
(1.30)
(0.484)***
(0.561)***
(0.64)***
(1.39)
(0.473)***
(0.586)***
R2
0.72
0.85
0.85
0.85
0.68
0.84
0.87
0.85
log headcount ratio
log squared poverty gap Table 5.2 Regressions of poverty measures
Developing countries, 2010 Regressions of poverty measures
Developing countries, 2010 (
)
Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjuste
$2-a-day line. ln y=log mean household consumption/income per capita; share d1= share of decile 1
the household consumption/income per capita distribution; share d1-d6=cumulative share of deciles
to 6. Robust standard deviations are shown under the coefficients. *** significant at 1% level. Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
$2-a-day line. ln y=log mean household consumption/income per capita; share d1= share of decile 1 in
the household consumption/income per capita distribution; share d1-d6=cumulative share of deciles 1
to 6. Robust standard deviations are shown under the coefficients. *** significant at 1% level. Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
$2-a-day line. ln y=log mean household consumption/income per capita; share d1= share of decile 1 in
the household consumption/income per capita distribution; share d1-d6=cumulative share of deciles 1
to 6. Robust standard deviations are shown under the coefficients. *** significant at 1% level. 61 61 Alvaredo-Gasparini 5.2. Poverty by region Poverty has a clear regional component: Table 5.3 reveals that Eastern Europe and
Central Asia is always the region with the lowest income poverty, followed by Middle
East and North Africa and Latin America and the Caribbean. Poverty in South Asia is
substantially larger than in Eastern Asia when weighting by population, but roughly
similar when ignoring weights. All income poverty measures are substantially higher in
Sub-Saharan Africa than in the rest of the developing world. Table 5.3
Poverty indicators by region
Developing countries, 2010
Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita. East Asia &
Pacific
Eastern
Europe &
Central Asia
Latin
America &
Caribbean
Middle East
& North
Africa
South Asia
Sub-Saharan
Africa
Weighted
$1.25 line
Headcount
12.5
1.0
5.5
2.4
31.0
48.5
Poverty gap
2.8
0.3
2.9
0.6
7.1
20.9
Squared poverty gap
0.9
0.2
2.1
0.2
2.4
11.8
$2 line
Headcount
29.7
2.4
10.4
12.0
66.7
69.9
Poverty gap
9.7
0.7
4.7
2.8
23.4
35.7
Squared poverty gap
4.2
0.3
3.2
1.0
10.5
22.4
Unweighted
$1.25 line
Headcount
17.9
1.6
8.6
3.5
17.6
41.5
Poverty gap
5.1
0.5
4.0
0.9
3.9
16.6
Squared poverty gap
2.3
0.2
2.6
0.3
1.3
8.8
$2 line
Headcount
38.4
5.2
15.6
13.5
43.5
62.2
Poverty gap
13.8
1.5
7.0
3.5
14.1
30.2
Squared poverty gap
6.7
0.6
4.4
1.4
6.1
18.1 (
)
Note: poverty computed over the distribution of consumption/income per capita. Figure 5.4 unveils the considerable heterogeneity within each geographic region. When using the $2 line, the poverty headcount ratio ranges in EAP from 1.4 (Malaysia)
to 70.6 (Timor-Leste), in ECA from 0.1 (Slovenia) to 35.6 (Georgia), in LAC from 1.2
(Uruguay) to 80.1 (Haiti), in MENA from 1.6 (Jordan) to 45.6 (Yemen), in SA from 6.8
(Maldives) to 76.5 (Bangladesh), and in SSA from 1.5 (Seychelles) to 94.5 (Liberia). Figure 5.5 displays a map of the poverty levels in the world that illustrates the regional
differences, as well as the within-region heterogeneities. 62 62 Alvaredo-Gasparini Figure 5.4
Poverty headcount ratio
Developing countries, 2010
Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
$2-a-day line. 5.2. Poverty by region 0
10
20
30
40
50
60
70
80
90
100
Latin America &
the Caribbean
Sub-Saharan Africa
Eastern Europe &
Central Asia
East Asia &
Pacific
Middle East &
North Africa
South Asia Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjuste
$2-a-day line. There is a considerable degree of spatial correlation of poverty measures across
countries. The Moran scatterplot is a way to illustrate that spatial correlation (Figure
5.6). The horizontal axis shows the normalized headcount ratio of a country ($2 line),
while the vertical axis depicts a weighted average of its neighbors´ normalized poverty
rates, where neighborhood is defined in terms of geographical proximity. The graph
suggests a strong positive correlation between a country poverty incidence rate and
that of its neighbors (the Moran correlation coefficient is 0.507, significant at 1%). Almost 80% of the countries are either in the HH cells (high poverty for the country
and its neighbors) or in the LL cells. 63 63 Alvaredo-Gasparini Figure 5.6
Spatial correlation of poverty rates
Moran´s scatterplot
Developing countries, 2010
Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
$2-a-day line. z is the normalized poverty headcount ratio (the value minus the mean, divided by the
standard deviation), Wz is the weighted average of the normalized poverty headcount ratios of a
country´s neighbors, where the weights W are defined in terms of contiguity. HH
LH
HL
LL
-1
0
1
2
3
Wz
-1
0
1
2
3
z
Moran scatterplot (Moran's I = 0.507) ,
Moran scatterplot (Moran's I = 0.507) Source: own calculations based on PovcalNet (2013). (
)
Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
$2-a-day line. z is the normalized poverty headcount ratio (the value minus the mean, divided by the
standard deviation), Wz is the weighted average of the normalized poverty headcount ratios of a
country´s neighbors, where the weights W are defined in terms of contiguity. Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
$2-a-day line. z is the normalized poverty headcount ratio (the value minus the mean, divided by the
standard deviation), Wz is the weighted average of the normalized poverty headcount ratios of a
country´s neighbors, where the weights W are defined in terms of contiguity. 5.2. Poverty by region The poverty gap indicator has an intuitive-appealing interpretation: when normalized
by the poverty line and the total population of a country, it gives the total cost needed
to end poverty, in the particular case in which cash transfers could be perfectly
targeted to poor people in the amount just needed to reach the poverty line, and no
changes in behavior take place. Table 5.4 shows the unweighted mean across
countries of the cost of eliminating poverty as percentage of GDP under this scenario
in each region. Although the context is clearly unrealistic, the figures give a rough idea
of the magnitude of the task of fighting poverty in each region of the developing world
in relation to the available economic resources. While eliminating poverty with the $2
line in this scenario would require on average less than 1 GDP point in the economies
of ECA and between 1 and 2 points in MENA and LAC, the size of the effort is larger in
Asia and orders of magnitude greater in Sub-Saharan Africa. 64 64 Alvaredo-Gasparini Table 5.4
The cost of eliminating poverty
Total poverty gap as percentage of GDP
Unweighted means by region, 2010
Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita. Region
$ 1.25
$ 2
East Asia and Pacific
0.6
2.9
Eastern Europe and Central Asia
0.1
0.3
Latin America and the Caribbean
0.7
1.7
Middle East and North Africa
0.2
1.0
South Asia
1.2
6.6
Sub-Saharan Africa
8.1
23.0
Poverty lines Note: poverty computed over the distribution of consumption/income per capita. International surveys, such as the Gallup Poll, provide an opportunity to alleviate some
of the typical comparability problems of household surveys, since survey design and
questionnaires are identical across countries. However, as discussed above, these
surveys have still small samples, and measurement errors are presumably large, given
that only one income question is included. The correlation between headcount ratios
computed with the Gallup Poll and PovcalNet is 0.32, significant at 2%, while the rank
Spearman correlation is 0.61, significant at 1%. Figure 5.7 shows the cumulative density function in some regions of the world, based
on Gallup data. There is first-order stochastic dominance of the Western Europe
distribution over the rest, while the South Asian distribution is dominated by the rest. The curves of ECA and EAP cross each other, although they do so at high-income levels. 86 Chapter 12 in this volume also covers the issue of global poverty in the developing world. In our
chapter the interest is more focused on the country trends, and then we make more use of unweighted
statistics. 6. Poverty: trends In the last decades the developing world has made undeniable progress toward the
goal of ending absolute poverty. The evidence suggests that the first goal of the
Millennium Development Goals – to halve extreme poverty from 1990 to 2015 – was
already met in 2010 for the aggregate of developing countries. However, the strong
decline in global absolute poverty hides substantial heterogeneities across economies
and over time. In this section we document and characterize trends in income poverty
in the countries of the developing world since the early 1980s to 2010, and trace those
changes to economic growth and distributional changes.86 The literature on international poverty trends can be divided into two main strands. The first one makes comparisons based exclusively on household survey microdata. This ambitious road that requires access to surveys for many countries over time has
been taken by several initiatives at the regional level, and by the World Bank at a
global scale, mainly through the work of Martin Ravallion and Shaohua Chen. The
second strand combines estimates of the national income distributions with GDP or
aggregate consumption data drawn from National Accounts to anchor the mean. Bourguignon and Morrison (2002), Bhalla (2002), Karshenas (2003) and Sala-i-Martin
(2006) are examples of this literature. In this section we mainly document the results
of the first approach. 5.2. Poverty by region Figure 5.7
Distribution functions
Source: own estimates based on microdata from Gallup World Poll 2006. Note: cumulative distribution functions of per capita household income. 0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0
5000
10000
15000
20000
25000
30000
South Asia
East Asia & Pacific
East Europe & Central Asia
Western Europe Figure 5.7
Distribution functions Source: own estimates based on microdata from Gallup World Poll 2006. Note: cumulative distribution functions of per capita household income. 65 65 Alvaredo-Gasparini Alvaredo-Gasparini 6. Poverty: trends 87 The cumulative distribution function of 2010 lies always below the functions corresponding to all
previous years. The first-order stochastic dominance assures that the result of lower poverty in 2010 is
robust to the choice of poverty line and valid for a broad class of poverty measures (Atkinson, 1987). 6.1. Trends in income poverty Data from PovcalNet reveals a widespread fall in absolute income poverty in the
developing countries over the last three decades (Figure 6.1). Only for a few nations
poverty in 2010 was not lower than in 1981; that set includes some economies in
Eastern Europe, Central Asia and Sub-Saharan Africa, and only a few in Latin America
and the Caribbean. The poverty decline in the 2000s was even more generalized: in
only 8 out of 121 countries did poverty increase between 1999 and 2010 (5 in Sub-
Saharan Africa). 66 66 Alvaredo-Gasparini Figure 6.1
Poverty headcount ratio
Developing countries
Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
$2-a-day line. Poverty 1981-2010
Poverty 1999-2010
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Headcount ratio 2010
Headcount ratio 1981
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Headcount ratio 2010
Headcount ratio 1999 Figure 6.1
Poverty headcount ratio
Developing countries
Poverty 1981-2010 Figure 6.1
Poverty headcount ratio
Developing countries
Poverty 1981-2010 Figure 6.1
Poverty headcount ratio
Developing countries
Poverty 1981-2010 Poverty 1981-2010
Pover
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Headcount ratio 2010
Headcount ratio 1981
0
10
20
30
40
50
60
70
80
90
100
0
Headcount ratio 2010 Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
$2-a-day line. Source: own calculations based on PovcalNet (2013). (
)
Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
$2-a-day line. Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjuste
$2-a-day line. The proportion of the population in the developing world living with less than $1.25
per day decreased from 52% in 1981 to 20.8% in 2010, which implies a trend decline of
around one point per year (Figure 6.2).87 This is a remarkable achievement that should
not be overlooked. It would be hard to find other episodes in history where the extent
of extreme poverty was reduced so massively in such a short period of time. Poverty headcount ratio y
Weighted and unweighted means, developing countries, 1981-2010
Source: own calculations based on PovcalNet (2013). 87 The cumulative distribution function of 2010 lies always below the functions corresponding to all
previous years. The first-order stochastic dominance assures that the result of lower poverty in 2010 is
robust to the choice of poverty line and valid for a broad class of poverty measures (Atkinson, 1987). 0
10
20
30
40
50
60
70
80
1981
1984
1987
1990
1993
1996
1999
2002
2005
2008
2010
Unweighted - $ 1.25 line
Unweighted - $ 2 line
Weighted - $ 1.25 line
Weighted - $ 2 line y
Weighted and unweighted means, developing countries, 1981-2010 y
Weighted and unweighted means, developing countries, 1981-2010 Weighted and unweighted means, developing countries, 1981-2010 Source: own calculations based on PovcalNet (2013). 0
10
20
30
40
50
60
70
80
1981
1984
1987
1990
1993
1996
1999
2002
2005
2008
2010
Unweighted - $ 1.25 line
Unweighted - $ 2 line
Weighted - $ 1.25 line
Weighted - $ 2 line Source: own calculations based on PovcalNet (2013). 0
10
20
30
40
50
60
70
80
1981
1984
1987
1990
1993
1996
1999
2002
2005
2008
2010
Unweighted - $ 1.25 line
Unweighted - $ 2 line
Weighted - $ 1.25 line
Weighted - $ 2 line Source: own calculations based on PovcalNet (2013). 67 Alvaredo-Gasparini Note: poverty computed over the distribution of consumption/income per capita. However, this extraordinary result should be put in perspective. First, even after this
global social improvement one of every five persons in the developing world still lives
in extremely deprived conditions (less than $1.25 a day), while four out of ten people
have household per capita consumption levels lower than a frugal $2 per day. However, this extraordinary result should be put in perspective. First, even after this
global social improvement one of every five persons in the developing world still lives
in extremely deprived conditions (less than $1.25 a day), while four out of ten people
have household per capita consumption levels lower than a frugal $2 per day. Second, the positive performance of China is key for the global result. Ignoring China,
the poverty decline is less impressive: the headcount ratio fell 16 instead of 31 points
in three decades (Table 6.1).88 In fact, when ignoring China it is not clear that the
developing world could meet the MDG for poverty reduction in 2015. While the
population-weighted mean of the poverty incidence rate dropped 31 points between
1981 and 2010, the unweighted mean declined about 10 points, and the median went
down just 8 points. The extreme poverty rate ($1.25) of a typical developing country
was reduced from 29.5% in 1981 to 19% in 2010, which represents a fall of around a
third of a point per year, a figure less impressive than the corresponding one for the
global poverty rate (one point a year). In fact, this decline took place only since the
late-1990s. 88 When ignoring China the distribution of 2010 still dominates (first-order stochastic dominance) 1981
although the distance between the two cumulative distributions is smaller. The curve for 1999 lies
below 1981 and 1990 but only for poverty lines lower than $3 a day. y
Weighted and unweighted means, developing countries, 1981-2010 A typical developing country did not experience any improvement for
almost two decades: the unweighted poverty rate for the developing world was 29.5%
in 1981, 29.8% in 1990 and 28.8% in 1996. From then on poverty declined more
consistently, especially between 2002 and 2008, when for a typical developing
economy the rate of poverty reduction was almost one point a year. 68 68 Alvaredo-Gasparini Table 6.1
Change in poverty measures (points)
Developing countries, 1981-2010 Table 6.1
Change in poverty measures (points)
Developing countries, 1981-2010
Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita. Poverty measure
1981-
1990
1990-
1999
1999-
2010
1981-
2010
Poverty measure
1981-
1990
1990-
1999
1999-
2010
1981-
2010
Headcount - $1.25 line
Poverty gap - $1.25 line
Unweighted
Unweighted
Mean
0.3
-1.9
-8.9
-10.5
Mean
0.2
-1.6
-4.2
-5.7
Median
4.9
0.0
-12.9
-8.0
Median
0.3
0.3
-4.0
-3.4
Weighted
Weighted
Mean
-9.0
-8.9
-13.4
-31.2
Mean
-6.4
-3.7
-4.8
-14.9
Without China
-2.8
-3.1
-10.0
-15.9
Without China
-1.6
-1.5
-3.7
-6.8
Headcount - $2 line
Poverty gap - $2 line
Unweighted
Unweighted
Mean
0.7
-0.6
-11.0
-11.0
Mean
0.3
-1.5
-6.5
-7.7
Median
-0.3
-3.4
-17.1
-20.7
Median
2.3
0.8
-9.2
-6.1
Weighted
Weighted
Mean
-4.9
-7.1
-16.6
-28.5
Mean
-6.6
-5.4
-8.8
-20.8
Without China
-1.2
-1.1
-10.9
-13.1
Without China
-1.7
-1.8
-6.4
-9.9
Headcount - $4 line
Poverty gap - $4 line
Unweighted
Unweighted
Mean
0.9
2.2
-11.4
-8.3
Mean
0.4
-0.4
-9.0
-9.0
Median
2.1
-2.1
-16.3
-16.3
Median
-0.4
-1.4
-12.3
-14.1
Weighted
Weighted
Mean
-1.7
-1.3
-14.0
-17.0
Mean
-8.4
-4.6
-12.1
-25.1
Without China
-1.2
1.7
-8.3
-7.8
Without China
-1.4
-0.5
-7.8
-9.7 (
)
Note: poverty computed over the distribution of consumption/income per capita. The fall in poverty appears less startling when using higher poverty lines. While the
headcount ratio with the $1.25 line fell 60% from 1981 to 2010, it declined 41% when
measured with the $2 line and 20% with the $4 line. In fact, while the MDG goal of
halving $1-a-day poverty from the value in 1990 was already met in 2010, the
assessment is different when using the $2 line: the headcount ratio in 2010 was about
2/3 of the value in 1990. 89 See Reddy and Pogge (2010), Chen and Ravallion (2012) and Cockburn et al. (2012). y
Weighted and unweighted means, developing countries, 1981-2010 The performance of the developing world in terms of poverty reduction also looks
much less spectacular when considering poverty counts, instead of the typical
measures that are invariant to the size of the population. In fact, for the majority of
the developing countries (63%) the number of poor people was higher in 2010 than in
1981. Even during the booming 2000s the poverty count increased in 30% of the
nations. The number of poor people was in the late 2000s only slightly lower than in
the early 1980s (2,585 million in 1981 and 2,394 million in 2010); ignoring China the
poverty count actually increased in 422 million (from 1,613 to 2,035). Some authors
have suggested that the difference in the assessment of world poverty when
alternatively using the headcount ratio or the number of poor people may be behind
some of the controversies in the public debate about globalization and the social
performance of the world in the last decades.89 Since the calculation of global poverty with microdata is very cumbersome, and
requires having a large number of comparable household surveys, some authors 69 69 Alvaredo-Gasparini estimate the changes in poverty with National Accounts data, anchoring the mean of
the income distribution to output or domestic consumption taken from National
Accounts, using secondary distributive data and making assumptions about the
functional form of the income distribution, typically the lognormal parameterization
(Ahluwalia et al., 1979; Chotikapanich et al., 1997; Bhalla, 2002; Sala-i-Martin, 2006;
Pinkovskiy and Sala-i-Martin, 2009).90 This methodology allows ambitious calculations
in terms of coverage, but it faces some obvious caveats as changes in National
Accounts aggregates may be misleading proxies for changes in household per capita
income, and the secondary distributive data in which the estimates are usually based
are mined with several comparability problems.91 According to the estimates by Pinkovskiy and Sala-i-Martin (2009) (PSM) using a
sample of 191 countries, and distributive data from the WIID, world poverty rates ($2
line) went down from 45.2% in 1970 to 37.8% in 1981, 24.9% in 1990, 16.8% in 1999,
and 13% in 2006. y
Weighted and unweighted means, developing countries, 1981-2010 That pattern implies a fall substantially faster than what is estimated
with household surveys data alone in the 1980s and 1990s, but slower in the 2000s.92
For instance, while the magnitude of the poverty incidence rates in PSM for the $3 line
is similar than in PovcalNet with the $1.25 line, the declines have been different: in
PSM the headcount ratio fell at annual 2.6% and 3.5% in the 1980s and 1990s,
respectively, while the rates were 2.1% and 2.5% with PovcalNet data. Instead, in the
2000s poverty fell at annual 3.1% in PSM and at annual 4.4% in PovcalNet. 90 It should be noticed that the World Bank poverty estimates also use National Accounts (NAS) data,
although to a limited extent. For instance, for countries with only one household survey, poverty is
estimated by applying the NAS consumption estimates to the available distribution data, assuming the
Lorenz curve remains fixed. 91 An intermediate alternative is proposed by Karshenas (2003), who calibrates survey means using
National Accounts statistics. Calibrated survey means are read off the fitted curve for the mean of
household survey consumption conditional on the NAS mean. 93 See Datt and Ravallion (1992), Kakwani and Subbarao (1990), Kakwani (2000) and Mahmoudi (200
for different proposals. 92 Dhongde and Minoiu (2010) explore several factors behind the different results in Chen and Ravallio
(2010) and Pinkovskiy and Sala-i-Martin (2009).
93 90 It should be noticed that the World Bank poverty estimates also use National Accounts (NAS) data,
although to a limited extent. For instance, for countries with only one household survey, poverty is
estimated by applying the NAS consumption estimates to the available distribution data, assuming the
Lorenz curve remains fixed.
91 An intermediate alternative is proposed by Karshenas (2003), who calibrates survey means using
National Accounts statistics. Calibrated survey means are read off the fitted curve for the mean of
household survey consumption conditional on the NAS mean.
92 Dhongde and Minoiu (2010) explore several factors behind the different results in Chen and Ravallion
(2010) and Pinkovskiy and Sala-i-Martin (2009).
93 See Datt and Ravallion (1992), Kakwani and Subbarao (1990), Kakwani (2000) and Mahmoudi (2001)
for different proposals. y pp y g
p
,
g
Lorenz curve remains fixed.
91 An intermediate alternative is proposed by Karshenas (2003), who calibrates survey means using
National Accounts statistics. Calibrated survey means are read off the fitted curve for the mean of
household survey consumption conditional on the NAS mean.
92 Dhongde and Minoiu (2010) explore several factors behind the different results in Chen and Ravallion 92 Dhongde and Minoiu (2010) explore several factors behind the different results in Chen and Ravallion
(2010) and Pinkovskiy and Sala-i-Martin (2009).
93 See Datt and Ravallion (1992), Kakwani and Subbarao (1990), Kakwani (2000) and Mahmoudi (2001) 94 Since the decompositions could be carried out changing the base year, the table shows the averages
over the two exercises. To obtain the results we use lineal approximations to the Pen curve based on
information on mean consumption per capita by decile from PovcalNet. Table 6.2 Decomposition of poverty changes
Developing countries, 1990-2010 Decomposition of poverty changes Developing countries, 1990-2010 Note: the columns show unweighted means across a sample of 76 developing countries of the change in
poverty headcount ratio ($2 line), and the growth and redistribution effects from the poverty change
decomposition. Note: the columns show unweighted means across a sample of 76 developing countries of the change in
poverty headcount ratio ($2 line), and the growth and redistribution effects from the poverty change
decomposition. 6.2. Decomposing poverty changes Researchers frequently use decompositions of changes in poverty into growth and
redistribution effects in order to characterize poverty trends.93 The growth effect
refers to the poverty change between two years that would have occurred if the mean
income had changed as it did, but the shape of the distribution had stayed fixed. On
the other hand, the redistribution effect records the poverty change between two
years that would have occurred if the shape of the distribution had changed in the way
it did, but the mean had remained fixed. Of course, this is just a mechanical exercise,
as indicators of economic growth, and changes in inequality and poverty are just three
different ways of aggregate information on income dynamics, and therefore they are
all jointly determined by the general equilibrium of the economy. In that sense the
decompositions are helpful to illustrate the way incomes have changed and affected 70 70 Alvaredo-Gasparini poverty, but they are silent on the fundamental factors underlying poverty changes,
and on the policies recommended to reduce deprivations more effectively. The change in the growth-inequality-poverty triangle was very different in the 2000s
compared to the previous decade. Table 6.2 illustrates this difference showing the
unweighted mean of the growth and redistribution effects of changes in poverty for a
sample of 76 developing countries with information on deciles shares in PovcalNet.94 The mild fall in the poverty headcount ratio ($2 line) in the 1990s can be decomposed
into a poverty-decreasing growth effect that outweighed a poverty-increasing
redistribution effect. On average (unweighted) mean consumption grew at an annual
0.2 per cent, and the Gini increased about 0.3 points per year, implying a very modest
decline in poverty. In contrast, in the 2000s both effects contributed to a falling
poverty. A robust increase of mean consumption (more than 3 per cent a year) and a
slow fall in inequality (around 0.1 Gini points a year) combined to yield a substantial
reduction in indicators of material deprivation. The growth effect was particularly
large, accounting for 90 per cent of the fall in the headcount ratio ($2 line). This result
does not mean that distributional changes are not important, but instead that they
have not been the main drivers of poverty reduction in the past. Table 6.2
Decomposition of poverty changes
Developing countries, 1990-2010
Source: own calculations based on data from PovcalNet. 6.2. Decomposing poverty changes Note: the columns show unweighted means across a sample of 76 developing countries of the change in
poverty headcount ratio ($2 line), and the growth and redistribution effects from the poverty change
decomposition. Actual
change
Growth
Redistribution
1990-1999
-0.3
-1.5
1.2
1999-2010
-10.1
-9.0
-1.1
1990-2010
-10.5
-10.4
-0.1
Effects Table 6.2
Decomposition of poverty changes
Developing countries, 1990-2010
Source: own calculations based on data from PovcalNet. Note: the columns show unweighted means across a sample of 76 developing countries of the change in
poverty headcount ratio ($2 line), and the growth and redistribution effects from the poverty change
decomposition. Actual
change
Growth
Redistribution
1990-1999
-0.3
-1.5
1.2
1999-2010
-10.1
-9.0
-1.1
1990-2010
-10.5
-10.4
-0.1
Effects 6.3. The cost of closing the poverty gap Although still a challenging problem, eliminating absolute extreme income poverty is
an increasingly affordable target. Based on PovcalNet data we computed the
population-weighted poverty gap index in the developing world as a share of global
GDP. This fraction, that indicates the fiscal effort required to end poverty in a scenario
of perfect targeting, absent behavioral responses, has been substantially falling over
time as poverty decreased and global GDP went up. The resources needed to close the
poverty gap with the $1.25 line as a share of global GDP declined from 1.3% in 1981 to 71 Alvaredo-Gasparini 0.2% in 2010 (the corresponding values for the $2 line are 3.6% and 1%).95 As a share
of the GDP in the developing world the cost of closing the poverty gap fell from 1.9% in
1981 to 0.4% in 2010 (5.4% and 1.8% for the $2 line). Kanbur and Sumner (2011) highlight the fact that while in 1990 over 90 per cent of the
world’s extremely poor people ($1.25) lived in countries classified as low-income
countries (LICs), by the late 2000s this share dropped to less than 30 per cent. From
the fact that most of the world’s poor live in middle-income countries with the
domestic financial capacity to end at least extreme poverty, Sumner (2012) concludes
that poverty reduction is increasingly becoming a domestic issue of national
distribution and domestic political economy, rather than primarily an aid and
international issue. Table 6.3 suggests than on average (unweighted) across developing
countries the redistributive national effort to end extreme poverty fell from 8.2% of
GDP in 1981 to 4% in 2010. The median value is much lower, and has fallen from 1.9%
in 1981 to 0.5% in 2010. The third column shows the share of countries where the cost
of eliminating extreme income poverty is less than 1 GDP point. That share
substantially increased in the 2000s, from 41.3% in 1999 to 55.4% in 2010. Similarly,
the share of countries for which closing the poverty gap is fiscally very burdensome (in
the table more than 3 GDP points) fell from more than 50% in 1990 to about 30% in
2010. Sumner (2012) reports similar trends, although a lower proportion of countries
with poverty gap/GDP less than 1%. Ravallion (2010) also notes that most middle-
income countries would require very small additional taxation to end poverty. 95 This computation assumes zero poverty in the high-income countries, when deprivation is measured
with the international lines. Note: poverty computed over the distribution of consumption/income per capita. Unweighted statistic Source: own calculations based on PovcalNet (2013). Source: own calculations based on PovcalNet (2013). 96 The contrast between East Asia and the rest of the developing world is evident in terms of progress
towards the first goal of the MDGs. EAP already met the goal in the early 2000s, far ahead of the rest. 6.3. The cost of closing the poverty gap Table 6.3
Poverty gap as share of GDP
Mean, median, share of countries with gap/GDP less than 1% and greater than 3%
Developing countries, 1981-2010
Source: own calculations based on PovcalNet (2013). Mean
Median
% less
1 GDP point
% more
3 GDP points
Mean
Median
% less
1 GDP point
% more
3 GDP points
1981
8.2
1.9
42.4
45.7
19.9
5.4
22.9
60.4
1984
8.4
2.2
41.3
45.7
20.5
6.6
19.8
60.4
1987
8.2
2.3
39.1
46.7
19.9
7.1
20.8
60.4
1990
8.1
3.3
39.1
51.1
19.6
7.0
24.0
59.4
1993
9.2
2.4
40.2
47.8
22.0
7.5
25.0
59.4
1996
9.2
2.0
40.2
42.4
21.6
7.3
24.0
60.4
1999
6.9
2.0
41.3
39.1
17.9
5.9
25.0
60.4
2002
5.9
1.4
41.3
40.2
15.7
4.8
27.1
58.3
2005
5.2
1.0
48.9
35.9
14.0
3.7
32.3
55.2
2008
4.3
0.7
50.0
33.7
11.6
3.0
40.6
50.0
2010
4.0
0.5
55.4
31.5
11.1
3.0
42.7
50.0
Poverty line $1.25
Poverty line $2 Poverty gap as share of GDP
Mean, median, share of countries with gap/GDP less than 1% and greater than 3%
Developing countries, 1981-2010 72 Alvaredo-Gasparini Alvaredo-Gasparini 6.4. Regional trends The patterns in income poverty over time have been heterogeneous across the
geographic regions of the developing world (Table 6.4). At least three facts are worth
stressing: (i) the remarkable decline in poverty in Asia over the last three decades, (ii)
the lack of significant progress in the rest of the regions in the 1980s and 1990s, and
(iii) the generalized fall in poverty in the 2000s. Arguably, the main fact about poverty dynamics in the last three decades has been the
noteworthy decline in Asia. The share of people living with less than $2 a day went
down from 92.4% to 29.7% between 1981 and 2010 in East Asia and Pacific, and from
87.2% to 66.7% in South Asia.96 The performance of EAP is enhanced by the presence
of China, but also the unweighted mean dramatically fell in this region (from 70.4% in
1981 to 38.4% in 2010). The fall in the unweighted mean was similar in South Asia
(from 80.3% to 43.5%). Almost all countries in Asia experienced drops in income
poverty over the period 1981-2010. The reductions were on average larger in the
2000s than in the previous decades. For instance, in South Asia the unweighted mean
fell 5.6 points in the 1980s, 10.9 in the 1990s and 20.4 in the 2000s. In the rest of the developing world the performance was weak and even negative in
the 1980s and 1990s. Income poverty rose in Latin America in the 1980s, in Eastern
Europe and Central Asia in the 1990s, and in Sub-Saharan Africa in both decades. In
contrast, all regions experienced falling poverty in the 2000s. The reductions were
large, and in general outweighed the weak performance of the previous decades. For
instance, on average (unweighted) income poverty ($2 line) went down 72% in ECA,
43% in MENA, 34% in LAC and 12% in SSA. The proportion of countries where the
headcount ratio fell more than 5% in the 2000s is above 90% in all these regions, with
the exception of SSA, where the corresponding proportion is 66%. 73 73 Alvaredo-Gasparini Table 6.4
Poverty headcount ratio, developing world 1981-2010
Weighted and unweighted means
Source: own calculations based on PovcalNet (2013). Note: the columns show unweighted means across a sample of 76 developing countries of the change in
the poverty headcount ratio ($2 line), and the growth and redistribution effects from the poverty
change decomposition. 6.4. Regional trends 1981
1990
1999
2010
Unweighted mean; $1.25 line
East Asia and Pacific
53.4
44.3
33.3
17.9
Eastern Europe and Central Asia
4.0
4.5
7.5
1.4
Latin America and the Caribbean
13.2
14.4
13.7
8.6
Middle East and North Africa
10.3
7.2
6.8
3.5
South Asia
59.7
51.3
37.8
17.6
Sub-Saharan Africa
49.3
54.0
52.2
41.5
Unweighted mean; $2 line
East Asia and Pacific
70.4
65.8
56.2
38.4
Eastern Europe and Central Asia
10.1
10.2
17.0
4.8
Latin America and the Caribbean
24.3
25.9
23.8
15.6
Middle East and North Africa
28.6
24.0
23.5
13.5
South Asia
80.3
74.7
63.9
43.5
Sub-Saharan Africa
67.1
71.5
71.0
62.2
Population-weighted mean; $1.25 line
East Asia and Pacific
77.2
56.2
35.6
12.5
Eastern Europe and Central Asia
1.9
1.9
3.8
0.7
Latin America and the Caribbean
11.9
12.3
11.9
5.5
Middle East and North Africa
9.6
5.7
5.0
2.4
South Asia
61.1
53.8
45.1
31.0
Sub-Saharan Africa
51.4
56.5
57.9
48.5
Population-weighted mean; $2 line
East Asia and Pacific
92.4
81.0
61.7
29.7
Eastern Europe and Central Asia
8.3
6.9
12.1
2.3
Latin America and the Caribbean
23.7
22.5
22.0
10.4
Middle East and North Africa
30.0
23.4
21.9
12.0
South Asia
87.2
83.6
77.8
66.7
Sub-Saharan Africa
72.2
75.9
77.5
69.9 The contrast between Asia and the rest of the developing world in terms of poverty
reduction is even more dramatic when the calculations are carried out anchoring the
mean of the income distribution to GDP from National Accounts. Figure 6.3 shows
regional estimates taken from Pinkovskiy and Sala-i-Martin (2009), where the sharp
declines in poverty in East and South Asia stand out. According to these estimates
there would be poverty convergence across the regions of the developing world, with
the exception of Sub-Saharan Africa. 74 74 Alvaredo-Gasparini Figure 6.3
Poverty headcount ratio
Developing countries, 1970-2006
Source: Pinkovskiy and Sala-i-Martin (2009). Note: poverty line $2 a day. 6.4. Regional trends 0
10
20
30
40
50
60
70
80
90
1970
1972
1974
1976
1978
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
East Asia
South Asia
Sub-Saharan Africa
Latin America
Middle East and North Africa Figure 6.3
Poverty headcount ratio
Developing countries, 1970-2006 Figure 6.3
Poverty headcount ratio
Developing countries, 1970-2006 Figure 6.3
Poverty headcount ratio
Developing countries, 1970-2006 0
1970
1972
1974
1976
1978
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004 Source: Pinkovskiy and Sala-i-Martin (2009). Source: Pinkovskiy and Sala-i-Martin (2009). Note: poverty line $2 a day. Regions have been different in terms of the growth-inequality-poverty triangle (Table
6.5). The growth effect was strong in Asia dwarfing a much smaller (and sometimes
poverty-increasing) redistribution effect. The increase in poverty in ECA in the 1990s is
associated with both negative growth and inequality rise, while the fall in poverty in
the following decade is mostly accounted by neutral positive growth. In Latin America
growth contributed in both decades, but only in the 2000s did the redistribution effect
become poverty-reducing. Finally, in Africa (MENA and SSA) the growth effect in the
2000s was the major contributing factor to the fall in poverty. Table 6.5
Decomposition of poverty changes
Developing countries
Source: own calculations based on data from PovcalNet. Note: the columns show unweighted means across a sample of 76 developing countries of the change in
the poverty headcount ratio ($2 line), and the growth and redistribution effects from the poverty
change decomposition. Actual
Actual
change
Growth
Redistribution
change
Growth
Redistribution
EAP
MENA
1990-1999
-10.5
-10.7
0.2
1990-1999
-1.5
-0.6
-1.0
1999-2010
-19.4
-19.2
-0.2
1999-2010
-10.1
-8.0
-2.1
1990-2010
-30.0
-30.3
0.3
1990-2010
-11.6
-8.4
-3.3
ECA
SA
1990-1999
8.6
6.1
2.5
1990-1999
-9.0
-10.9
2.0
1999-2010
-11.5
-10.5
-1.0
1999-2010
-13.2
-12.0
-1.1
1990-2010
-2.9
-3.9
1.0
1990-2010
-22.1
-23.3
1.2
LAC
SSA
1990-1999
-3.2
-5.3
2.1
1990-1999
-1.8
-1.4
-0.4
1999-2010
-7.9
-5.3
-2.6
1999-2010
-5.9
-6.0
0.0
1990-2010
-11.1
-10.2
-0.9
1990-2010
-7.7
-7.8
0.0
Effects
Effects Table 6.5
Decomposition of poverty changes
Developing countries Note: the columns show unweighted means across a sample of 76 developing countries of the change in
the poverty headcount ratio ($2 line), and the growth and redistribution effects from the poverty
change decomposition. 97 See Ahuja et al. (1997) and Nissanke and Thorbecke (2010) as examples of a vast literature.
98 p
p
g
$ /
y
p
y
99 During the early and mid-1990s poverty declined substantially, but then in the late 1990s to the early
2000s the downward trend stalled. Li et al. (2013) argue that further reductions in poverty became more
challenging due to several factors, for example, the fact that a high proportion of the remaining poverty
was geographically dispersed and transient, and also because poverty had become less responsive to
macroeconomic growth (World Bank, 2009). Policies adopted after 2002, such as the minimum living
guarantee program, the new rural cooperative medical system, and the new rural pension system, have
addressed some of these factors. 6.4. Regional trends 75 75 Alvaredo-Gasparini In the rest of this section we take a closer look at the changes in poverty reported by
the literature in each geographic region of the developing world. In the rest of this section we take a closer look at the changes in poverty reported by
the literature in each geographic region of the developing world. 98 According to World Bank (2012), poverty in East Asia and Pacific “will continue to decline, and th
share of people living on $2/day will reach 24.5 per cent by the end of 2013.” 97 See Ahuja et al. (1997) and Nissanke and Thorbecke (2010) as examples of a vast literature.
98 According to World Bank (2012), poverty in East Asia and Pacific “will continue to decline, and the
share of people living on $2/day will reach 24.5 per cent by the end of 2013.”
99 During the early and mid-1990s poverty declined substantially, but then in the late 1990s to the early
2000s the downward trend stalled. Li et al. (2013) argue that further reductions in poverty became more
challenging due to several factors, for example, the fact that a high proportion of the remaining poverty
was geographically dispersed and transient, and also because poverty had become less responsive to
macroeconomic growth (World Bank, 2009). Policies adopted after 2002, such as the minimum living
guarantee program, the new rural cooperative medical system, and the new rural pension system, have
addressed some of these factors. East Asia and Pacific As mentioned above, East Asia and Pacific has achieved an impressive record in terms
of poverty reduction. The fall in the indicators of material deprivation has been strong,
sustained over the two last decades and widespread across countries.97 The poverty
headcount ratio with the $2 line fell from 92.4% in 1981 to 81% in 1990, 61.7% in 1999
and 29.7% in 2010.98 China’s progress against absolute poverty was a key factor behind this dynamics
(World Bank, 2009; Ravallion and Chen, 2007; Minoiu and Reddy, 2008). Rural areas
accounted for the bulk of the gains to the poor, although migration to urban areas
helped; rural economic growth was far more important to national poverty reduction
than urban economic growth; agriculture played a far more important role than the
secondary or tertiary sources of GDP, mainly from the efficiency gains after the
decollectivization of agriculture. Ravallion and Chen (2007) claim that, though they
cannot offer a rigorous test against alternative explanations, “the halving of the
national poverty rate in the first few years of the 1980s was largely attributable to
picking these blow-lying fruits of agrarian reform”. Provinces starting with relatively
high inequality saw slower progress against poverty, due both to lower growth and a
lower growth elasticity of poverty reduction. While in 1990 the incidence of poverty in
China was roughly 25 points higher than in the rest of the developing world, by the end
of the 2000s, it had fallen more than 10 points below the average.99 Between 1990 and 2010 the headcount ratio fell from 60% to 12% in China ($1.25
line), and from 54% to 18% in Indonesia, the two most populated countries in the
region. The rest of the economies experienced similar patterns. The headcount ratio
dropped from 58% to 15% in Cambodia, from 12% to 0.4% in Thailand, and from 73%
to 14% in Vietnam. The reductions were less spectacular, but anyway significant in the Pacific countries
(Micronesia, Philippines, Papua New Guinea, Timor-Leste). According to Asian
Development Bank (2012) using the national poverty lines, poverty declined in all
economies except Mongolia, Micronesia, Samoa, Timor-Leste, Tonga and Tuvalu. With 76 Alvaredo-Gasparini Alvaredo-Gasparini respect to the poverty reduction target of the Millennium Development Goals, of the
ten economies for which data are available, 6 have achieved the poverty target, and
Cambodia is very close to reaching it by 2015. 100 For an analysis of the growth elasticities of poverty reduction, see World Bank (2005), chapter 2.
101 In the Caribbean poverty is estimated based on consumption expenditures, but surveys are still
scarce, so poverty changes are difficult to monitor. Eastern Europe and Central Asia The evidence clearly suggests that poverty in Eastern Europe and Central Asia
increased during the 1990s and decreased during the 2000s. The collapse of output in
many of these countries following the dismantling of the Soviet Union, along with
hyperinflation that wiped out savings, resulted in a dramatic drop in living standards
for the majority of people and the emergence of poverty as a major issue. However,
according to Simai (2006) poverty in the region was not a new phenomenon: most of
the countries began their transformation with extensive hidden unemployment and at
least one-tenth of its population below the subsistence level. The transition also
involved setbacks in non-monetary dimensions of wellbeing. Past achievements in
social welfare came under pressure, the most striking being the reduction in life
expectancy: in 1995, life expectancy for males in the Russian Federation was just 58
years, 10 years less than that of men in China (UN, 2004). Table 6.4 reminds the reader that poverty rates in these countries have always been
much lower than in the other developing regions, irrespective of the line considered
and of the weighted or unweighted averages, and despite the fact that low-income CIS
(Georgia, Uzbekistan, Moldova, Armenia, Kygryz Republic, Tajikistan) display figures
well above the regional mean. When growth resumed after the Russian crisis in 1998,
poverty started to fall.100 It was not until 2004 that the region as a whole returned to
the level of GDP recorded in 1990 (World Bank, 2005). Much of the poverty reduction
initially occurred in the populous middle-income countries (Kazakhstan, Russian
Federation, Ukraine), but eventually it spread almost everywhere. Scholars have
identified four main explanations for the poverty dynamics: (i) the positive growth in
output and wages; (ii) the decline in inequality, with incomes of poorer households
increasing faster than average in some countries (CIS) but not in others (Poland,
Romania); (iii) the increasing role of public transfers, with benefits improving in
coverage and adequacy; and (iv) private remittances, which in many cases far
exceeded public funding. East Asia and Pacific Lao PDR, Philippines, and Timor-Leste
are progressing more slowly toward that goal. Latin America and the Caribbean Poverty estimates in Latin America, available since the 1970s, were always mostly
based on income, not consumption.101 Despite the fact that statistics were initially 77 Alvaredo-Gasparini weak, there is agreement among researchers that during the 1970s economic growth
pushed poverty down in the region (Altimir, 1979, 1996). In contrast, the “lost decade"
of the 1980s was characterized by recurrent crises and output stagnation, that brought
about a weak social performance. Latin American economic growth resumed in the
1990s, but in the context of growing inequalities, a combination that resulted in a
modest decline in poverty (Londoño and Székely, 2000). The exceptional economic
conditions in the 2000s and the consolidation of more ambitious social policies implied
a sharp drop in poverty indicators in that decade. Gasparini et al. (2013) estimate that
the income poverty headcount ratio ($2.5 line) fell from 36% in the early 1970s to
27.3% in 1980, slowly increased to reach 27.8% in 1992, mildly decreased to 24.9% in
2003, and was reduced dramatically to 16.3% by 2009.102 The number of poor people
in the region is estimated to have fallen from 119 million in 1992 to 89 million in 2009
(Gasparini et al., 2013). Other indicators of income poverty and of various types of
material deprivation are consistent with these results. Data from SEDLAC confirms that
the reduction in poverty continued in the period 2009-2013, despite a deceleration in
the GDP growth rates. The improvements in social indicators in the 2000s have been
linked to at least two factors: on the one hand, most of the region’s economies
experienced robust growth together with upswings in employment and labor income;
on the other, all countries boosted social spending and put ambitious social protection
systems into place or expanded the scope of their existing systems (López Calva and
Lustig, 2010; Cruces and Gasparini, 2012). The performance in terms of poverty reduction has been heterogeneous across the
regions in Latin America. While Central America experienced a slow decline in income
deprivation over the last two decades, in the rest of the regions the rapid poverty fall
of the 2000s contrasts with the sluggish or even negative performance of the 1990s. SEDLAC data indicate that using the international poverty line of $2.5, the average
(unweighted) poverty rate in Southern South America increased from 17.7% in 1992 to
18.5% in 2003, and then dropped to 9.1% by 2010. 102 Estimates are based on SEDLAC statistics, Londoño and Székely (2000), Wodon et al. (2001) and
official poverty estimates from all countries in Latin America. Latin America and the Caribbean In the same period the average
poverty rate in the Andean region first rose from 30.2% to 33%, and then declined to
17.5%. In contrast, poverty in Central America fell more uniformly over the two
decades: 33.3% in 1992, 28.6% in 2003 and 23.1% in 2010. More than half of the population in Latin America lives in Brazil or Mexico. After a
decline in the early 1990s, income poverty in Brazil remained stable for about a
decade: the poverty incidence rate with the $2.5 line was 27.8% in 1995 and 27.1% in
2003. After that plateau, the country experienced a fast and sustained reduction in
income poverty, reaching 13.9% in 2010. The Mexican economy was hit by a serious
crisis in the mid-1990s (the so-called Tequila crisis) that raised income poverty from
17.8% in 1992 to 43.4% in 1995 (SEDLAC data, $2.5 line). From that peak, income
poverty initiated a consistent decline to reach a value of 12% in 2006, with no gains in
the second half of the 2000s. 78 78 Alvaredo-Gasparini Middle East and North Africa As discussed in section 4 it is useful to divide the last four decades in MENA into three
periods. The first one, spanning until the mid-1980s, was characterized by strong
economic growth: the average per capita income growth for the region between 1975
and 1984 was over 4.5%. Adams and Page (2003) argue that given MENA´s relatively
equal income distribution, this economic growth had a powerful impact on reducing
poverty in the region. However, assessing that progress is difficult since pre-1985
household surveys are only available for Tunisia and Egypt. Iqbal (2006) reports that in
Tunisia poverty fell from 51% in 1965 to 16% in 1985, while Egypt’s achievement was
also impressive, with poverty declining from 82% to 53% between 1975 and 1985. Page (2007) estimates a fall in the incidence of poverty ($2 line) in the region from 57%
in the late 1970s to 22% in the early 1990s. The second period, covering the late 1980s and most of the 1990s, was characterized
by a sluggish economic performance, in part due to low prices for hydrocarbons,
declining remittances and aid flows, as well as a low payoff to the reforms that were
implemented. On average for the developing MENA countries, real per capita incomes
went up by less than 1.5% per year. The proportion of those living with less than $2
per day stayed roughly unchanged around 22% for a decade (PovcalNet). Iqbal (2006)
reports that “by 2001, approximately 52 million people were poor, an increase in
absolute numbers of approximately 11.5 million people, compared with the situation
in 1987”. The 1990s were a lost decade for economic growth and poverty reduction in
the developing economies of the MENA region (Page, 2007). The downward pattern in poverty appeared to have resumed in the 2000s. The
incidence of poverty ($2 line) fell from 22% in 1999 to 12% in 2010. According to
PovcalNet data the number of people living with less than $2 a day increased by 7
million in the 1990s, and then was reduced by 20 million in the 2000s (from 60 to 40
million). While in the 1990s poverty went down in a third of the MENA countries, in
the 2000s it went down in all economies, with the possible exception of Yemen. South Asia South Asia achieved impressive economic growth in the past fifteen years. Since 1996
until the recent global crisis, GDP growth in the region exceeded 5 percent per year. As
a consequence, poverty rates were considerably reduced. In Bangladesh, India and
Nepal, absolute poverty (headcount ratio, $1.25 line) fell by 18, 15 and 43 percentage
points, respectively, between 1996 and 2010. In Pakistan, it fell by 22 percentage
points between 2002 and 2010 (yet, one in 10 children still dies before his fifth
birthday and only 57 percent of children complete primary school). Devarajan and Nabi
(2006) optimistically expected the region to have single-digit poverty rates in 2015 if
growth rates accelerated to 10 percent per year. This has indeed happened already in
Sri Lanka (where poverty dropped from 17% in 1996 to 4% in 2010) as well as in 79 Alvaredo-Gasparini Maldives (where, according to the 2006 census, about 1 per cent of the population was
living on less than $1 a day). Maldives (where, according to the 2006 census, about 1 per cent of the population was
living on less than $1 a day). Growth has been instrumental in reducing poverty rates, but, as Ghani (2010) pointed
out from a gloomier perspective, poverty rates were not falling fast enough to reduce
the total number of poor people. Those living on less than $1.25 a day increased from
549 million in 1981 to 595 million in 2005. In India, where around three-quarters of
these poor live, the number increased from 420 million in 1981 to 455 million in 2005
(Ravallion et al. 2009). The situation seems to have slightly improved according to the
most recent observations, but the question remains: why has India not performed as
well as China in this dimension? One explanation may be related to the already
mentioned Indian paradox: survey-based households’ income growth has been much
lower than National Accounts-based GDP growth. 103 According to the $4 line, more than 90 per cent of the SSA population is actually considered poor. Sub-Saharan Africa The economic and social performance of Sub-Saharan African countries has been
frustrating. Five decades after most nations gained independence, poverty is still at
very high levels in SSA, in fact the highest in the world. Fortunately, it seems that
finally, after years of impoverished economic performance, the last decade shows
some signs of improvement. Based on figures from PovcalNet, the incidence of poverty
increased between 1981 and 1999 from 51% to 58% for the $1.25 per day line, and
from 72% to 77% for the $2 line (the unweighted statistics are not very different).103
Because of the increase in population the number of people living with less than $1.25
a day almost doubled in Sub-Saharan Africa during those years, from 205 million to 377
million. These results are even more disappointing when compared to the rest of the
developing world. The share of the world’s poor living in Sub-Saharan Africa rose from
11% in 1981 to 22% in 1999. Artadi and Sala-i-Martin (2003) find that the SSA poverty
rate was stable in the 1970s, around a value of 49%, and soared in the 1980s and early
1990s reaching 60% in 1995, following the deterioration of the aggregate per capita
income of Sub-Saharan economies. They report a nearly 50% reduction in the
worldwide poverty rate between 1980 and 2000, that contrast with a 27% increase in
SSA. Following the dismal performance of the 1980s and early 1990s, SSA witnesses some
economic and social progress. Poverty declined considerably from their heights of the
mid-1990s; in fact the reduction in extreme poverty from that date to the late 2000s
was similar than in the rest of the developing world, excluding China. Chen and
Ravallion (2012) stressed the fact that for the first time since 1981 the share of people
in Sub-Saharan Africa living below $1.25 a day was less than 50%. Changes in poverty
have been closely linked to economic growth. According to Fosu (2010), poverty has
declined in SSA by about 0.5 percentage points per year since the mid-1990s, quite 80 Alvaredo-Gasparini comparable with South Asia’s record. Sala-i-Martin and Pinkovskiy (2010) estimate that
African poverty has been falling steadily since 1995. According to these authors the
African poverty rate in 2006 was 31.8%, 30 per cent lower than in 1995, and 28 per
cent lower than in 1990. Sub-Saharan Africa The decline in poverty in the 2000s has been quite extended:
as reported above, 2/3 of the SSA countries in PovcalNet have experienced reductions
larger than 5%. Sala-i-Martin and Pinkovskiy (2010) find that “poverty fell for both
landlocked as well as coastal countries; for mineral-rich as well as mineral-poor
countries; for countries with favorable or with unfavorable agriculture; for countries
regardless of colonial origin; and for countries with below or above median slave
exports per capita during the African slave trade. Hence, the substantial decline in
poverty is not driven by any particular country or set of countries”. However, Fosu
(2009) highlights the considerable heterogeneity across country experiences. For
example, while high economic growth in Botswana has been transformed to only a
minimal decline in poverty, Ghana has succeeded in translating its relatively modest
growth to considerable poverty reduction. Fosu (2009) attributes this contrast to the
difference in the levels of income inequality between the two countries. Despite the encouraging signs of progress in the fight against poverty in Sub-Saharan
Africa, Chen and Ravallion (2012) alert that the lags in survey data availability and
problems of comparability and coverage, rise up concerns about how robust this
positive trend will prove to be. 104 The analysis is restricted to countries with initial poverty above 5%. 105 The first panel in Figure 6.4 suggests an increase in the dispersion of absolute changes at higher
values of the initial poverty level. A quantile regression analysis confirms that the slope of the regression
line is negative and significant for the lower quantiles (the high-performers in terms of poverty
reduction), and statistically non-significant for the higher quantiles. The systematic difference in the
slopes across quantiles suggests that poverty convergence depends on factors beyond the initial poverty
level. *=significant at 10%, **=significant at 5%, ***=significant at 1%. Note: standard errors in parenthesis. Note: standard errors in parenthesis. Source: own calculations based on PovcalNet (2013). 6.5. Poverty convergence Given the heterogeneity among economies in terms of social improvement, a natural
question is whether countries starting out with a high incidence of material
deprivation tend to be the ones with higher rates of poverty reduction. Figure 6.4
suggests some signs of poverty convergence in absolute terms but not in
proportionate terms.104 The 1981 poverty level ($2 line) is negatively correlated with
the annualized change over the period 1981-2010, but not with the proportionate
change (annualized log difference). In the first panel the regression line has a slope of
-0.009 with a t-ratio of -4.51, based on a robust (White) standard error. This result
means that on average the absolute fall in poverty has been larger in countries with
higher poverty incidence, but since the difference across countries is small, there is
absence of poverty convergence in proportionate terms. 81 81 Alvaredo-Gasparini Figure 6.4
Poverty convergence among developing countries
1981-2010
Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
$2-a-day per person line. Absolute changes
Proportionate changes
-.1
-.05
0
.05
annual growth rate in headcount ratio 1981-2010
0
20
40
60
80
100
headcount ratio 1981
-3
-2
-1
0
1
annual change in headcount ratio 1981-2010
0
20
40
60
80
100
headcount ratio 1981 Poverty convergence among developing co
1981-2010
Absolute changes
-3
-2
-1
0
1
annual change in headcount ratio 1981-2010
0
20
40
60
80
100
headcount ratio 1981 Proportionate changes
-.1
-.05
0
.05
annual growth rate in headcount ratio 1981-2010
0
20
40
60
80
100
headcount ratio 1981 Source: own calculations based on PovcalNet (2013). (
)
Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted
$2-a-day per person line. (
)
Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjuste
$2-a-day per person line. The results for other poverty indices, lines and time periods are similar (Table 6.6). The
coefficients for the absolute change in poverty are negative but small, while the
coefficients for the proportionate change are in general positive, but in most cases
non-significant.105 Source: own calculations based on PovcalNet (2013). 106 See Chen and Ravallion (1997), World Bank (2000), Ravallion (2001, 2007, 2012), Fields (2002),
Bourguignon (2003), Besley and Burguess (2003), Kraay (2006), Fosu (2011) as examples of a large
literature. Table 6.6
Poverty convergence among developing countries
Coefficients of initial poverty on a poverty change regression Table 6.6
Poverty convergence among developing countries
Coefficients of initial poverty on a poverty change regression Source: own calculations based on PovcalNet (2013)
Index
Poverty line
Absolute
changes
Proportionate
changes
Absolute
changes
Proportionate
changes
Absolute
changes
Proportionate
changes
Headcount
$ 1.25
-0.017
0.001
-0.018
0.010
-0.016
0.023
(0.002)***
(0.004)
(0.003)***
(0.004)*
(0.004)***
(0.006)***
Headcount
$ 2
-0.009
0.006
-0.008
0.016
-0.008
0.028
(0.002)***
(0.004)
(0.002)***
(0.004)**
(0.004)**
(0.007)***
Poverty gap
$ 1.25
-0.021
-0.007
-0.026
-0.001
-0.024
0.014
(0.002)***
(0.004)
(0.003)***
(0.005)
(0.005)***
(0.007)**
Poverty gap
$ 2
-0.016
0.001
-0.018
0.010
-0.016
0.024
(0.002)***
(0.004)
(0.003)***
(0.005)*
(0.004)***
(0.008)***
Squared poverty gap
$ 1.25
-0.024
-0.013
-0.032
-0.008
-0.030
0.006
(0.002)***
(0.004)**
(0.004)***
(0.006)
(0.007)***
(0.006)
Squared poverty gap
$ 2
-0.020
-0.005
-0.024
0.002
-0.022
0.014
(0.002)***
(0.004)
(0.003)***
(0.004)
(0.005)***
(0.005)***
1990-2010
1981-2010
1999-2010 *=significant at 10%, **=significant at 5%, ***=significant at 1%. 82 Alvaredo-Gasparini Ravallion (2012) argues that the lack of poverty convergence (in proportionate terms),
despite evidence for mean convergence and for the poverty-reduction impact of
growth, suggests that the dynamic processes for growth and poverty reduction depend
directly on the initial level of poverty. He shows evidence on two adverse effects of
being a country with high poverty levels: first, these countries tend to grow at a slower
pace, controlling for the initial mean, and second a high poverty rate implies lower
“productivity” of economic growth in terms of poverty reduction (lower poverty-
growth elasticity). 6.6. Poverty and growth The dynamics of poverty are closely related to income growth. The economic literature
has gathered abundant evidence supporting the fact that absolute poverty tends to fall
with economic growth.106 Moreover, the longer the growth spells under consideration,
the larger the share of the variance in poverty that is accounted for by the growth
component (Ferreira, 2010). Figure 6.5 illustrates this relationship by showing poverty
along with two alternatives measures of mean income: per capita gross national
income (GNI) constructed from National Accounts, and per capita consumption
obtained from household surveys. The figure shows the unweighted mean of these
variables among developing countries in the period 1981-2010. On average, the
economic performance of the developing countries was weak in the 1980s and early
1990s, hindering the perspectives of poverty reduction. Since the mid-1990s economic
growth resumed in most countries in the developing world, a process that accelerated
in the 2000s, leading to a sustained decrease in all measures of income poverty. At the
end of the decade that pattern was slowed down, but not halted, by the poor
economic performance of several developing countries associated to the international
crisis. 83 83 Alvaredo-Gasparini Figure 6.5
Per capita GNI, per capita consumption and poverty headcount ratio
Unweighted mean, developing countries, 1981-2010 Figure 6.5
Per capita GNI, per capita consumption and poverty headcount ratio
Unweighted mean, developing countries, 1981-2010 Figure 6.5
Per capita GNI, per capita consumption and poverty headcount ratio
Unweighted mean, developing countries, 1981-2010
Source: own calculations based on PovcalNet (2013). Note: per capita gross national income in constant 2005 PPP dollars, per capita consumption from
household surveys in constant 2005 dollars. Unweighted averages across developing countries, series
displayed with mean=100. Headcount poverty ratio $2-a-day line, unweighted averages across
developing countries. 35
37
39
41
43
45
47
49
51
53
55
80
90
100
110
120
130
140
1981
1984
1987
1990
1993
1996
1999
2002
2005
2008
2010
Headcount ratio
GNI and consumption
GNI pc
Consumption pc
Poverty headcount ($2 line) Per capita GNI, per capita consumption and poverty headcount ratio
Unweighted mean, developing countries, 1981-2010
35
37
39
41
43
45
47
49
51
53
55
80
90
100
110
120
130
140
1981
1984
1987
1990
1993
1996
1999
2002
2005
2008
2010
Headcount ratio
GNI and consumption
GNI pc
Consumption pc
Poverty headcount ($2 line) Source: own calculations based on PovcalNet (2013). 6.6. Poverty and growth Source: own calculations based on PovcalNet (2013). Note: per capita gross national income in constant 2005 PPP dollars, per capita consumption from
household surveys in constant 2005 dollars. Unweighted averages across developing countries, series
displayed with mean=100. Headcount poverty ratio $2-a-day line, unweighted averages across
developing countries. Figure 6.6 is another illustration of the relationship between poverty reduction and
economic growth. The figure shows that the change in poverty is closely negatively
related to economic growth, either in per capita consumption expenditures drawn
from household surveys or in GNI from NAS. Notice that in both panels the fitted line
approximately crosses the (0,0) point. Figure 6.6 is another illustration of the relationship between poverty reduction and
economic growth. The figure shows that the change in poverty is closely negatively
related to economic growth, either in per capita consumption expenditures drawn
from household surveys or in GNI from NAS. Notice that in both panels the fitted line
approximately crosses the (0,0) point. Figure 6.6 Figure 6.6
Growth and poverty reduction in the developing countries, 1981-2010
Source: own calculations based on PovcalNet (2013). Note: the horizontal axis shows the annual growth rate between 1981 and 2010 in per capita
consumption from household surveys in constant 2005 dollars (panel 1) and in per capita gross national
income in constant 2005 PPP dollars (panel 2); the vertical axis shows the annual growth change in the
poverty headcount ratio ($1.25 line) in the same period. The size of each circle is proportional to the
country population in 2010. 1. Poverty reduction and growth in per capita consumption
2. Poverty reduction and growth in per capita GNI
-.2
-.1
0
.1
annual growth rate in poverty headcount ratio
-.05
0
.05
.1
per capita GNI annual growth rate
-.2
-.1
0
.1
annual growth rate in poverty headcount ratio
-.05
0
.05
.1
per capita consumption annual growth rate Growth and poverty reduction in the developing countries, 1981-2010
1. Poverty reduction and growth in per capita consumption
2. Poverty reduction and growth in per capita GNI 1. Poverty reduction and growth in per capita consumption 2. Poverty reduction and growth in per capita GNI 1. Poverty reduction and growth in per capita consumption
-.2
-.1
0
.1
annual growth rate in poverty headcount ratio
-.05
0
.05
.1
per capita consumption annual growth rate Source: own calculations based on PovcalNet (2013). Note: the horizontal axis shows the annual growth rate between 1981 and 2010 in per capita
consumption from household surveys in constant 2005 dollars (panel 1) and in per capita gross national
income in constant 2005 PPP dollars (panel 2); the vertical axis shows the annual growth change in the
poverty headcount ratio ($1.25 line) in the same period. The size of each circle is proportional to the
country population in 2010. 84 84 Alvaredo-Gasparini Since the positive correlation between poverty reduction and growth is a well-
established result, research in this area is mainly focused on estimating the magnitude
of the corresponding elasticity, an issue that may have relevant policy implications. “If
(the elasticity) is reasonably high, then poverty reduction strategies almost exclusively
relying on economic growth are probably justified. If it is low, however, ambitious
poverty reduction strategies might have to combine both economic growth and some
redistribution” (Bourguignon, 2003). 107 The sample includes 725 spells in 76 countries for which consistent information for the whole period
is available. Results do not significantly vary when we restrict the analysis to more recent periods which
allows a larger sample of countries. Figure 6.6 The growth elasticity of poverty reduction is
typically estimated by regressing the annualized proportional change in a poverty
indicator against the annualized growth rate of mean income (per capita income or
consumption from surveys, or per capita GDP, GNI, or aggregate private consumption
from NAS) in a sample of growth spells. In a regression without controls the resulting
coefficient is the total elasticity, as opposed to a partial elasticity that can be obtained
by holding inequality and other factors constant. Based on a dataset for 67 countries over the period from 1981 to 1994, and using the
$1 line, Ravallion and Chen (1997) find a central estimate for the poverty-growth
elasticity of -3.1. Values estimated by other authors are somewhat lower (in absolute
terms): around -2 in World Bank (2000), -1.6 in Bourguignon (2003), -2.6 in Adams
(2004), and -1.8 in Ferreira and Ravallion (2009). More recently, Ravallion (2012)
reports an elasticity of −1.4 for the $1.25 line. The elasticity is lower when using the
growth rate of consumption per capita from NAS (−0.8), and also lower when using a
higher poverty line. The confidence intervals around the regression coefficient are
typically wide. Ravallion (2007) reports that the 95% confidence interval implies that
for a poverty level of 40%, an annual growth rate of 2% is consistent with poverty
reductions ranging from 1% to 7%. Estimates of the total growth elasticity of poverty reduction over the period 1981-2010
obtained with the latest version of PovcalNet data are shown in Table 6.7.107 All the
elasticities are negative and significant at 1%. The growth elasticity of poverty
reduction, as measured by the proportion of individuals below $1.25 a day, is around -
1.5. The results in Table 6.7 confirm that elasticities are lower in absolute value when
considering a higher poverty line. The result of lower elasticities using GNI from
National Accounts holds but only until the 2000s, suggesting a change in the relative
trends between consumption reported in household surveys and output estimated in
NAS. In general, the absolute value of the elasticities estimated with both sources have
increased in the 2000s compared to previous decades, suggesting that growth was
better translated to the poor in that period of falling inequality. For instance, while the
elasticity, using the $1.25 line and consumption per capita, was -1.53 for the period
1981-2010, it amounted to -1.83 when restricting the analysis to the 2000s. Figure 6.6 85 Alvaredo-Gasparini Table 6.7
Total poverty-growth elasticities
Developing countries
Source: own calculations based on PovcalNet (2013). Note: all elasticities are significant at 1% level. 1981 - 2010
1990-2010
1999-2010
1981 - 2010
1990-2010
1999-2010
1.25
-1.53
-1.56
-1.83
-1.46
-1.51
-2.16
2
-1.39
-1.41
-1.68
-1.17
-1.19
-1.91
4
-1.30
-1.38
-1.10
-0.78
-0.81
-1.21
1.25
-1.75
-1.77
-1.87
-1.44
-1.47
-2.09
2
-1.50
-1.47
-1.85
-1.35
-1.30
-1.83
4
-1.41
-1.47
-1.39
-0.95
-0.98
-1.54
1.25
-2.18
-2.25
-1.87
-1.50
-1.52
-1.87
2
-1.60
-1.52
-1.86
-1.44
-1.29
-1.91
4
-1.40
-1.43
-1.54
-1.04
-1.04
-1.71
Consumption per capita
GNI per capita
Index
Poverty line
Headcount
Poverty gap
Squared PG Table 6.7
Total poverty-growth elasticities
Developing countries So far, we have reported total elasticities, which can be seen as summary measures of
the co-movements of poverty and growth. The literature has tried to improve this
characterization by adding other variables into the analysis, typically measures of
inequality and development. The empirical evidence supports the intuition that higher
inequality tends to reduce the absolute value of the elasticity, by shifting the gains
from growth away from the poor (Ravallion, 1997, 2001; Kraay, 2006).108 For example,
based on a sample of 65 countries during 1981-2005, and using the $1 poverty line,
World Bank (2005) reports that the poverty-growth elasticity is highest among low-
inequality countries (with a value of approximately -4.0 for countries with Ginis in the
mid-20s) and lowest among high-inequality countries (close to -1.0 for countries with a
Gini coefficient around 60). The change in inequality is also found as a significant direct
determinant of the elasticity. For instance, Ravallion (2001) finds that the median rate
of reduction in the poverty headcount ratio ($1 line) among growing economies was
10% per year among countries with falling inequality, and 1% per year for those
countries with rising inequality. Using a sample of 114 growth spells from the mid-1980s to the mid-1990s in 26
developing countries Bourguignon (2003) finds an elasticity of -1.6 in the model
without controls and -2 when controlling for the change in the Gini. The latter
specification increases the R2 from 0.266 to 0.419, suggesting that the heterogeneity in
distributional changes is as much responsible for the variation in poverty changes
across growth spells as the heterogeneity in the speed of growth. 108 Although the intuition is compelling, the result is theoretically ambiguous. See Ravallion (2007) for a
proof, and Bourguignon (2003) for a case in which assuming log-normality yields an unambiguous result. Figure 6.6 Bourguignon (2003)
also finds positive and significant coefficients for the interaction terms between the
growth rate and both (i) the initial level of inequality, and (ii) the level of development
(proxied by the poverty line over mean income). He also reports negative coefficients
for the interaction terms between the change in the Gini and those two variables. The 86 Alvaredo-Gasparini first four columns in Table 6.8 in general confirm the results in Bourguignon (2003),
using a larger dataset spanning three decades.109 Table 6.8
Regressions for annualized proportional change in the poverty headcount ratio
Developing countries, 1981-2010 Table 6.8
Regressions for annualized proportional change in the poverty headcount ratio
Developing countries, 1981-2010
Source: own calculations based on PovcalNet (2013). Note: sample of 76 countries with information for the period 1981-2010. Poverty indicator=headcount
ratio, $2-a-day line. Growth rates computed from per capita consumption in household surveys. Unconditional
(1)
(2)
(3)
(4)
(5)
(6)
g = growth rate in per capita consumption
-1.391
-1.467
-2.879
-2.910
-2.111
-2.073
(0.147)***
(0.144)***
(0.570)***
(0.590)***
(0.206)*** (0.188)***
dGini = change in Gini coefficient
1.488
1.566
3.195
(0.265)***
(0.279)***
(1.206)***
g * initial Gini coeficient
0.022
0.023
(0.013)*
(0.014)*
g * (poverty line/mean consumption)
0.903
0.879
(0.094)***
(0.101)***
dGini* initial Gini coefficient
-0.019
(0.027)
dGini * (poverty line/mean consumption)
-1.666
(0.345)***
g * initial poverty rate
0.021
0.021
(0.003)*** (0.003)***
log initial poverty rate
-1.559
(0.927)*
Intercept
0.150
-0.008
0.187
0.064
-0.255
4.108
(0.225)
(0.203)
(0.154)
(0.208)
(0.122)**
(2.655)
Observations
725
695
695
695
725
725
R2
0.414
0.502
0.583
0.610
0.508
0.516
Conditional on
Inequality and development
Poverty Table 6.8
Regressions for annualized proportional change in the poverty headcount ratio
Developing countries, 1981-2010 Table 6.8
Regressions for annualized proportional change in the poverty headcount ratio
Developing countries, 1981-2010
Source: own calculations based on PovcalNet (2013). Note: sample of 76 countries with information for the period 1981-2010. Poverty indicator=headcount
ratio, $2-a-day line. Growth rates computed from per capita consumption in household surveys. 109 Instead, unlike Bourguignon (2003) we fail to find a coefficient close to -1 for the interaction between
the growth rate and the theoretical value of the poverty-growth elasticity under the lognormal
assumption. 6.7. Relative poverty So far we have presented evidence on absolute poverty, an option that requires
keeping the real value of the poverty line fixed over time. The alternative is relative
poverty, where the line is adjusted to reflect changes in a general measure of
economic well-being. Although the measurement of relative poverty has not been
common in the developing world, sustained economic growth have triggered the
update of absolute poverty lines in some countries (e.g. China and India), and foster
discussions on relative poverty. The measurement of relative, instead of absolute,
poverty, can be justified on two grounds (Sen, 1983; Chen and Ravallion, 2008). First,
the very concept of poverty may depend on social norms that are likely different
across countries and over the development process. Second, even for a fixed norm, an
absolute line in the space of welfare may require a relative line in the space of
consumption if individual welfare also depends on relative consumption with respect
to the rest of the society where the person lives. The typical relative poverty line is set as a constant proportion of the mean (or
median) of the distribution, implying that poverty does not fall (increase) when all
incomes grow (fall) at the same rate. This result is considered problematic by many
authors who prefer weakly versions in which the cost of inclusion is not a constant
proportion of mean income. For instance, following Atkinson and Bourguignon (2001),
Ravallion and Chen (2012) propose a poverty line that is fixed up to a critical value of
the mean, where it rises with elasticity less than one. In particular, Ravallion and Chen
(2012) set the line for country i at time t as zit=max[$1.25, ($1.25+Mit)/2], where Mit is
the country and date specific mean. Figure 6.7 reveals an overall trend decline in the
incidence of weakly relative poverty in the developing world. The fall has been
relatively meager, so that the number of poor by this measure actually increased from
2.3 billion in 1981 to 2.7 billion in 2008. While the coefficient in an OLS regression
between the proportionate rate of poverty reduction (annualized differences in the
logs) and the rate of growth in the mean is -1.89 (standard error=0.23) for the absolute
measure of poverty, it becomes -0.38 (standard error=0.08) for the relative measure. Figure 6.6 Unconditional
(1)
(2)
(3)
(4)
(5)
(6)
g = growth rate in per capita consumption
-1.391
-1.467
-2.879
-2.910
-2.111
-2.073
(0.147)***
(0.144)***
(0.570)***
(0.590)***
(0.206)*** (0.188)***
dGini = change in Gini coefficient
1.488
1.566
3.195
(0.265)***
(0.279)***
(1.206)***
g * initial Gini coeficient
0.022
0.023
(0.013)*
(0.014)*
g * (poverty line/mean consumption)
0.903
0.879
(0.094)***
(0.101)***
dGini* initial Gini coefficient
-0.019
(0.027)
dGini * (poverty line/mean consumption)
-1.666
(0.345)***
g * initial poverty rate
0.021
0.021
(0.003)*** (0.003)***
log initial poverty rate
-1.559
(0.927)*
Intercept
0.150
-0.008
0.187
0.064
-0.255
4.108
(0.225)
(0.203)
(0.154)
(0.208)
(0.122)**
(2.655)
Observations
725
695
695
695
725
725
R2
0.414
0.502
0.583
0.610
0.508
0.516
Conditional on
Inequality and development
Poverty Regressions for annualized proportional change in the poverty headcount ratio
Developing countries, 1981-2010 (
)
Note: sample of 76 countries with information for the period 1981-2010. Poverty indicator=headcount
ratio, $2-a-day line. Growth rates computed from per capita consumption in household surveys. The results suggest the possibility of a double dividend from reducing inequality: first,
given a growth rate, lower inequality is associated directly with less poverty, and
second, less inequality means more poverty reaction to a given growth rate, that is an
acceleration of poverty reduction for a given rate of economic growth. Ravallion (2007)
illustrates the relevance of lower inequality for the perspectives of poverty reduction
assuming a country with a poverty incidence rate of 40% and a growth rate of 2% per
year: with an initial Gini coefficient of 30 it would take 11 years to reduce poverty by
half, while with a Gini of 60 it would take 35 years. In a more recent study Ravallion (2012) finds that the (absolute) growth elasticity of
poverty reduction tends to be lower in countries with a higher initial poverty rate (see
also the two last columns in Table 6.8). Ravallion (2012) finds a large attenuating effect
of a higher initial poverty rate on the elasticity: at an initial poverty rate of 10% the
elasticity is −2.2, while it falls to −0.5 at a poverty rate of 80%. The results are robust to 87 Alvaredo-Gasparini the inclusion of additional interaction effects with the initial Gini coefficient, the partial
elasticity of poverty reduction holding the Lorenz curve constant, the primary school
enrollment rate, life expectancy, the price of investment goods, and regional dummies. Figure 6.6 Unfortunately, while the characterizations of the growth elasticity of poverty reduction
discussed in this section are useful, the literature is still far from being able to clearly
identify the structural factors that are behind the differences in the elasticities, and
therefore is relatively silent about the specific policies that could foster a larger impact
of economic growth on poverty. 6.7. Relative poverty Only East Asia has experienced a marked reduction in the incidence of relative poverty:
from 81% in 1981 to 42% in 2008. In fact it is the only region in which the poverty 88 Alvaredo-Gasparini count was reduced according to this measure. Weakly relative poverty measures rose
or stayed unchanged in most regions in the 1990s, and slowly went down in the 2000s. Figure 6.7
Poverty headcount ratio with absolute and weakly relative poverty lines
Developing countries, 1981-2008
Source: Ravallion and Chen (2012). In Table 6.9 we extend the estimates of Ravallion and Chen (2012) to 2010, and add
estimates of strongly relative poverty (50 per cent of mean income) and anchored
poverty, computed by “anchoring” the relative lines (50 per cent of mean income) to
the values in 1990, updating them only by domestic price changes (see chapter 9 in
this volume). Our view of poverty trends widely varies across methodologies: while the
unweighted anchored poverty rate fell 32% between 1990 and 2010, the strongly
relative poverty rate went up 2%. The differences are even larger when considering
the population-weighted statistics. 0
10
20
30
40
50
60
70
80
1981
1984
1987
1990
1993
1996
1999
2002
2005
2008
Absolute ($ 2 per day)
Weakly relative Figure 6.7
Poverty headcount ratio with absolute and weakly relative poverty lines
Developing countries, 1981-2008
Source: Ravallion and Chen (2012). 0
10
20
30
40
50
60
70
80
1981
1984
1987
1990
1993
1996
1999
2002
2005
2008
Absolute ($ 2 per day)
Weakly relative Figure 6.7
Poverty headcount ratio with absolute and weakly relative poverty lines
Developing countries, 1981-2008 g
Poverty headcount ratio with absolute and weakly relative poverty lines
Developing countries, 1981-2008 Absolute ($ 2 per day)
Weakly relative Source: Ravallion and Chen (2012). In Table 6.9 we extend the estimates of Ravallion and Chen (2012) to 2010, and add
estimates of strongly relative poverty (50 per cent of mean income) and anchored
poverty, computed by “anchoring” the relative lines (50 per cent of mean income) to
the values in 1990, updating them only by domestic price changes (see chapter 9 in
this volume). Our view of poverty trends widely varies across methodologies: while the
unweighted anchored poverty rate fell 32% between 1990 and 2010, the strongly
relative poverty rate went up 2%. The differences are even larger when considering
the population-weighted statistics. Note: weakly relative poverty computed according to Ravallion and Chen (2012). Strongly relative lines
set at 50% of mean income. Anchored poverty computed by “anchoring” the relative lines (50% of mean
income) to the values in 1990, and updating them only by domestic price changes. 7. Concluding remarks There has been a remarkable improvement in the availability of information for
distributive analysis in the last decades due to increasing efforts by researchers,
national governments and international organizations. To be sure, the picture of
inequality and poverty in the developing world is substantially sharper now than in the
late 1990s, when the first volume of this Handbook was written. There remain,
however, enormous data limitations that make that picture only a very rough
approximation of reality. Household surveys are lacking in some countries and are
carried out very occasionally in others. Changes in methodology over time are
frequent; a fact that generally implies improvement in the data collection, but that at
the same time introduces comparability issues with previous surveys that are difficult
to deal with. Household surveys have endemic problems in capturing some income
and consumption items and in dealing with selective compliance and under-reporting
issues. The gaps with National Accounts aggregates, usually variable over time, are a
disturbing sign of measurement errors and conceptual issues. Comparability across
countries is another big problematic issue, as there are few efforts among national
agencies to standardize surveys or at least some criteria to gather and process
information. Another issue of concern is the difficulty in obtaining statistical
confidence intervals for the distributive statistics, either because agencies do not
report them, do not provide information on sampling issues, or do not release the
microdata. In sum, in order to be able to characterize and track distributive changes in
the developing world with more accuracy we need more efforts to extend the
coverage and frequency of household surveys, and improve their reliability and
comparability across countries. There is still a long way to go to get an accurate
assessment of poverty and inequality in the developing world. From the data available, some general facts emerge. High poverty and inequality are
pervasive characteristics of the developing world, but are not immutable features of
these economies. The evidence suggests a robust decline in the levels of income
absolute poverty in the developing world, driven mainly by East Asia in the 1980s and
1990s, but generalized to the rest of the developing countries in the 2000s. Income
poverty has been reduced in most countries and in the world as whole, making the
achievement of the first MDG possible. Despite these positive results there are reasons
for concern. 6.7. Relative poverty Table 6.9
Absolute and relative poverty
Developing countries, 1990-2010
Source: own calculations based on data from PovcalNet (sample of 76 countries). N
kl
l
i
d
di
R
lli
d Ch
(2012) S
l
l
i
li
absolute
($2)
weakly
relative (RC)
strongly
relative
anchored
absolute
($2)
weakly
relative (RC)
strongly
relative
anchored
1990
40.6
46.5
28.0
28.0
64.7
54.1
19.7
19.7
1993
39.8
47.9
29.0
32.9
62.7
53.7
21.1
19.1
1996
38.6
47.4
29.1
32.1
58.0
51.4
22.0
17.1
1999
38.3
47.5
30.1
31.9
56.7
50.2
22.9
16.9
2002
36.7
46.9
30.1
29.6
52.5
49.5
23.7
15.2
2005
32.3
45.0
29.5
24.8
45.6
46.5
23.7
12.3
2008
28.7
43.2
29.0
19.9
42.1
46.1
24.9
9.9
2010
27.7
42.4
28.6
18.9
39.4
45.6
26.6
9.5
weighted
unweighted Table 6.9
Absolute and relative poverty
Developing countries, 1990-2010 Note: weakly relative poverty computed according to Ravallion and Chen (2012). Strongly relative lines
set at 50% of mean income. Anchored poverty computed by “anchoring” the relative lines (50% of mean
income) to the values in 1990, and updating them only by domestic price changes. Note: weakly relative poverty computed according to Ravallion and Chen (2012). Strongly relative lines
set at 50% of mean income. Anchored poverty computed by “anchoring” the relative lines (50% of mean
income) to the values in 1990, and updating them only by domestic price changes. 89 Alvaredo-Gasparini Alvaredo-Gasparini 7. Concluding remarks The task of fighting poverty continues to be very challenging: around 1.2
billion people survive with less than $1.25 a day, an extremely low standard. Also,
most of the people who succeeded in jumping the $1.25 line in the last decades are
still poor by the standards of middle-income developing countries, and remain highly
vulnerable if economic conditions worsen. Moreover, due to economic growth,
concerns about relative poverty may be increasingly important, and the evidence in
that sense is less optimistic. The evidence on income relative inequality suggests that on average the developing
countries are somewhat more unequal than three decades ago. The patterns have
been different by period and region. Relative income inequality rose in the late 1980s 90 90 Alvaredo-Gasparini and in the 1990s. The changes were larger in Eastern Europe and Central Asia,
probably as a result of the transition from a central-planned to a more market-
oriented economy; in East Asia, likely as a consequence of the economic take-off; and
in Latin America probably as the result of recurrent macroeconomic crises and some
structural transformations. Distributive changes in the 2000s become more equalizing
in most of the developing world, but the changes were rather moderate and with
considerable heterogeneity across countries. In fact, in this decade of widespread
social improvement around a third of the countries did not experience falling
inequality levels. Reducing inequality certainly remains a top concern in the developing
countries of the world. 91 91 Alvaredo-Gasparini References Acar, S. and Dogruel, F. (2012). Sources of inequality in selected MENA countries. Structural Change and Economic Dynamics 23(3), pp. 276-285. Asian Development Bank (2012). Key Indicators for Asia and the Pacific. 43rd
edition. Adams R. Jr. and Page, J. (2003). Poverty, inequality and growth in selected Middle East
and North Africa countries, 1980–2000. World Development 31(12), pp. 2027-2048. Ahluwalia, M., Cartner, N. and Chenery, H. (1979). Growth and poverty in developing
countries. Journal of Development Economics 6(3), pp. 299-341. Ahuja, V., Bidani, B., Ferreira, F., and Walton, M. (1997). Everyone's miracle? Revisiting
poverty and inequality in East Asia. Directions in Development. The World Bank. Alatas, V. and Bourguignon, F., (2000). The evolution of the distribution of income
during Indonesian fast growth: 1980 –1996, Princeton University, Princeton
(unpublished). Alejo, J. (2012). Educación y Desigualdad: una metodología de descomposición basada
en dos interpretaciones de la ecuación de Mincer. Evidencia para Argentina. Anales de
la XLVII Reunión Anual de la Asociación Argentina de Economía Política, Trelew,
Argentina. Altimir, O. (1979). La Dimensión de la pobreza en América Latina. Cuadernos de la
CEPAL 27. Altimir, O. (1996). Cambios de la desigualdad y la pobreza en la América Latina. El
Trimestre Económico 61(1). Alvaredo, F. (2011). A Note on the Relationship between Top Income Shares and the
Gini Coefficient; Economics Letters, 110(3): 274-277 Alvaredo, F., Atkinson, A. B., Piketty, T. and Saez, E. The World Top Incomes Database,
http://topincomes.parisschoolofeconomics.eu, 25/03/2013. Alvaredo, F. and Atkinson, A. B. (2010). Colonial Rule, Apartheid and Natural
Resources: Top Incomes in South Africa 1903-2007. CEPR DP 8155; series updated in
the World Top Incomes Database. Alvaredo, F. and Cano, L. (forthcoming). High incomes in Ecuador. Alvaredo, F. and Londoño Vélez, J. (2013). High Incomes and Personal Taxation in a
Developing Economy: Colombia 1993-2013. Commitment to Equity-CEQ Working
Paper n. 12. Amiel, Y. and Cowell, F. (1999). Thinking about Inequality: Personal judgment and
income distributions. Cambridge: Cambridge University Press. Anand, S. and Segal, P. (2008). What do we know about global income inequality? Journal of Economic Literature 46(1), pp. 57-94. 92 92 Alvaredo-Gasparini Alvaredo-Gasparini Anderson, E. (2005). Openness and inequality in developing countries: A review of
theory and recent evidence. World Development 33(7), pp. 1045-1063. Arif, G. and Farooq, S. (2011). Poverty, Inequality and Unemployment in Pakistan. Pakistan Institute of Development Economics and Islamic Development Bank working
paper. Aron, J., Kahn, B. and Kingdon, G. References (2009) South Africa economic policy under
democracy. Oxford University Press, Oxford. Artadi, E. and Sala-i-Martin, X. (2003). The economic tragedy of the XXth Century:
Growth in Africa. NBER Working Paper 9865. Asian Development Bank (2012). Asian development outlook 2012. Confronting rising
inequality in Asia. ADB. Atkinson, A. (1987). On the measurement of poverty. Econometrica 55(4), pp. 749-764. Atkinson, A. and Bourguignon, F. (2001). Poverty and inclusion from a world
perspective. In Stiglitz and Muet (eds). Governance, equity and global markets. Oxford:
Oxford University Press. Atkinson, A. and Brandolini, A. (2001). Promise and pitfalls in the use of “secondary”
data-sets: Income inequality in OECD countries as a case study. Journal of Economic
Literature 39(3), pp. 771-199. Atkinson, A. and Brandolini, A. (2004). Global world inequality: absolute, relative or
intermediate?” Paper Presented for the 28th Generation Conference of the
International Association for Research in Income and Wealth, Cork, Ireland. Atkinson, A. and Brandolini, A. (2010). On analyzing the world distribution of income. World Bank Economic Review 24(1), pp. 1-37. Atkinson, A. and Lugo, M. (2010). Growth, poverty and distribution in Tanzania. Atkinson, A. B. and Micklewright, J. (1992). Economic transformation in Eastern Europe
and the distribution of income. Cambridge Books, Cambridge University Press. Atkinson, A. B. and Morelli, S. (2011). Economic crisis and inequality. Human
Development Research Paper 2011/06, UNDP. Atkinson, A. and Morelli, S. (2012). Chartbook of economic inequality: 25 countries,
1911-2010. Manuscript. Atkinson, A. B., and Piketty, T. eds. (2007). Top Incomes over the Twentieth Century: A
Contrast between Continental European and English-Speaking Countries. Oxford
University Press. Atkinson, A. B., and Piketty, T. eds. (2010). Top Incomes: A Global Perspective. Oxford
and New York: Oxford University Press. Banerjee, A. and Piketty, T. (2010). Top Indian Incomes 1922-2000; in Atkinson, A. B. and Piketty, T. (editors) Top Incomes: A Global Perspective, Oxford University Press,
chapter 1, pp. 1-39. 93 Alvaredo-Gasparini Alvaredo-Gasparini Bastagli, F., Coady, D. and Gupta, S. (2012). Income inequality and fiscal policy. IMF
Staff Discussion Note, SDN/12/08, June. Barros, R. P., Carvalho, M., Franco, S. and Mendoça, R. (2006). Uma análise das
principais causas da queda recente na desigualdade de renda brasileira. Revista
Econômica 8(1). Baulch, B. and McCulloch, N. (2000). Simulating the impact of policy upon chronic and
transitory poverty in rural Pakistan. Econometrics 0004003, EconWPA. Benabou, R. (1996). Inequality and growth. In NBER Macroeconomics Annual 1996,
Volume 11, NBER. Besley, T. and Burgess, R. (2003). References Halving global poverty. Journal of Economic
Perspectives 17(3), pp. 3-22. Bhalla, S. (2002). Imagine there’s no country: poverty, inequality and growth in the era
of globalization. Institute for International Economics. Bibi, S. and Nabli, M. (2010). Equity and inequality in the Arab region. Policy Research
Report 33, Economic Research Forum. Bigsten, A. and Shimeles, A. (2003). Prospect for pro-poor growth strategies in Africa. WIDER Research Paper 42/2004. Birdsall, N. and Nellis, J. (2003). Winners and losers: assessing the distributional impact
of privatization. World Development, 31(10), pp. 1617-1633. Bourguignon, F. (2003). The growth elasticity of poverty reduction: Explaining
heterogeneity across countries and time periods. In Eichler and Turnovsky (eds). Inequality and Growth: Theory and Policy Implications. CESifo Seminar Series, London:
MIT Press. Bourguignon, F. and Bussolo, M. (2012). Income distribution and computable general
equilibrium models: macro-micro modelling. In Dixon and Jorgenson (eds). Handbook
of Computable General Equilibrium Modeling, Volume 1A. North Holland Press. Bourguignon, F., Bussolo, M. and Pereira da Silva, L. (2008). The Impact of
Macroeconomic Policies on Poverty and Income Distribution. Macro-Micro Evaluation
Techniques and Tools. Washington, DC: The World Bank. Bourguignon, F., Ferreira, F. and Leite, P. (2008). Beyond Oaxaca‐Blinder: Accounting
for differences in households income distributions. Journal of Economic Inequality,
6(2), pp. 117-148. Bourguignon, F., Ferreira, F. and Lustig, N. (2005). The microeconomics of income
distribution dynamics in East Asia and Latin America. Washington, DC: The World Bank. Bourguignon, F., Levin, V. and Rosenblatt, D. (2004). Declining international inequality
and economic divergence: reviewing the evidence through different lenses. Économie
internationale 100, pp. 13-25. Bourguignon, F. and Morrison, C. (2002). Inequality among world citizens: 1820-1992. American Economic Review 92(4), pp. 727-744. Burkhauser, R., Feng, S., Jenkins, S. and Larrimore, J., (2012). "Recent Trends in Top
Income Shares in the United States: Reconciling Estimates from March CPS and IRS Tax 94 Alvaredo-Gasparini Return Data," The Review of Economics and Statistics, 94(2): 371-388. Cameron, L. (2002), Growth with or without equity? The distributional impact of
Indonesian development. Asian-Pacific Economic Literature, 16: 1–17. Caselli, F., Esquivel, G. and Lefort, F. (1996). Reopening the convergence debate: a new
look at cross-country growth empirics. Journal of Economic Growth 1, 363-389. Caselli, F. and Michaels, G. (2013). Do oil windfalls improve living standards? Evidence
from Brazil. American Economic Journal vol. 5(1), pp. 208-38. Chakravarty, S. (1987). Development Planning: The Indian Experience. Oxford
University Press, New Delhi. Chen, S. and Ravallion, M. (2001). References How did the world's poorest fare in the
1990s? Review of Income and Wealth 47(3), pp. 283-300. Chen, S. and Ravallion, M. (2007). Absolute poverty measures for the developing
world, 1981-2004. Proceedings of the National Academy of Sciences of the United
States of America, 104/43, pp. 16757-62. Chen, S. and Ravallion, M. (2008). China is poorer than we thought, but no less
successful in the fight against poverty. World Bank Policy Research Working Paper No. 4621. Chen, S. and Ravallion, M. (2010). The developing world is poorer than we thought, but
no less successful in the fight against poverty. The Quarterly Journal of Economics
125(4), pp. 1577-1625. Chen, S. and Ravallion, M. (2012). More relatively-poor people in a less absolutely-poor
world. The World Bank Policy Research Working Paper 6114. Chotikapanich, D., Valenzuela, R. and Prasada Rao, D. (1997). Global and regional
inequality in the distribution of income: Estimation with limited and incomplete data. Empirical Economics 22(4), pp. 533-546. Christiansen, L., Demery, L. and Paternostro, S. (2002). Growth, distribution and
poverty in Africa: Messages from the 1990s. The World Bank Policy Research Paper
2810. Chusseau, N. and Hellier, J. (2012). Globalization and inequality: Where do we stand? ECINEQ Working Paper 279. Cockburn, J., Duclos, J. and Zabsonré, A. (2012). Is the value of humanity increasing? A
critical-level enquiry. FERDI Working Paper 52. Conte, A. and Vivarelli, M. (2007). Globalization and employment: Imported skill biased
technological change in developing countries. IZA Discussion Paper 2797. Cornia, G. (1996). Transition and income distribution: Theory, evidence and initial
interpretation. UNU-WIDER Research in Progress 1. Cornia, G. (2011). Economic integration, inequality and growth: Latin America vs. the
European Economies in Transition. Review of Economics and Institutions 2(2). Cornia, G. and Kiiski, S. (2001). Trends in income distribution in the post-world war II
period: Evidence and interpretation. UNU–WIDER Discussion Paper, 2001/89. 95 Alvaredo-Gasparini Cornia, G. and Reddy, S. (2001). The impact of adjustment related social funds on
income distribution and poverty. UNU/WIDER Discussion Paper 2001/1. Cornia, G. and Reddy, S. (2001). The impact of adjustment related social funds on
income distribution and poverty. UNU/WIDER Discussion Paper 2001/1. Cruces, G. and Gasparini, L. (2012). Políticas sociales para la reducción de la
desigualdad y la pobreza en América Latina y el Caribe. Diagnóstico, propuesta y
proyecciones en base a la experiencia reciente. References Premio Fundación Vidanta 2012,
Contribuciones a la reducción de la pobreza y la desigualdad en América Latina y el
Caribe, Fundación Vidanta, México. Datt, G. and Ravallion, M. (1992). Growth and redistribution components of changes in
poverty measures: A decomposition with applications to Brazil and India in the 1980s. Journal of Development Economics 38(2), pp. 275-295. Deaton, A. (1997). The analysis of household surveys: A microeconometric approach to
development policy. Washington D.C: The World Bank. Deaton, A. (2003). How to monitor poverty for the millennium development goals. Journal of Human Development 4(3), pp. 353-378. Deaton, A. (2005). Measuring poverty in a growing world (or measuring growth in a
poor world). Review of Economics and Statistics 87(1), pp. 1-19. Deaton, A. (2010). Price indexes, inequality, and the measurement of world poverty. American Economic Review 100(1), pp. 5-34. Deaton, A. and Dupriez, O. (2008). Poverty PPPs around the world: an update and
progress report. Development Data Group, World Bank. Deaton, A. and Dupriez, O. (2011). Purchasing power parity exchange rates for the
global poor. Applied Economics 3(2), pp. 137–166. Deaton, A. and Heston, A. (2010). Understanding PPPs and PPP-based national
accounts. Macroeconomics 2(4), pp. 1-35. Deaton, A. and Zaidi, S. (2002). Guidelines for constructing consumption aggregates for
welfare analysis. Living Standards Measurement Study Working Paper 135, The World
Bank. Deininger, K. and Squire, L. (1996). A new data set measuring income inequality. World
Bank Economic Review 10(3), pp. 565-591. De Ferranti, D., Perry, G., Ferreira, F. and Walton, M. (2004). Inequality in Latin
America, breaking with history? Washington, DC: The World Bank. Devarajan S. and Nabi, I. (2006). Economic Growth in South Asia: Promising, Un-
equalizing,...Sustainable? South Asia Region, World Bank. Dhongde, S. and Minoui, C. (2010). Global poverty estimates: Present and future. ECINEQ Working Papers 181. Dhongde, S. and Miao, X. (2013). Cross-country convergence in income inequality. ECINEQ Working Papers 290. Dollar, D. and Kraay, A. (2002). Growth is good for the poor. Journal of Economic
Growth 7(3), pp. 195-225. 96 Alvaredo-Gasparini Alvaredo-Gasparini Essama-Nssah, B. (2012). Identification of sources of variation in poverty outcomes. The World Bank Policy Research Working Papers 5954. Feenstra, R. (2008). Offshoring in the Global Economy: Lecture 1: Microeconomic
Structure; Lecture 2: Macroeconomic Implications. The Ohlin Lectures, presented at
the Stockholm School of Economics on September 17-18, 2008. Ferreira, F. (1999). Economic transition and the distributions of income and wealth. The Economics of Transition 7(2), pp. 377-410. Garcia, M. and Moore, C. (2012). The cash dividend: The rise of cash transfer program
in Sub-Saharan Africa. Washington DC: The World Bank. References Ferreira, F. (2010). Distributions in motion: Economic growth, inequality, and poverty
dynamics. The World Bank Policy Research Working Paper 5424. Ferreira, F. (2012). Inequality of opportunity around the world: what do we know so
far? Inequality in Focus 1(1), pp. 8-11, The World Bank. Ferreira, F., Leite, P. and Wai-Poi, M. (2010). Trade Liberalization, Employment Flows,
and Wage Inequality in Brazil. In Nissanke and Thorbecke (eds). The poor under
globalization in Asia, Latin America and Africa. Oxford: Oxford University Press. Ferreira, F. and Ravallion, M. (2009). Poverty and inequality: The global context. In
Salverda, Nolan and Smeeding (eds). The Oxford Handbook of Economic Inequality. Oxford: Oxford University Press. Fields, G. (2002). Distribution and Development: A New Look at the Developing World. Cambridge: MIT Press. Fiszbein, A. and Schady, N. (2009). Conditional cash transfers. Reducing present and
future poverty. The World Bank Policy Research Report. Fleisher, Belton M., Sabirianova, K. and Wang, X. (2005). Returns to skills and the
speed of reforms: evidence from Central and Eastern Europe, China, and Russia. Journal of Comparative Economics, vol. 33(2), pp. 351-370, June. Forbes, K. (2011). Inequality in crisis and recovery : revealing the divides: the case of
Brazil. ILO Working Papers 469849, International Labour Organization. Fortin, N., Lemieux, T. and Firpo, S. (2011). Decomposition Methods in Economics. In
Ashenfelter and Card (eds). Handbook of Labor Economics, Volume 4, Part A. Fosu, A. (2009). Inequality and the impact of growth on poverty: Comparative evidence
for Sub-Saharan Africa. Journal of Development Studies 45(5), pp. 726–745. Fosu, A. (2010). The global financial crisis and development: Whither Africa? UNU-
WIDER Working Paper 2010/124. Fosu, A. (2011). Growth, inequality, and poverty reduction in developing countries. Recent global evidence. UNU-WIDER Working Paper 2011/1. Galbraith, J. and Kum, H. (2005). Estimating the inequality of household incomes:
toward a dense and consistent global data set. Review of Income and Wealth 51(1), pp. 115-143. Garcia, M. and Moore, C. (2012). The cash dividend: The rise of cash transfer programs
in Sub-Saharan Africa. Washington DC: The World Bank. 97 97 Alvaredo-Gasparini Gasparini, L., Cicowiez, M. and Sosa Escudero, W. (2013). Pobreza y desigualdad en
América Latina. Conceptos, herramientas y aplicaciones. Editorial Temas. Gasparini, L., Cruces, G. and Tornarolli, L. (2011). Recent trends in income inequality in
Latin America. Economia 10(2), pp. 147-201. Gasparini, L., Galiani, S., Cruces, G. and Acosta, P. (2011). References Educational upgrading and
returns to skills in Latin America: evidence from a supply-demand framework, 1990-
2010. IZA Discussion paper 6244. Gasparini, L. and Gluzmann, P. (2012). Estimating income poverty and inequality from
the Gallup World Poll: the case of Latin America and the Caribbean. Journal of Income
Distribution 21(1). Gasparini, L. and Lustig, N. (2011). The rise and fall of income inequality in Latin
America”. In Ocampo and Ros (eds). The Oxford Handbook of Latin American
Economics, Chapter 27. Oxford: Oxford University Press. Gentilini, U. and Sumner, A. (2012). Poverty where people live: what do national
poverty lines tell us about global poverty? International Policy Centre for Inclusive
Growth Working Paper 98. Gertler, P. (2004). Do conditional cash transfers improve child health? Evidence from
PROGRESA’s control randomized experiment. American Economic Review 94(2), pp. 336-341. Ghani, E. (2010). The poor half billion in South Asia. What is holding back lagging
regions? Oxford University Press. Gluzmann, P. (2012). Desigualdad del ingreso y del bienestar subjetivo. Análisis y
comparaciones internacionales. Ph.D. dissertation, Universidad Nacional de La Plata. Go, D., Nikitin, D., Wang, X. and Zou, H. (2007). Poverty and inequality in Sub-Saharan
Africa: Literature survey and empirical assessment. Annals of Economics and Finance,
Society for AEF, 8(2), pp. 251-304. Goldberg, P. and Pavcnik, N. (2007). Distributional Effects of globalization in developing
countries. Journal of Economic Literature 45(1), pp. 39-82. Goldin, C. and Katz, L. (2008). The race between education and technology. Cambridge:
Harvard University Press. Goñi-Pacchioni, J., López, H. and Servén, L. (2011). Fiscal redistribution and income
inequality in Latin America. World Development 39(9), pp. 1558-1569. Gorodnichenko, Y. and Sabirianova, K. (2005). Returns to schooling in Russia and
Ukraine: A semiparametric approach to cross-country comparative analysis. Journal of
Comparative Economics 33(2), pp. 324–350. Gosh, J. (2012). Unequal in Asia. Frontline, 29(15) Jul 28-Aug 10, pp. 127-128. Harrison, A., McLaren, J. and McMillan, M. (2011). Recent perspectives on trade and
inequality. Annual Review of Economics 3(1), pp. 261-289. Hellier, J. and Lambrecht, S. (2012). Inequality, growth and welfare: The main links. ECINEQ Working Paper 258. 98 Alvaredo-Gasparini Helpman, E. and Trajtenberg, M. (1998). A time to sow and a time to reap: Growth
based on general purpose technologies. In Helpman (ed). General Purpose
Technologies and Economic Growth. Cambridge: MIT Press. Helpman, E. and Trajtenberg, M. (1998). A time to sow and a time to reap: Growth
based on general purpose technologies. In Helpman (ed). References General Purpose
Technologies and Economic Growth. Cambridge: MIT Press. Heston, A., Summers, R. and Aten, B. (2012). Penn World Table Version 7.1, Center for
International Comparisons of Production, Income and Prices at the University of
Pennsylvania, July. Heston, A., Summers, R. and Aten, B. (2012). Penn World Table Version 7.1, Center for
International Comparisons of Production, Income and Prices at the University of
Pennsylvania, July. Hussain, A. (2008). Power dynamics, institutional instability and economic growth: The
case of Pakistan. The Asia Foundation. IDB (1999). América Latina frente a la desigualdad. Informe de Progreso Económico y
Social, Banco Interamericano de Desarrollo. Iqbal, F. (2006). Sustaining gains in poverty reduction and human development in the
Middle East and North Africa. Washington, DC: The World Bank. International Labor Organization (ILO) (2010). World Social Security Report 2010/11:
Providing Coverage in Times of Crisis and Beyond; Geneva. Inchauste, G., Olivieri, S., Saavedra, J. and Winkler, H. (2012). What Is Behind the
Decline in Poverty Since 2000? Evidence from Bangladesh, Peru and Thailand. The
World Bank Policy Research Working Paper 6199. Ivaschenko, O. (2002). Growth and Inequality: Evidence from transitional economies. CESIFO Working Paper 746. Kakwani, N. (2000). On measuring growth and inequality components of poverty with
application to Thailand. Journal of Quantitative Economics 16, pp. 67-8. Kakwani, N., Khandker, S. and Son, H. (2003). Poverty equivalent growth rate: With
applications to Korea and Thailand. Technical report, Economic Commission for Africa. Kakwani, N., and Krongkaew, M. (2000). Economic growth, poverty and income
inequality in the Asia-Pacific region. Journal of the Asia Pacific Economy 5(1-2), pp. 9-
13. Kakwani, N. and Pernia, E. (2000). What is pro-poor growth? Asian Development
Review 18(1), pp. 1-16. Kakwani, N. and Subbarao, K. (1990). Rural poverty and its alleviation in India. Economic and Political Weekly 25. Kanbur, R. and Sumner, A. (2011). Poor countries or poor people? Development
assistance and the new geography of global poverty. Cornell University Working Paper
2011-08. Karshenas, M. (2003). Global poverty: National Accounts based versus survey based
estimates. Development and Change 34(4), pp. 683-712. Khan, A. R. (2008). Measuring inequality and poverty in Bangladesh: an assessment of
the survey data. The Bangladesh Development Studies. 31(3/4), pp. 1-34 Klasen, S. (1997). Poverty, inequality and deprivation in South Africa: an analysis of the
1993 SALDRU survey. Social Indicators Research, 41(1-3), pp. 51-94. 99 Alvaredo-Gasparini Klasen, S. (2005). Measuring poverty and deprivation in South Africa. References Review of Income
and Wealth, 46(1), pp. 33-58. Korinek, A., Mistiaen, J. and Ravallion, M. (2006). Survey nonresponse and the
distribution of income. Journal of Economic Inequality 4 (2). Kraay, A. (2006). When is growth pro-poor? Evidence from a panel of countries. Journal of Development Economics 80(1), pp. 198–227. Krongkaew, M. (1994). Income distribution in East Asian developing countries: An
update. Asia Pacific Economic Literature 8(2), pp. 58–73. Krongkaew, M. and Zin, R. (2007). Income distribution and sustainable economic
development in East Asia: A Comparative Analysis. IDEAs Working Paper 02/2007. Kuznets, S. (1955). Economic growth and income inequality. American Economic
Review 45(1), pp. 1-28. Leibbrandt, M., Woolard, I. and Woolard, C., (2009). A long-run perspective on
contemporary poverty and inequality dynamics. In J. Aron, B. Kahn and G. Kingdon
(editors) South Africa economic policy under democracy. Oxford University Press,
Oxford, Chapter 10. Leibbrandt, M., Woolard, I., McEwen, H., and Koep, C. (2010). Better employment to
reduce inequality further in South Africa. In Tackling inequalities in Brazil, China, India
and South Africa, OECD, Paris. Leibbrandt, M., Woolard, I., Finn, A. and Argent, J. (2010). Trends in South African
income distribution and poverty since the fall of apartheid. OECD Social, Employment
and Migration Working Papers n. 101. Leigh, A. (2007). How closely do top income shares track other measures of inequality? The Economic Journal, 117: F619–F633. Lewis, A. (1954). Economic development with unlimited supplies of labor. Manchester
School of Economics and Social Studies 22, pp. 139–192. Li, H., Squire, L. and Zou, H. (1998). Explaining international and intertemporal
variations in income inequality. Economic Journal 108(446). Li, S. Chuliang, L. and Sicular, T. (2013). Overview: income inequality and poverty in
China, 2002–2007. In Li Shi, Hiroshi Sato, and Terry Sicular (eds.). Rising Inequality in
China: Challenge to the Harmonious Society, Cambridge University Press. Lind, J. and Mehlum, H. (2010). With or without U? The appropriate test for a U shaped
relationship. Oxford Bulletin of Economics and Statistics 72(1), pp. 109-118. Loayza, N. and Raddatz, C. (2010). The composition of growth matters for poverty
alleviation. Journal of Development Economics 93(1), pp. 137-151. Londoño, J. and Székely, M. (2000). Persistent poverty and excess inequality: Latin
America, 1970-1995. Journal of Applied Economics 3(1), pp. 93-134. López Calva, L. and Lustig, N. (eds.) (2010). Declining inequality in Latin America: A
decade of progress? Brookings Institution and UNDP. 100 Alvaredo-Gasparini Lustig, N. (1990). References Economic crisis, adjustment and living standards in Mexico, 1982-85. World Development, 18(10), pp. 1325-1342. Lustig, N. (1990). Economic crisis, adjustment and living standards in Mexico, 1982-85. World Development, 18(10), pp. 1325-1342. Lustig, N. (1995). Coping with Austerity: Poverty and Inequality in Latin America. Washington, DC: Brookings Institution. Lustig, N. (2012). Fiscal policy and income redistribution in Latin America: Challenging
the conventional wisdom. Tulane University Economics Working Paper 1124. Mahmoudi, V. (2001). Growth-Equity decomposition of a change in poverty: An
application to Iran. University of Essex. Mavridis, D. and Mosberger, P. (forthcoming). Capital and labor: top income shares in
Hungary, 1915-2010. Mazundar, D. (2012) Decreasing poverty and increasing inequality in India; in OECD
(editor), Tackling Inequalities in Brazil, China, India and South Africa: The Role of
Labour Market and Social Policies, OECD Publishing, chapter 4, pp. 157-207. McGrath, M. D. and Whiteford, A. (1994). The distribution of income in South Africa,
Human Science Research Council, Pretoria. McGrath, M. D. (1983). The distribution of personal income in South Africa in selected
years over the period from 1945 to 1980. Ph D thesis, University of Natal, Durban. Milanovic, B. (1995). Poverty, inequality and social policy in transition economies. The
World Bank Policy Research Working Paper 9. Milanovic, B. (1998). Income, inequality, and poverty during the transition from
planned to market economy. World Bank Regional and Sectoral Studies. The World
Bank, Washington, D.C. Milanovic, B. (1999). Explaining the increase in inequality during transition. Economics
of Transition 7(2), pp. 299-341. Milanovic, B. (2002). True world income distribution, 1988 and 1993: first calculation
based on household surveys alone. Economic Journal 112(476), pp. 51-92. Milanovic, B. (2003). Is Inequality in Africa really different? The World Bank Policy
Research Working Paper 3169. Milanovic, B. (2005). Worlds apart: measuring international and global inequality. Princeton: Princeton University Press. Milanovic, B. (2011). A short history of global inequality: The past two centuries. Explorations in Economic History 48(4), pp. 494-506. Milanovic, B. (2012a). Global income inequality by the numbers: In history and now. An
overview. The World Bank Policy Research Working Paper 6259. Milanovic, B. (2012b). Global inequality recalculated and updated: The effect of new
PPP estimates on global inequality and 2005 estimates. Journal of Economic Inequality
10(1), pp. 1-18. 101 Alvaredo-Gasparini Milanovic, B. and Ersado, L. (2010). Reform and inequality during the transition. An
analysis using panel household survey data, 1990-2005. UNU/WIDER Working Paper
62. Milanovic, B. and Ersado, L. (2010). References Reform and inequality during the transition. An
analysis using panel household survey data, 1990-2005. UNU/WIDER Working Paper
62. Minoiu, C. and Reddy, S. G. (2008). Chinese poverty: assessing the impact of alternative
assumptions. Review of Income and Wealth, 54(4), pp. 572-596. Miranti, R., Vidyattama, Y., Hansnata, E., Cassells, R. and Duncan, A. (2013). Trends in
Poverty and Inequality in Decentralising Indonesia. OECD Social, Employment and
Migration Working Papers No. 148. Mitra, P. and Yemtsov, R. (2006). Increasing inequality in transition economies: Is there
more to come? The World Bank Policy Research Working Paper 4007. Moradi, A. and Baten, J. (2005). Inequality in Sub-Saharan Africa: new data and new
insights from anthropometric estimates. World Development 33(8), pp. 1233-1265. Morley, S. (2001). The income distribution problem in Latin America and the Caribbean. Santiago: CEPAL. Nattrass, N. and Seekings, J. (1997). Citizenship and Welfare in South Africa:
Deracialisation and Inequality in a Labour-Surplus Economy. Canadian Journal of
African Studies, 31(3), pp. 452-481. Nissanke, M. and Thorbecke, E. (editors) (2010). The poor under globalization in Asia,
Latin America and Africa. Oxford University Press, 2010. Okojie, C. and Shimeles, A. (2006). Inequality in Sub-Saharan Africa. The Inter-Regional
Inequality Facility. London: DFID. Page, J. (2007). Boom, bust, and the poor: Poverty dynamics in the Middle East and
North Africa, 1970–1999. The Quarterly Review of Economics and Finance 46(5), pp. 832-851. Palma, J. (2011). Homogeneous middles vs. heterogeneous tails, and the end of the
“inverted-U”: the share of the rich is what it´s all about. Cambridge Working Papers in
Economics 1111. Parker, S. and Skoufias, E. (2001). Conditional cash transfers and their impact on child
work and school enrollment: evidence from the PROGRESA program in Mexico. Economia 2(1). Piketty, T. and Qian, N. (2010). Income inequality and progressive income taxation in
China and India, 1986-2015. In A. B. Atkinson and T. Piketty (eds.), Top Incomes: A
Global Perspective, Oxford University Press. Pinkovskiy, M. (2013). World welfare is rising: Estimation using nonparametric bounds
on welfare measures. Journal of Public Economics 97(1), pp. 176-195. Pinkovskiy, M. and Sala-i-Martin, X. (2009). Parametric estimations of the world
distribution of income. NBER Working Paper 1543. Pissarides, C. (1997). Learning by trading and the returns to human capital in
developing countries. World Bank Economic Review 11(1), pp. 17-32. 102 Alvaredo-Gasparini Rama, M. (2003). Globalization and workers in developing countries. The World Bank
Policy Research Working Paper 2958. Ravallion, M. (1997). References Can high-inequality developing countries escape absolute
poverty? Economics Letters 56(1), pp. 51-57. Ravallion, M. (2001). Growth, inequality and poverty: Looking beyond averages. World
Development 29(11), pp. 1803–1815. Ravallion, M. (2003). Inequality convergence. Economic Letters 80(3), pp. 351-356. Ravallion, M. (2004a). Pro-poor growth: a primer. The World Bank Policy Research
Working Paper 3242. Ravallion, M. (2004b). Competing concepts of inequality in the globalization debate. The World Bank Policy Research Working Paper 3243. Ravallion, M. (2007). Inequality is bad for the poor. In Jenkins and Micklewright (eds). Inequality and poverty re-examined. Oxford: Oxford University Press. Ravallion, M. (2010). Price levels and economic growth: making sense of the PPP
changes between ICP rounds. The World Bank Policy Research Working Paper 5229. Ravallion, M. (2012). Why don’t we see poverty convergence? American Economic
Review 102(1). Ravallion, M. (2013). How long will it take to lift one billion people out of poverty? The
World Bank Policy Research Working Paper 6325. Ravallion, M. and Chen, S. (1997). What can new survey data tell us about recent
changes in distribution and poverty? World Bank Economic Review 11(2), pp. 357-382. Ravallion, M. and Chen, S. (2003). Measuring pro-poor growth. Economic Letters 78(1),
pp. 93-99. Ravallion, M., and Chen, S. (2007). China’s (uneven) progress against poverty. Journal
of Development Economics 82(1), pp. 1-42. Ravallion, M. and Chen, S. (2011). Weakly relative poverty. Review of Economics and
Statistics 93(4), pp. 1251-1261. Ravallion, M., Chen, S. and Sangraula P. (2009). Dollar a day revisited. World Bank
Economic Review 23(2), pp. 163-184. Ravallion, M., Datt, G. and van de Walle, D. (1991). Quantifying absolute poverty in the
developing world. Review of Income and Wealth 37(4), pp. 345-361. Reddy, S. and Minoiu, C. (2007). Has world poverty really fallen? Review of Income and
Wealth 53(3), pp. 484-502. Reddy, S. and Pogge, T. (2010). How not to count the poor. In Anand, Segal and Stiglitz
(eds.). Debates on the measurement of poverty. Oxford: Oxford University Press. Robinson, J. (2009). The political economy of inequality. ERF Working Paper 493. Sala-i-Martin, X. (2006). The world distribution of income: falling poverty and …
convergence, period. The Quarterly Journal of Economics 121(2), pp. 351-397. 103 Alvaredo-Gasparini Sala-i-Martin, X. and Pinkovskiy, M. (2010). African poverty is falling ... Much faster
than you think! NBER Working Paper 15775. Sala-i-Martin, X. and Pinkovskiy, M. (2010). African poverty is falling ... Much faster
than you think! NBER Working Paper 15775. Salehi-Isfahani, D. References (2010). Human development in the Middle East and North Africa. UNDP Human Development Research Paper 2010/26. Sen, A. (1976). Real national income. Review of Economic Studies 43(1), pp. 19-39. Sen, A. (1983). Poor, relatively speaking. Oxford Economic Papers 35(2), pp. 153-69. Sharma, M., Inchauste, G. and Feng, J. (2011). Rising inequality with high growth and
falling poverty. An eye on East Asia and Pacific; no. 9, World Bank. Shorrocks, A. and Wan, G. (2008). Ungrouping income distributions: Synthesising
samples for inequality and poverty analysis. In Basu and Kanbur (eds). The Oxford
Handbook of Arguments for a Better World: Essays in Honor of Amartya Sen. Volume I:
Ethics, Welfare and Measurement. Oxford: Oxford University Press. Simai, M. (2006). Poverty and inequality in Eastern Europe and the CIS Transition
Economies. DESA Working Paper No. 17. Simkins, C. (1991). The Urban Foundation income distribution model: Phase One. Mimeo. Johannesburg: Urban Foundation. Skoufias, E. and Suryahadi, A., (2000). Changes in Regional Inequality and Social
Welfare in Indonesia between 1996 and 1999, Working Paper, Social Monitoring and
Early Response Unit (SMERU) Research Institute, Jakarta. Soares, S., Guerreiro Osório, R., Veras Soares, F., Medeiros, M. and Zepeda, E. (2009). Conditional cash transfers in Brazil, Chile and Mexico: impacts upon inequality. Estudios Económicos 0 (Special i), pp. 207-224. Solt, F. (2009). Standardizing the world income inequality database. Social Science
Quarterly 90(2), pp. 231-242. Standing, G. and Vaughan-Whitehead, D. (1995). Minimum wages in Central and
Eastern Europe : from protection to destitution International Labour Office. Central and
Eastern European Team. Sumner, A. (2012). From deprivation to distribution: is global poverty becoming a
matter of national inequality? IDS Working Paper 2012-394. Székely, M. and Hilgert, M. (1999). What’s behind the inequality we measure : an
investigation using Latin American data. Inter-American Development Bank, Research
Department Working Paper 409. Tabatabai, H. (1996). Statistics on poverty and income distribution: an ILO compendium
of data. Geneva: International Labor Office. Terreblanche, S. (2002). A history of inequality in South Africa, 1652-2002. University of
Natal Press, Pietermaritzburg. Tinbergen, J. (1975). Income Differences: Recent Research. Amsterdam: North-Holland. 104 Alvaredo-Gasparini Todd, P. and Wolpin, K. (2006). Assessing the impact of a school subsidy program in
Mexico: Using a social experiment to validate a dynamic behavioral model of child
schooling and fertility. American Economic Association 96(5), pp. 1384-1417. Todd, P. and Wolpin, K. (2006). Zin, R.H.M. (2005). Income distribution in East Asian developing countries: recent
trends. Asian-Pacific Economic Literature, 19(2), 36-54. References 105 105 Table A.1
List of developing countries by region and population, 2010 List of developing countries by region and population, 2010 List of developing countries by region and population, 2010
Source: population (in million) is taken from the United Nations Demographic Yearbook. Eastern Europe and Central Asia
Latin America and the Caribbean
Middle East and North Africa
Countries
Pop. Data
Countries
Pop. Data
Countries
Pop. Data
Countries
Pop. Data
Countries
Pop. Data
Countries
Pop. Data
Countries
Pop. Data
American Samoa
0.066
Albania
3.181
X
Antigua and Barbuda
0.087
Algeria
34.428
X
Afghanistan
32.518
Angola
18.038
X
Nigeria
150.666
X
Cambodia
13.823
X
Armenia
3.079
X
Argentina
39.714
X
Djibouti
0.856
X
Bangladesh
145.478
X
Benin
8.356
X
Rwanda
10.004
X
China
1324.655
X
Azerbaijan
8.763
X
Belize
0.322
X
Egypt
78.323
X
Bhutan
0.701
X
Botswana
1.955
X
São Tomé & P. 0.160
X
Fiji
0.844
X
Belarus
9.602
X
Bolivia
9.618
X
Iran
72.289
X
India
1190.864
X
Burkina Faso
15.515
X
Senegal
11.787
X
Indonesia
234.951
X
Bosnia and Herzegovina
3.774
X
Brazil
191.543
X
Iraq
30.178
X
Maldives
0.308
X
Burundi
7.943
X
Seychelles
0.087
X
Kiribati
0.097
Bulgaria
7.623
X
Chile
16.796
X
Jordan
5.787
X
Nepal
28.905
X
Cameroon
18.759
X
Sierra Leone
5.612
X
Korea, Dem. Rep. 24.126
Croatia
4.434
X
Colombia
45.006
X
Lebanon
4.167
Pakistan
167.442
X
Cape Verde
0.487
X
Somalia
8.922
Lao
6.022
X
Czech Republic
10.424
X
Costa Rica
4.522
X
Libya
6.150
Sri Lanka
20.217
X
Central African R. 4.238
X
South Africa
48.793
X
Malaysia
27.502
X
Estonia
1.341
X
Cuba
11.267
Morocco
31.321
X
Chad
10.654
X
South Sudan
8.977
Marshall Islands
0.053
Georgia
4.384
X
Dominica
0.068
Syria
19.638
X
Comoros
0.697
X
Sudan
32.438
X
Micronesia
0.110
X
Hungary
10.038
X
Dominican Republic
9.665
X
Tunisia
10.329
X
Congo, Dem. R. 62.475
X
Swaziland
1.032
X
Mongolia
2.667
X
Kazakhstan
15.674
X
Ecuador
14.057
X
West Bank and Gaza
3.937
X
Congo, Rep. Note: an “X” in the Data column marks that the country has distributive information in PovcalNet. Source: population (in million) is taken from the United Nations Demographic Yearbook. References Assessing the impact of a school subsidy program in
Mexico: Using a social experiment to validate a dynamic behavioral model of child
schooling and fertility. American Economic Association 96(5), pp. 1384-1417. United Nations-UNECE (2004) Economic Survey of Europe, 2004 No. 1, chapter 7. United Nations-UNECE (2004) Economic Survey of Europe, 2004 No. 1, chapter 7. van der Berg, S., and Louw, M. (2004). Changing patterns of South African income
distribution: towards time series estimates of distribution and poverty. South African
Journal of Economics, 72(3), pp. 546-572. Van de Walle, N. (2008). The institutional origins of inequality in Sub-Saharan Africa. Cornell University Mario Einaudi Center for International Studies Working Paper 6-08. Voitchovsky, S. (2009). Inequality, Growth and Sectoral Change. Chapter 22 in Nolan,
Salverda and Smeeding (eds.), Oxford Handbook of Economic Inequality. Whiteford, A. and Van Seventer, D. (2000). South Africa’s changing income distribution
in the 1990s. Studies in Economics & Econometrics 24(3), pp. 7-30. Winters, A., McCulloch, N. and McKay, A. (2004). Trade liberalization and poverty: The
Evidence so far. Journal of Economic Literature 42, pp. 72-115. Wodon, Q., Castro, R. and Lee, K. (2001). Poverty in Latin America: Trends (1986-1998)
and determinants. Latin American Journal of Economics - formerly Cuadernos de
Economía 38(114). Wood, A. (1997). Openness and wage inequality in developing countries: The Latin
American challenge to East Asian conventional wisdom. World Bank Economic Review
11(1), pp. 33-57. World Bank (2000). World Development Report: Attacking Poverty. New York: Oxford
University Press. World Bank (2005). ECAPOV II. Growth, Poverty and Inequality in Eastern Europe and
Former Soviet Union. Washington, DC: The World Bank. World Bank (2006). World development report: Equity and development. World Bank
and Oxford University Press. World
Bank
(2006). Can
South
Asia
reduce
poverty
in
a
generation? http://go.worldbank.org/NF9FFJTM20 World Bank (2008). Global Purchasing Power Parities and Real Expenditures. 2005
International Comparison Program. Washington, DC: The World Bank. World Bank (2009). The World Development Report 2009: Reshaping Economic
Geography. Washington, D.C.: The World Bank. World Bank (2012). East Asia and Pacific Data Monitor, October. World Bank (2013). The East Asian Miracle: Economic Growth and Public Policy. Oxford
University Press. Zin, R.H.M. (2005). Income distribution in East Asian developing countries: recent
trends. Asian-Pacific Economic Literature, 19(2), 36-54. Zin, R.H.M. (2005). Income distribution in East Asian developing countries: recent
trends. Asian-Pacific Economic Literature, 19(2), 36-54. References 3.836
X
Tanzania
42.268
X
Myanmar
47.250
Kosovo
1.747
El Salvador
6.130
X
Yemen
22.627
X
Côte d'Ivoire
18.987
X
Togo
5.777
X
Palau
0.020
Kyrgyz Republic
5.319
X
Grenada
0.104
Eritrea
4.948
Uganda
31.339
X
Papua New Guinea
6.549
Latvia
2.266
X
Guatemala
13.691
X
Ethiopia
79.446
X
Zambia
12.380
X
Philippines
90.173
X
Lithuania
3.358
X
Guyana
0.752
X
Gabon
1.450
X
Zimbabwe
12.452
Samoa
0.182
Macedonia
2.053
X
Haiti
9.736
X
Gambia
1.636
X
Solomon Islands
0.510
Moldova
3.570
X
Honduras
7.303
X
Ghana
23.264
X
Thailand
68.268
X
Montenegro
0.629
X
Jamaica
2.687
X
Guinea
9.559
X
Timor-Leste
1.078
X
Poland
38.126
X
Mexico
110.627
X
Guinea-Bissau
1.454
X
Tonga
0.103
Romania
21.514
X
Nicaragua
5.636
X
Kenya
38.455
X
Tuvalu
0.010
Russian Federation
141.950
X
Panama
3.406
X
Lesotho
2.127
X
Vanuatu
0.228
Serbia
7.350
X
Paraguay
6.230
X
Liberia
3.658
X
Vietnam
85.122
X
Slovak Republic
5.407
X
Peru
28.463
X
Madagascar
19.546
X
Slovenia
2.021
X
St. Kitts and Nevis
0.051
Malawi
14.005
X
Tajikistan
6.691
X
St. Lucia
0.170
X
Mali
14.460
X
Turkey
70.924
X
St. Vincent and G. 0.109
Mauritania
3.295
X
Turkmenistan
4.918
X
Suriname
0.515
X
Mauritius
1.269
Ukraine
46.258
X
Trinidad and Tobago
1.331
X
Mozambique
22.333
X
Uzbekistan
27.303
X
Uruguay
3.334
X
Namibia
2.200
X
Venezuela
27.935
X
Niger
14.450
X
Sub-Saharan Africa
South Asia
East Asia and Pacific Alvaredo-Gasparini Figure 5.5 Figure 5.5 Map of poverty in the developing world, circa 2010 Map of poverty in the developing world, circa 2010 Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. < 5
5 - 10
10 - 15
15 - 20
20 - 30
30 - 40
40 - 50
50 - 60
60 - 70
70 - 80
> 80
High Incomes
No Data
Poverty
headcount (%) Source: own calculations based on PovcalNet (2013). No Data Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. (
)
Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjuste 107
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Tomruk piyasası aktörlerinin dikili ağaç satış yönetmeliklerinde yapılan değişikliklere ilişkin algıları
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Turkish Journal of Forestry
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Turkish Journal of Forestry | Türkiye Ormancılık Dergisi
2023, 24(4): 378-389 | Research article (Araştırma makalesi) Turkish Journal of Forestry Perceptions of log market actors on revisions to the regulations of the sale of
standing tree Abstract: The standing tree sales method is a sales method used in Türkiye as well as all over the world. The Standing Sales
Regulation, which first came into force in 1996, has been amended several times. However, the effects of these amendments are
not clear. Although the amendments made with the enactment of Decree No. 6877 were aimed at solving the existing problems, it
is unclear whether this amendment really solved the problems or not. This study sought to determine how the changes made by
identifying the differences between Decree No. 6521 and Decree No. 6877 affect all stakeholders in the standing sale mechanism
and whether the problems have been resolved. For this purpose, the opinions of the stakeholders were obtained by means of a
questionnaire survey conducted among forest cooperatives, forest villagers, employees of forest enterprises and enterprises
processing wood-based products in the Kastamonu Regional Forestry Directorate, which was defined as the study area. Face-to-
face interviews with the stakeholders were used to collect the data. Percentage and tabular methods were used to express the
opinions of the interest groups, and the Kruskal-Wallis analysis and Mann-Whitney U test were used to determine whether their
opinions on the changes differed. The study's results indicate that the recent regulatory changes have influenced interest groups in
varying ways. The yield percentage calculation has improved, but transportation issues remain continuous. The compulsory
employment of Forest Engineers in standing sales is not accepted except by forest enterprises. In addition, in the production of
forest products, the employment of workers from outside the region where production takes place is not accepted by other interest
groups except the forestry administration, and the production periods should not be fixed but should be determined according to
the quantity to be produced. q
y
p
Keywords: Log market, Standing tree sales, Regulation no. 6521, Regulation no. 6877, Kastamonu, Türkiye Tomruk piyasası aktörlerinin dikili ağaç satış yönetmeliklerinde yapılan
değişikliklere ilişkin algıları Alper Aközlüa
, Gökhan Şena,* Özet: Dikili ağaç satış metodu tüm dünyada olduğu gibi Türkiye’de de uygulanan bir satış yöntemidir. İlk kez 1996’da yürürlüğe
giren dikili satış yönetmeliği birçok kez değişikliğe uğramıştır. Buna karşın bu değişikliklerin etkilerinin neler olduğu belirli
değildir. 6877 Sayılı Tamim’in yürürlüğe girmesi ile oluşan değişiklikler ile mevcut sorunların çözümü hedeflenmişse de bu
değişimin gerçekten sorunlara çözüm bulup bulmadığı bir muallaktır. Bu çalışmada 6521 sayılı tamim ile 6877 sayılı tamim
arasındaki farklılıklar belirlenerek yapılan değişikliklerin dikili satış mekanizmasındaki tüm ilgi gruplarını nasıl etkilediği ve
sorunların çözüme kavuşup kavuşmadığı belirlenmeye çalışılmıştır. Bu amaçla çalışma alanı olarak belirlenen Kastamonu Orman
Bölge Müdürlüğü’nde orman kooperatifleri, orman köylüleri, orman işletmesi çalışanları ve odun kökenli ürün işleyen işletmeler
ile yapılan anket çalışması ile ilgi gruplarının görüşleri belirlenmiştir. Verilerin elde edilmesinde ise yüz yüze anket yöntemi
uygulanmıştır. İlgili grupların görüşlerinin ifade edilmesinde yüzde ve tablo yöntemleri kullanılırken, değişimler hakkındaki
görüşlerinin farklılık gösterip göstermediği aşamasında Kruskal-Wallis analizi, Mann-Whitney U testinden yararlanılmıştır. Çalışma sonuçları, tamimlerde yapılan değişimlerin ilgi gruplarını farklı yönde etkilediğini göstermektedir. Bu değişikliklerden
verim yüzdesi hesabının iyileştiği, transportta yaşanan sorunların halen tam olarak giderilemediği, dikili satışlarda zorunlu Orman
Mühendisi çalıştırılmasının orman işletmeleri hariç pek kabul görmediği belirtilmektedir. Bununla birlikte paydaşların iş güvenliği
konusundaki farkındalığının halen çok yüksek olmadığı, işletmeler hariç diğer paydaşlar tarafından peyderpey satış ve vadeli ödeme
yöntemlerinin olumlu karşılandığı, orman ürünü üretimlerinde yerel alan dışından işçi çalıştırılmasının pek kabul görmediği ve
üretim sürelerinin işin miktarına göre düzenlenmesi gerektiği paydaşlar tarafından belirtilmektedir. A
ht
k li
l
T
k i
Dikili ğ
t
6521
l t
i
6877
l t
i
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Tü ki ş
g
g
ğ p y
ş
Anahtar kelimeler: Tomruk piyasası, Dikili ağaç satışı, 6521 sayılı tamim, 6877 sayılı tamim, Kastamonu, Türkiye @
* Corresponding author (İletişim yazarı): gsen@kastamonu.edu.tr
✓ ✓ Received (Geliş tarihi): 24.10.2023, Accepted (Kabul tarihi): 18.11.2023
a Kastamonu Üniversitesi, Orman Fakültesi, Orman Mühendisliği Bölümü,
37150, Merkez, Kastamonu
@
a Kastamonu Üniversitesi, Orman Fakültesi, Orman Mühendisliği Bölümü,
37150, Merkez, Kastamonu
@
* Corresponding author (İletişim yazarı): gsen@kastamonu.edu.tr
✓ Received (Geliş tarihi): 24.10.2023, Accepted (Kabul tarihi): 18.11.2023
Citation (Atıf): Aközlü, A., Şen, G., 2023.
Tomruk piyasası aktörlerinin dikili ağaç satış
yönetmeliklerinde yapılan değişikliklere ilişkin
algıları. Turkish Journal of Forestry, 24(4): 378-
389.
DOI: 10.18182/tjf.1395550 1. Giriş New
York eyaletinde özel orman sahipleri yaptıkları bireysel
kontratlar ile satışlarını gerçekleştirirken devlet ormanlarında
bu işlem halka açık bir ihale süreci ile gerçekleştirilmektedir
(DEC, 2023). İrlanda’da ise 3 farklı satış yöntemi
kullanılmaktadır. Bunlar dikili satış, rampada/yol kenarında
satış ve kapı satışıdır (satılan ürün satıcının adresine kadar
götürülür) (Teagasc, 2023). Yüzde 80 oranında özel ormana
sahip olan Avusturya’da (Schenker, 1995) ise yapılan orman
ürünü satışlarının büyük çoğunluğu (%85) alivreli satış
yöntemi ile gerçekleştirilmektedir. Ancak 200 hektar ve
üzerindeki ormanlardan yapılacak satışlarda ise dikili satış
yöntemi kullanılmaktadır (Puwein, 2007). Kongo’da genelde
tüm hakları ile ormanlar yüklenicilere verilerek satış işlemi
yapılmakta (Counsell, 2006), Almanya, Rusya ve İngiltere
gibi ülkelerde ise dikili satış yöntemi kullanılmaktadır (Ünal
ve Karakaya, 2002). İngiltere’de ayrıca, depolardan ihale ile
ve sunta, kağıt vb. üretimi yapan şirketlere uzun vadeli
sözleşmeler ile de satışlar gerçekleştirilebilmektedir (FE,
2023). Fransa’da ise doğrudan satışlar (%26,8), tedarik
sözleşmeleri (18,7) ve açık artırmalı satışlar (%54,5) ile
orman ürünlerinin satışları gerçekleştirilmektedir (Elyakime
ve Cabanettes, 2009). Türkiye’de işletme ve üretim bakımından ormancılık
sektöründe tekel bir yapının olması beraberinde bazı
sorunları da getirmektedir. Bu durum da OGM’nin halen ne
üretirsem onu satarım anlayışı ile pazarlama faaliyetlerini
sürdürmesine neden olmaktadır (Demirel, 2006). Buna karşın
uluslararası ticaret boyut değiştirmiş ülkeler ekonomik
çıkarları için gümrük vergi ve fonlarının kaldırılması gibi
yeni kararlar almıştır (Türker ve Yazıcı, 1998). Bunun
yanında Avrupa Birliği (AB) ile gümrük birliği anlaşmasının
yapılması, Sovyetler Birliği’nin dağılması ve bu ülkelerden
Türkiye’ye bol miktarda düşük fiyatlı odun hammaddesi
girişinin gerçekleşmesi (Daşdemir, 2003) OGM’nin tekel
özelliğini sekteye uğratmaya başlamıştır. Bu gelişmeler
ormancılık sektöründe üretimde kalitenin
artırılması,
maliyetlerin düşürülmesi, tüketici istek ve taleplerinin
dikkate
alınması
ve
yeni
pazarlama
anlayışlarının
oluşturulması gerekliliğini ortaya çıkarmıştır (Demirel,
2006). Değişen piyasa yapısının ve satışların artırılması amacı
ile tomruk satışlarında dikili satış uygulamasına geçilmesi
öngörülmüştür. Maliyetlerin düşürülecek olması, alıcının
istediği gibi ürünü işleyebilmesine olanak sağlaması, üretim
sürecindeki ekonomik kayıpları en aza indirmesi, işçilerin
sigortalanmasının sağlanması gibi nedenler bu yöntemin
uygulamaya başlamasında etkili olmuştur (Daşdemir, 2011). Dikili ağaç satışı uygulaması ilk defa 1996 yılında çıkarılan
5038 sayılı yönetmelikle başlamıştır. Ancak orman idaresinin
koruma,
takip
ve
kontrol
bakımından
endişelerinin
bulunması, köylülerin sosyo-ekonomik konularda itilafa
düşme endişesi, dikili satış fiyatı ve verim yüzdelerinin doğru
belirlenememe ihtimali, alınan dikili üretiminin köylüye
yaptırılıp
yaptırılmayacağının
belirli
olmaması,
kooperatifleri
başvuracaklar
listesine
almaması
gibi
nedenlerden dolayı dikili satış yöntemi yaygınlaşamamıştır
(Kaplan, 2005). Görülen bu eksikliklerin giderilmesi
amacıyla 6057 sayılı tamim, ilk tamimin yayınlanmasından
iki yıl sonra yürürlüğe girmiştir. 1. Giriş kanunlar ile odun ve diğer orman ürünlerinin üretimi, hasadı
ve ticareti belirli kurallara ve izinlere bağlı hale gelmiştir. Ormanlar, geçmişte insanların faydalanmasına açık bir
şekilde varlığını sürdürmekte idi. Çıkarılan çeşitli yasalar
sonrasında
ormanlardan
yapılan
faydalanmalar
sınırlandırılmış ve bazı kurallara bağlı hale getirilmiştir. Bu Orman ürünlerinin üretim ve satışı ile ilgili olarak dünya
üzerinde çeşitli sistemler uygulanmaktadır. Bu sistemlerin
temelinde de ormanların sahipliliği oldukça büyük etki
göstermektedir. Özellikle özel orman sahipleri pazarlama ve
satış konusunda daha esnek iken, devlete ait ormanlarda ve
a Kastamonu Üniversitesi, Orman Fakültesi, Orman Mühendisliği Bölümü,
37150, Merkez, Kastamonu
@
* Corresponding author (İletişim yazarı): gsen@kastamonu.edu.tr
✓ Received (Geliş tarihi): 24.10.2023, Accepted (Kabul tarihi): 18.11.2023
Citation (Atıf): Aközlü, A., Şen, G., 2023. Tomruk piyasası aktörlerinin dikili ağaç satış
yönetmeliklerinde yapılan değişikliklere ilişkin
algıları. Turkish Journal of Forestry, 24(4): 378-
389. DOI: 10.18182/tjf.1395550 @
* Corresponding author (İletişim yazarı): gsen@kastamonu.edu.tr
✓ ✓ Received (Geliş tarihi): 24.10.2023, Accepted (Kabul tarihi): 18.11.2023 Turkish Journal of Forestry 2023, 24(4): 378-389 379 tarafından işletileceği ve devlet ormanlarına ait her çeşit
işlerin Orman Genel Müdürlüğü (OGM) tarafından
yapılacağı” ifade edilmiştir (Daşdemir, 2011). Günümüzde
Türkiye’de özel orman sahipleri ya da tapulu arazilerinde
yetiştirdikleri orman ürünlerini kişiler istedikleri şekilde
satma hakkına sahiptir. Devlet ormanlarında üretilen odun
ürünlerinin satış yöntemleri ise üretim öncesi ve sonrası
döneme göre yapılmaktadır. Üretimden sonra yapılan satışlar
açık arttırmalı satış, yükselterek açık artırmalı satış,
eksilterek açık artırmalı satış, teklif usulü gizli arttırmalı satış
(kapalı zarf), pazarlıklı satış ve tahsisli satış şeklinde
yapılırken, üretimden önce ise dikili ağaç satışları ve alivreli
satışlar
şeklinde
uygulanmaktadır
(Miraboğlu,
1983;
Daşdemir, 2011). devlet
işletmeciliğinde
yönetilen
orman
alanlarından
yapılacak üretim ve satış faaliyetleri daha sıkı bir denetim
altında ve belirli yasa ve yönetmeliklere bağlı kalınarak
yapılmaktadır. Orman ürünleri satış yöntemlerinde özel orman sahipleri
direkt olarak yüklenici ile yapacakları kontratlar ile satışlarını
gerçekleştirebilirler. Buna karşın devlet ormanlarında
satışların kesim yapıldıktan sonra mı yoksa ağaçlar halen
dikili halde iken mi satış yapılacağı önem kazanmaktadır. Bu
kapsamda
ülkeler
farklı
yöntemler
kullanmaktadır. Finlandiya’da kullanılan odunların %9’u devlet orman
işletmelerinden, %85’i özel orman işletmelerinden, %6’sı
endüstriyel
ormancılık
yapan
özel
işletmelerden
karşılanmaktadır. Finlandiya’daki yuvarlak odun satışlarının
%14,5’i dikili satış ve geri kalanı da alivreli satış olarak
gerçekleşmektedir (Melkas ve Oy, 2022). Litvanya’da hem
devlet hem de özel şahıslara ait ormanlar bulunmaktadır. Ancak üretim ve satış işlerinin tamamına yakını özel
işletmeler
tarafından
gerçekleştirilmektedir. Devlet
ormanlarından yapılan tomruk satışlarının %10’u dikili
satışlar,
geri
kalanı
ise
alivreli
satış
şeklinde
pazarlanmaktadır (Gaizutis, 2005; Toppinen vd., 2005). 1. Giriş Bu tamimde tahsisli dikili
ağaç satışlarının kooperatifler tarafından da alınabilmesinin
önü açılmıştır. Böylece orman köylülerinin dikili halde
ağaçları alıp, üretimini yapıp pazara sunma hakları
oluşmuştur (Ünal ve Karakaya, 2002). Ancak dikili ağaç
satışındaki aksaklıklar tam olarak yine çözülememiş ve bu
tamimlerin devamında 2004 yılında 6350 sayılı tamim, 2007
yılında 6521 sayılı tamim, 2013 yılında ise 6877 sayılı tamim
yürürlüğe girmiştir (OGM, 2013). Türkiye’de de diğer ülkelere benzer şekilde birden çok
satış yöntemi uygulanmaktadır. Osmanlı döneminde halk
ormanlardan istediği şekilde yararlanırken (Cibal-i Mübaha),
1870 yılında çıkarılan Orman Nizannamesi ile bu serbest
kullanım durumu sınırlandırılmıştır (Özdönmez vd., 1989). Satışlar ile alakalı olarak getirdiği yenilik ise kesilecek
ürünlerin damgalanma şartı ve üretilen tomrukların idarece
belirlenecek olan bir bedel üzerinden satılmasıdır (Diker,
1947). Ayrıca üretim işlerinin yükleniciler tarafından
yapılacağı dikili satışlarda uygulanmaya başlamıştır. 3116
Sayılı Orman Kanunu çıkarıldığında “devlet ormanlarının
devlet tarafından işletilmesi” hükmü getirilerek (31. Madde)
özel firmaların işlettiği ormanlar devlet işletmesine alınmıştır
(Daşdemir, 2011). 1945 yılında çıkartılan 4785 sayılı yasa ile ormanların
büyük bir bölümü devlet mülkiyeti altına alınmış, devlet
ormanı dışındaki mülkiyet hakkı neredeyse bitmiş ve orman
sınırı içine giren alan miktarı da artırılmıştır (Gümüş, 2004). Daha sonra 1956 yılında 6831 sayılı Orman Kanunu
çıkarılmış ve bu kanuna göre “devlet ormanlarının devlet Yürürlüğe
giren
her
bir
tamim
bir
öncekinin
aksaklıklarını
ve
eksikliklerini
gidermek
amacı
ile
yapılmaktadır. Buna karşın her bir değişiklik tomruk
piyasasındaki tüm paydaşları etkilemekte ve onlar için
olumlu ya da olumsuz yeni durumlar oluşturmaktadır. Bu Turkish Journal of Forestry 2023, 24(4): 378-389 380 nedenle bu değişimlerin etkilerinin belirlenmesi yapılacak
yeni çalışmalar içinde önemli olmaktadır. Dikili satışlar ile
ilgili çeşitli çalışmalar yapılsa da yasa veya yönetmeliklerin
değişimlerinin etkilerinin analiz edildiği başka bir çalışmaya
rastlanılamamıştır. Bu çalışmada, 6521 sayılı dikili satış
tamiminin yürürlükten kaldırılıp 6877 sayılı dikili satış
tamiminin yürürlüğe girmesi ile oluşan değişikliklerin dikili
ağaç satışı ile alakalı ilgi grupları üzerindeki etkilerinin
incelenmesi amaçlanmıştır. Çalışma sonuçlarının hem
literatürdeki eksikliğin doldurulmasında hem de karar verici
ve kanun yapıcılara önemli bir referans bilgileri sağlayacağı
düşünülmektedir. Şekil 1. Kastamonu coğrafi konumu 2.2.1. Örnek alan büyüklüğünün belirlenmesi Çalışmada öncelikle para, zaman, araç, gereç ve uzman
yetersizliği
vb. sebeplerle
örnek
alan
belirlemesi
gerçekleştirilerek
sınırlı
sayıdaki
örnek/grup
birimde
örneklem araştırmaları yapılmaktadır (Özdamar, 2002). Bu
kapsamda çalışmada öncelikle anket uygulaması yapılacak
Orman İşletme Müdürlükleri belirlenmiştir. Samatlar, Kastamonu sınırları içerisinde bulunan Orman
İşletme Müdürlükleri’nden işletmesinin bazı dönemlerde
Araç işletmesine bağlı olmasından dolayı Araçsam adı ile
değerlendirilmiştir. Karadere Orman işletme Müdürlüğü
sınırları içinde yerleşim yeri olmadığından dolayı da
Kastamonu Orman İşletme Müdürlüğü ile değerlendirilerek
araştırmada Kaskar olarak isimlendirilmiştir. KOBM’ne
bağlı Orman İşletme Müdürlükleri dikili satışta etkili
olabilecek belirlenen bazı değişkenlere göre (Çizelge 1)
Ranking metodu ile sıralanmıştır (Çizelge 2). Ranking
(sıralama) tekniğinde, karar elemanları karar verici tarafından
göreceli önem derecelerine göre sıralanmaktadır (Yılmaz,
2006). Bu teknik kapsamında orman işletme müdürlüklerinin
göreceli öncelik derecelerine göre hüküm verilmekte ve
sıralanmaktadır. Bu sıralama işlemi, “dokuz dereceli ölçek”
vasıtasıyla yapılmaktadır. Bu ölçekte; 1- Zayıf oranda
önemli, 3- Daha az önemli, 5- Orta derecede önemli, 7- Daha
çok önemli, 9- Aşırı derecede önemli olarak kabul
edilmektedir. Bunlar yanında “2, 4, 6 ve 8 değerleri” de orta
değerler olarak kullanılabilmektedir (Güngör ve Şen, 2018). Ranking’de en yüksek değerleri alan 3 Orman İşletme
Müdürlüğü örnek alan olarak belirlenmiş ve bu alanları
temsil edebilecek istatistiksel olarak anlamlı en küçük anket
sayıları belirlenmiştir. 2.1. Materyal 2.1. Materyal Bu çalışmanın materyalini, 2007 tarihli 6521 sayılı tamim
ile 2013 yılında yürürlüğe giren 6877 sayılı tamim arasındaki
değişikliklerin ilgi grupları üzerindeki etkilerini belirlemek
amacıyla Kastamonu ilinde yapılan anketlerden elde edilen
veriler, konu ile ilgili daha önce yapılmış çalışmalar ve diğer
ikincil veriler oluşturmaktadır. Şekil 1. Kastamonu coğrafi konumu Çalışma
Kastamonu
ilinde
gerçekleştirilmiştir. Kastamonu ili, Karadeniz bölgesinin batı kesiminde 41-42°
kuzey enlemleri ile 33-46° doğu boylamları arasında yer
almaktadır (Şekil 1). Yüzölçümü 13.108 km2 olan il, Türkiye
yüzölçümünün yaklaşık %1,7’sini oluşturmaktadır (Kravkaz-
Kuscu, 2018; Şen vd., 2022; TOB, 2023). İl genelinde en
yüksek sıcaklık ortalaması 16°C, en düşük sıcaklık
ortalaması ise 4,2°C’dir ve yıllık ortalama yağış miktarı
486,4 mm’dir (Kurnaz ve Güneş Şen, 2023). Kastamonu ili
tarımsal faaliyetlerin yüksek olduğu ve il yüzölçümünün
%64’ünün ormanlarla kaplı olduğu bir ildir. 695.763 hektarı
normal kapalı, 180.551 hektarı boşluklu kapalı olmak üzere
toplam orman alanı 876.314 hektardır. Bu ormanların
yönetimi 16 işletme müdürlüğü olan Kastamonu Orman
Bölge Müdürlüğü (KOBM) tarafından gerçekleştirilmektedir
(KOBM, 2023). Kastamonu ilinde Küre Dağları Milli Parkı
ve Ilgaz Dağı Milli Parkı olmak üzere iki adet milli park
bulunmaktadır (Ayan vd., 2007). Küre Dağları Milli Parkı
Platin Yaban Hayatı Sertifikasına sahiptir (Meydan ve
Öztürk, 2023). Kastamonu iline bağlı 20 ilçe ve 1.054 köy
bulunmaktadır (TÜİK, 2020). 2.2. Yöntem Bu çalışmada, 6521 sayılı dikili satış tamiminin
yürürlükten kaldırılıp 6877 sayılı dikili satış tamiminin
yürürlüğe girmesi ile oluşan değişikliklerin dikili ağaç satışı
ile alakalı ilgi gruplarının görüşlerinin belirlenmesi amacıyla
oluşturulan araştırma hipotezi aşağıdaki gibidir; H0a:
Tomruk
piyasası
aktörlerinin
dikili
satış
yönetmeliklerinde yapılan değişiklikler hakkındaki bilgi
düzeyleri ve görüşleri arasında fark yoktur. Turkish Journal of Forestry 2023, 24(4): 378-389 381 Çizelge 1. Anket yapılacak Orman İşletme Müdürlüğü seçiminde kullanılan değişkenler
Dikili satışa konu orman ürünü
miktarı/Toplam üretilen orman
ürünü miktarı (m3)
Kooperatif
sayısı (adet)
Odun kökenli
ürün işleyen
işletme sayısı
(adet)
Toplam köy
nüfusu
Dikili satışa
konu ürün
miktarı (m3)
Verimli orman
alanı (ha)
Değişken
1
2
3
4
5
6
Azdavay
0,12
5,00
5,00
6096
13770,00
29079,00
Cide
0,42
4,00
24,00
18657
32209,00
35643,50
Çatalzeytin
0,18
7,00
10,00
4075
10778,00
19309,00
Daday
0,18
35,00
5,00
7501
27518,00
52422,00
İhsangazi
0,13
9,00
3,00
3530
8025,00
19420,50
İnebolu
0,17
6,00
12,00
25543
14423,00
40341,10
Küre
0,13
14,00
21,00
14401
14891,00
28549,50
Taşköprü
0,08
61,00
25,00
19451
22018,00
82469,50
Tosya
0,40
24,00
350,00
10934
47050,00
55503,00
Pınarbaşı
0,11
3,00
5,00
3463
9376,00
28992,00
Bozkurt
0,27
7,00
10,00
4640
16520,00
21892,00
Hanönü
0,00
16,00
0,00
2270
347,00
19247,00
Araçsam
0,26
2,00
7,00
14661
31422,00
61997,00
Kaskar
0,17
68,00
150,00
31443
23840,50
66873,00
Çizelge 2. Anket yapılacak Orman İşletme Müdürlüğü seçimi için yapılan Normalizasyon sonuçları
Normalize
-1
Normalize
-2
Normalize
-3
Normalize
-4
Normalize
-5
Normalize
-6
Toplam
normalize
Genel
oranlama
Azdavay
0,05
0,02
0,01
0,04
0,05
0,05
0,21
0,04
Cide
0,16
0,02
0,04
0,11
0,12
0,06
0,51
0,08
Çatalzeytin
0,07
0,03
0,02
0,02
0,04
0,03
0,21
0,03
Daday
0,07
0,13
0,01
0,05
0,10
0,09
0,45
0,08
İhsangazi
0,05
0,03
0,00
0,02
0,03
0,03
0,17
0,03
İnebolu
0,06
0,02
0,02
0,15
0,05
0,07
0,39
0,06
Küre
0,05
0,05
0,03
0,09
0,05
0,05
0,33
0,05
Taşköprü
0,03
0,23
0,04
0,12
0,08
0,15
0,65
0,11
Tosya
0,15
0,09
0,56
0,07
0,17
0,10
1,14
0,19
Pınarbaşı
0,04
0,01
0,01
0,02
0,03
0,05
0,17
0,03
Bozkurt
0,10
0,03
0,02
0,03
0,06
0,04
0,27
0,05
Hanönü
0,00
0,06
0,00
0,01
0,00
0,03
0,11
0,02
Araçsam
0,10
0,01
0,01
0,09
0,12
0,11
0,43
0,07
Kaskar
0,06
0,26
0,24
0,19
0,09
0,12
0,96
0,16
1,00
1,00
1,00
1,00
1,00
1,00
6,00
1,00 elge 1. Anket yapılacak Orman İşletme Müdürlüğü seçiminde kullanılan değişkenler Belirlenen işletme müdürlüklerindeki 4 ilgi grubu ile
anket
yapılması
kararlaştırılmıştır. 2.2. Yöntem Aşağıda bu değişiklikler maddeler halinde
gösterilmiştir. alınmıştır. Aşağıda bu değişiklikler maddeler halinde
gösterilmiştir. 2.2. Yöntem Bunlar;
orman
kooperatifleri (ORKOP), orman köylüsü (OK), orman
işletme müdürlüğü çalışanları (OİMÇ) ve odun kökenli ürün
işleyen işletmeler (OKÜİ)’dir. Yapılacak anket sayıları eşitlik
1 ile belirlenmiştir. anketler hazırlanmıştır. Oluşturulan anket formları yapılan ön
uygulama ile denenmiş ve yenilenmiştir. Hazırlanan anketler
belirlenen ilgi gruplarına yüz yüze anket yöntemi ile
uygulanmış olup, bu anketlerde ilgi gruplarına dair
tanımlayıcı özellikleri ve değişen dikili ağaç satışı
tamimlerinin paydaşlar üzerine etkilerini belirlemeye yönelik
sorulara yer verilmiştir. n =
F∗t2∗P∗Q
(F∗m2)+(t2∗P∗Q)
(1) Çalışmadaki analizler SPSS 21 istatistik paket programı
ile gerçekleştirilmiştir. Gruplar arası denek sayılarının eşit
olmaması ve verilerin sıralı olması nedeniyle ilgi grupları
arasında farkların olup olmadığı tek yönlü varyans analizinin
parametrik olmayan karşılığı olan Kruskal-Wallis Analizi ile
incelenmiştir (Özdamar, 2002). İstatistiksel olarak gruplar
arasındaki farkın anlamlı olduğu durumlarda hangi gruplar
arasında farkın olduğunu bulmak için Mann-Whitney U testi
yapılmış ve sonrasında Bonferroni düzeltmesi yapılarak
grupların
etki
büyüklüğü
hesaplanmıştır. Bonferroni
düzeltmesi 3 ve daha fazla grup için yapılmaktadır. 3 grup
olduğunda her birinin kendi alfa yanılma yüzdesi olduğundan
0,05
üçe
bölünür
ve
anlamlılık
0,0167’ye
göre
değerlendirilir. 3’den fazla gruplarda ise bu 0,01 olarak kabul
edilir. Z değeri için etki büyüklüğü r = Z / √N formülüyle
bulunur (Cevahir, 2020). (1) Formülde n= Örnek büyüklüğü, F= Anakütle, P= Tahmini
oran, Q= 1-P, t= Güven düzeyi %95 (Tablo değeri: 1,96) ve
m= Hata payı %10’dır. Formüle göre belirlenen örneklem
büyüklükleri Çizelge 3’te gösterilmiştir. Anket güvenilirliğini
artırmak için ulaşılabilen daha fazla kişiyle anket yapılmıştır. 2.2.2 Anket formlarının geliştirilmesi ve değerlendirme
yöntemleri Anket formlarının belirlemesinde öncelikle 6521 ve 6877
sayılı dikili satış yönetmelikleri incelenerek farklılıkları
ortaya çıkarılmıştır. Devamında çalışmanın amacında
belirlenen hipotezlerin test edilmesine imkân verecek şekilde Turkish Journal of Forestry 2023, 24(4): 378-389 382 Çizelge 3. Çalışmadaki ana kütle, örneklem ve uygulanan anket sayıları
Anket yapılan işletme
müdürlükleri
OİMÇ
OK
OKÜİ
ORKOP
Ana
kütle
(Kişi)
Örnek
sayısı
Anket
sayısı
(Adet)
Ana kütle
(Kişi)
Örnek
sayısı
Anket
sayısı
(Adet)
Ana
kütle
(Kişi)
Örnek
sayısı
Anket
sayısı
(Adet)
Ana
kütle
(Kişi)
Örnek
sayısı
Anket
sayısı
(Adet)
Tosya
11
25
8
4.922
96
66
350
81
54
24
59
18
Taşköprü
14
11
8.158
67
25
18
61
32
Kaskar
17
11
11.460
60
150
63
68
20
Toplam
42
30
24.540
193
525
135
153
70
ORKOP: Orman kooperatifleri, OK: Orman Köylüsü, OİMÇ: Orman İşletme M0üdürlüğü Çalışanları, OKÜİ: Odun Kökenli Ürün İşleyen İşletmeler Çizelge 3. Çalışmadaki ana kütle, örneklem ve uygulanan anket sayıları
OİMÇ
OK alınmıştır. Değişiklik 1. ORKOP’ların %18,7’sinin ortak sayısı 10-20,
%34,3’ünün 21-40, %18,6’sının 41-60 ve %28,4’ünün de 61-
371 arasında değiştiği belirlenmiştir. ORKOP katılımcılarının
%90’ı kooperatiflerindeki üye sayısının yeterli olduğunu
ifade etmişlerdir. Katılımcıların faaliyet alanları tamamıyla
ormancılıktır ancak ek olarak sulama, tarım, akaryakıt ve
taşıma işleriyle de ilgilendiklerini belirtmişlerdir. Değişiklik 2. 3.1. İlgi gruplarının bazı özellikleri Değişiklik 1. Değişiklik 1. Anket
uygulaması
yapılan
ilgi
gruplarının
bazı
tanımlayıcı özellikleri her bir grup için ayrı ayrı verilmiştir. OK’deki katılımcıların %23,8’i (46 kişi) 20-40 yaş
aralığında, %66,3’ü (128 kişi) 41-60 yaş aralığında ve %9,9’u
da 61-80 yaş aralığındadır. OK katılımcılarının %20,7’si
okuryazar, %68,9’u ilköğretim mezunu, %9,8 oranında lise
ve dengi okul mezunu ve %0,5 oranında ise yüksekokul-
üniversite mezunudur. Katılımcıların %92,5’i en az üç iş
kolundan diğerleri ise iki iş kolundan gelir elde etmektedirler. Gelir elde edilen iş kollarının %17,9’unu tarım, %26,1’ini
hayvancılık,
%35,9’unu
ormancılık,
%12,6’sı
ticaret
oluşturmaktadır. %7,5’i ise emekli maaşı almakta olduğunu
belirtmişlerdir. OK katılımcılarının %43’ü (83 kişi) yıllık
9.000-15.000 TL, %31,1’i 16.000-20.000 TL, %13’ü 21.000-
29.000 TL, %9,3’ü 30.000-40.000 TL ve %3,6’sı ise
%41.000-50.000
TL
yıllık
gelir
elde
ettiklerini
belirtmişlerdir. Gelirlerinin tamamını ormancılıktan kazanan
kişilerin oranı sadece %3,6, %50’ye kadar olan kısmını
ormancılıktan sağlayanların oranı ise %72,6 olarak
belirtilmiştir. 6521/2- “Dikili ağaç satışında, dikili ağacın m3 satış
bedeli, satışa konu bölme veya bölmecikte pazarlama
imkânları da göz önüne alınarak üretilmesi öngörülen emvale
göre her ağaç türü için verim yüzdelerinin tespit edilmesi ve
bu
yüzdelere
göre
çıkması
gereken
ürünlerin
fiyatlandırılması yapılarak belirlenmektedir. Verim yüzdeleri
gerçeğe ne kadar yakın belirlenirse alıcı ve satıcı yönünden
doğru sonuca ulaşılacak ve dikili satışın yaygınlaşmasına
olumlu katkı sağlayacaktır. Verim yüzdesi tespitinde ayrıca
alt tabakadaki fertler ile şüceyrat odununun dikili damgaya
ve verim yüzdesine dâhil etmede özellikle dikkatli olunarak,
deneme ağaçları seçilirken objektif kriterler ve aşağıdaki
hususlara uyulacaktır.” 6877/A-2- “Dikili ağaç satışında maliyet bedeli,
muhammen bedel ve satış fiyatına esas teşkil etmesi
nedeniyle; satışa konu bölme veya bölmecikte ki her ağaç
türü için üretilmesi öngörülen ürünlerin verim yüzdesi
hassasiyetle belirlenir. Ancak, yapılan dikili damgada toplam
miktarın %5'inin altında olup piyasa değeri açısından özel
kıymet ifade etmeyen ağaç türlerinde ayrıca verim yüzdesi
tespit edilmeyebilir. Bu durumda söz konusu hacim benzer
ağaç türü hacmine ilave edilmek suretiyle değerlendirilir. Ayrıca verim yüzdesi tespitinde alt tabakadaki fertler ile
şüceyrat odununu dikili damgaya ve verim yüzdesine dâhil
etmede özellikle dikkatli olunarak, deneme ağaçları
seçiminde objektif kriterlere ve aşağıdaki hususlara uyulur.” ORKOP’ların ortak sayıları 10 ile 371 kişi arasında
değişmektedir. ORKOP’ların %18,7’sinin ortak sayısı 10-20,
%34,3’ünün 21-40, %18,6’sının 41-60 ve %28,4’ünün de 61-
371 arasında değiştiği belirlenmiştir. ORKOP katılımcılarının
%90’ı kooperatiflerindeki üye sayısının yeterli olduğunu
ifade etmişlerdir. Katılımcıların faaliyet alanları tamamıyla
ormancılıktır ancak ek olarak sulama, tarım, akaryakıt ve
taşıma işleriyle de ilgilendiklerini belirtmişlerdir. ORKOP’ların ortak sayıları 10 ile 371 kişi arasında
değişmektedir. Değişiklik 2. Anket yapılan orman idaresi personelinin %60’ının
meslekteki süreleri 2-5 yıl, %30’unun 6-8 yıl, %10’ununda
21-40 yıldır. 6521/2.1.1- “Satışa konu bölme veya bölmecikteki
ağaçların Dikili Kabuklu Gövde Hacmi (DKGH) ve çap
kademelerine dağılımı göz önüne alınarak DENEME AĞACI
HACİM BÜYÜKLÜĞÜ tespit edilir. Hacim büyüklüğü
satışa konu ağaçların temsiline imkan verecek şekilde 1 m3,
3 m3, 5 m3, 8 m3 gibi belirlenecektir. Burada dikkat edilmesi
gereken husus D.K.G.H miktarı arttıkça ve ağırlıklı çap grubu
kalınlaştıkça daha iyi temsil için deneme ağacı hacim
büyüklüğünün artması gerekmektedir.” 6521/2.1.1- “Satışa konu bölme veya bölmecikteki
ağaçların Dikili Kabuklu Gövde Hacmi (DKGH) ve çap
kademelerine dağılımı göz önüne alınarak DENEME AĞACI
HACİM BÜYÜKLÜĞÜ tespit edilir. Hacim büyüklüğü
satışa konu ağaçların temsiline imkan verecek şekilde 1 m3,
3 m3, 5 m3, 8 m3 gibi belirlenecektir. Burada dikkat edilmesi
gereken husus D.K.G.H miktarı arttıkça ve ağırlıklı çap grubu
kalınlaştıkça daha iyi temsil için deneme ağacı hacim
büyüklüğünün artması gerekmektedir.” OKÜİ katılımcılarının faaliyet alanları kereste ticareti,
mobilya
üretimi
ve
marangozlardan
oluşmaktadır. Katılımcıların %34,8’i 50-68, %31,9’u 42-49 ve %33,3’ü de
25-37 yaş aralığındadır. Bu kişilerin %45,9´u ilköğretim,
%43,0’ı lise ve dengi ve %8,1’i de yüksekokul-üniversite
mezunudur. %3’ü ise sadece okuryazardır. Bu kişilerin
%31,1’inin yıllık geliri 32.000-50.000 TL, %16,3’ünün
52.000-75.000 TL aralığında, %10,4’ ünün 80.000-94.000 TL
aralığında olduğu belirlenmiştir. Gelirleri hakkında bilgi
vermeyenlerin oranı ise %42,2’dir. 6877/1.2.1.1. “Satışa konu bölme veya bölmecikteki
ağaçların DKGH ve çap kademelerine dağılımı göz önüne
alınarak deneme ağacı hacim büyüklüğü tespit edilir. Hacim
büyüklüğü, satışa konu ağaçların temsiline imkan verecek
şekilde 1 m3, 3 m3, 5 m3, 8 m3 gibi belirlenir. Burada dikkat
edilmesi gereken husus DKGH miktarı arttıkça ve ağırlıklı
çap grubu kalınlaştıkça daha iyi temsil için deneme ağacı
hacim
büyüklüğünün
artması
gerekmektedir. Hacim
büyüklüğü tespit edilirken (deneme alanı alınanlar hariç)
alınacak olan deneme ağacı hacmi o ağaç türünün damgadaki
toplam miktarının %2’sinden aşağı olamaz. Ancak; kızılçam 6877/1.2.1.1. “Satışa konu bölme veya bölmecikteki
ağaçların DKGH ve çap kademelerine dağılımı göz önüne
alınarak deneme ağacı hacim büyüklüğü tespit edilir. Hacim
büyüklüğü, satışa konu ağaçların temsiline imkan verecek
şekilde 1 m3, 3 m3, 5 m3, 8 m3 gibi belirlenir. Burada dikkat
edilmesi gereken husus DKGH miktarı arttıkça ve ağırlıklı
çap grubu kalınlaştıkça daha iyi temsil için deneme ağacı
hacim
büyüklüğünün
artması
gerekmektedir. Hacim
büyüklüğü tespit edilirken (deneme alanı alınanlar hariç)
alınacak olan deneme ağacı hacmi o ağaç türünün damgadaki
toplam miktarının %2’sinden aşağı olamaz. Ancak; kızılçam 3. Bulgular 3.1. İlgi gruplarının bazı özellikleri Değişiklik 5. Değişiklik 5. Değişiklik 5. 3.2. 6521 ve 6877 sayılı dikili ağaç satış usul ve esasları
hakkındaki tamimler arasındaki farklılıklar 3.2. 6521 ve 6877 sayılı dikili ağaç satış usul ve esasları
hakkındaki tamimler arasındaki farklılıklar 3.2. 6521 ve 6877 sayılı dikili ağaç satış usul ve esasları
hakkındaki tamimler arasındaki farklılıklar Dikili satış uygulamasında yapılan değişiklikler hakkında
ilgi gruplarının görüşlerini belirlemek için öncelikle 6521 ve
6877 sayılı tamimler arasındaki değişiklikler belirlenmiştir. Tamimlerdeki farklılıklar belirlenirken uygulamada ilgi
gruplarına etki edebilecek seviyedeki değişiklikler dikkate Turkish Journal of Forestry 2023, 24(4): 378-389 383 Orman Kalkındırma Kooperatiflerinin tahsisli dikili ağaç
satışlarında üretim işçiliği üyelerince yapılacağı yazılı beyan
edilmesi halinde sigorta mevzuatı yönünden sorumlulukları
kendilerine ait olmak üzere Sosyal Sigortalar Kurumu’na
bildirimde bulunulmayacaktır.” Orman Kalkındırma Kooperatiflerinin tahsisli dikili ağaç
satışlarında üretim işçiliği üyelerince yapılacağı yazılı beyan
edilmesi halinde sigorta mevzuatı yönünden sorumlulukları
kendilerine ait olmak üzere Sosyal Sigortalar Kurumu’na
bildirimde bulunulmayacaktır.” Orman Kalkındırma Kooperatiflerinin tahsisli dikili ağaç
satışlarında üretim işçiliği üyelerince yapılacağı yazılı beyan
edilmesi halinde sigorta mevzuatı yönünden sorumlulukları
kendilerine ait olmak üzere Sosyal Sigortalar Kurumu’na
bildirimde bulunulmayacaktır.” tensil sahası ve endüstriyel plantasyon sahalarında,
meşcerenin homojen yapıya sahip olması halinde %1’den az
olmamak şartıyla belirlenebilir.” Değişiklik 3. Değişiklik 4. 6521/ (Tamimde yok)
6877/1.17. “Orman yön 6521/ (Tamimde yok)
6877/1.17. “Orman yönetiminin belgelendirildiği (Forest
Stewardship Council –FSC- ve Programme for the
Endorsement of Forest Certification -PEFC-) ormanlarda
yapılacak dikili ağaç satışlarında belgelendirmenin zorunlu
gerekleri şartnamelerin özel hükümler bölümünde belirtilir.” 6877/1.17. “Orman yönetiminin belgelendirildiği (Forest
Stewardship Council –FSC- ve Programme for the
Endorsement of Forest Certification -PEFC-) ormanlarda
yapılacak dikili ağaç satışlarında belgelendirmenin zorunlu
gerekleri şartnamelerin özel hükümler bölümünde belirtilir.” 6521/2.4-“Alıcı elde ettiği ürünleri nakletmek istediğinde
Orman İşletme Şefliğinin ölçü ve tespitine göre nakline izin
verilecektir. Peyderpey satış veya parçalı ölçüm yapılması
halinde; satışı yapılan dikili gövde hacminden çıkabilecek
ürün miktarı gözetilerek toplam dikili miktarın %75’ne
kadarının nakline izin verilecektir. Dikili miktarın %25’inden
az olmamak üzere kalan miktar öncekilerle birlikte işletme
şefliğince değerlendirilerek dikili kabuklu gövde hacminden
veya yüzde yüzün üzerinde verim yüzdesi tespiti yapılmış ise
bu miktardan az olması durumunda işletme şefliğince nakline
müsaade edilecektir. Dikili satış işleminin her aşaması
idarece kontrol edilerek, tüm ölçü tespit tutanaklarının birer
nüshası İşletme Müdürlüğüne gönderilecektir. Elde edilen
toplam ürün miktarı; dikili kabuklu gövde hacminden veya
yüzde yüzün üzerinde verim yüzdesi tespiti yapılmış ise
bundan fazla olması durumunda, son %25’lik kısma ait
bölüm nakledilmeden durum İşletme Müdürlüğünce Bölge
Müdürlüğüne bildirilir. Bölge Müdürlüğünce kurulacak
komisyon marifetiyle alıcının da katılabileceği şekilde en geç
bir hafta içerisinde durum incelenerek herhangi bir
usulsüzlük
olup
olmadığını
tespitle
işletmesine
bildirilecektir. Usulsüzlük yoksa ürünün nakline izin
verilecek olup aksi durumda şartname hükümlerine göre
işlem yapılır.” 6521/ (Tamimde yok) 6521/ (Tamimde yok) 6877/1.18. “Baltalık uygulamaları, sıklık bakımı gibi
önceden DKGH'nin sağlıklı olarak tespit edilemediği
çalışmalarda dikili ağaç satışı söz konusu olması durumunda
genel sahayı temsil edecek büyüklükte ve özellikte alınacak
deneme alanı ölçümleri genel sahaya uyarlanarak işlem
yapılır.” 6877/1.18. “Baltalık uygulamaları, sıklık bakımı gibi
önceden DKGH'nin sağlıklı olarak tespit edilemediği
çalışmalarda dikili ağaç satışı söz konusu olması durumunda
genel sahayı temsil edecek büyüklükte ve özellikte alınacak
deneme alanı ölçümleri genel sahaya uyarlanarak işlem
yapılır.” Değişiklik 7. Değişiklik 6. Değişiklik 6. Değişiklik 3. 6877/1.15. “Bölge Müdürlüğünün dikili ağaç satışlarına
ait, ihale onay yazısı veya tahsis fiyatına ait onay yazısının
İşletme Müdürlüğü kaydına girdiği tarihten itibaren 15 gün
içinde, İşletme Müdürlüklerince satışın konusu, alıcısı, yeri
ve mahiyeti hakkında SSGK bildirimde bulunulur. Ayrıca
müşterinin fiilen işe başladığı tarih de SGK’ya bildirilir. Ancak; tahsisli dikili ağaç satışlarında orman köylerini
kalkındırma
kooperatiflerinin
üretim
işçiliğini
kendi
üyelerince yapılacağını yazılı beyan etmesi halinde sigorta
mevzuatı yönünden sorumlulukları kendilerine ait olmak
üzere SGK'ya bildirimde bulunulmaz.” 6521/2.4- “Dikili satışa konu bölme ve bölmecikteki
verim yüzdesinin tespiti için gerekli iş ve işlemlere ait işçilik
giderleri 730/1000 ölçme ve diğer giderler hesabından
karşılanacaktır.” 6521/2.4- “Dikili satışa konu bölme ve bölmecikteki
verim yüzdesinin tespiti için gerekli iş ve işlemlere ait işçilik
giderleri 730/1000 ölçme ve diğer giderler hesabından
karşılanacaktır.” 6877/1.2.4. “Dikili ağaç satışına konu bölme ve
bölmecikteki verim yüzdesinin tespiti için gerekli iş ve
işlemlere ait işçilikler Genel Yönetim Giderleri-İşçi Ücret
Giderleri Hesabından (770.04 nolu hesap) idareye ait işçiler
tarafından yapılır. İdareye ait işçilerin yeterli olmaması
durumunda, OGM tarafından gönderilen talimata uygun
olarak verim yüzdelerinin tespiti konusunda hizmet alımı
yapılabilir.” Değişiklik 8. 3.3. İlgi gruplarının dikili satış tamim değişikliği hakkındaki
görüşleri 6521/ (Tamimde yok)
Ü 6521/ (Tamimde yok)
Ü (
y
)
6877/4.8.2. “Orman Ürünleri Satış Usul ve Esasları
Hakkında Yönetmeliğin 9 uncu maddesinin (1) inci fıkrasının
(ö) bendine göre “Yıllık odun hammadde işleme kapasitesi
yirmibeşbin metreküp ve üzeri olan her türlü fabrika ve
tesislere, yıllık kapasite kullanım miktarlarına göre Genel
Müdürlükçe belirlenen oranlarda odun ve dikili ağaçlar”
tahsisli satış usulüyle satılabilir. Yıllık odun hammadde
işleme kapasitesi 25 bin metreküp ve üzeri olan her türlü
fabrika ve tesislere yapılacak tahsisli satışlarda, dikili ağaç
satışı yaptıran gerçek ve tüzel kişiler üretim işçiliğini
kendileri yapabileceği gibi dışarıdan hizmet alımıyla da
yaptırabilirler. Dikili ağaçlardan elde edilen ürünlerin tahsis
yapılan fabrika veya tesislerde değerlendirilemeyecek
olanları hariç, tahsise konu fabrika ve tesislerin dışında bir
yere nakline müsaade edilmeyeceği gibi bu ürünlerin
işlenmeden devrine veya satışına da izin verilmez. Tahsisli
Dikili Ağaç Satış Esasları Bu madde kapsamındaki fabrika ve
tesislere yapılacak tahsisli satışlarda, gerçek dışı beyanda
bulunulması, sahte evrak ibraz edilmesi, dikili ağaç satışı
yaptıran gerçek ve tüzel kişilerin dikili ağaç satışından elde
ettiği ürünlerden kendi fabrika ve tesislerinde işlenebilecek
olanları tahsise konu fabrika ve tesislerde işlemeksizin
devretmesi veya satması durumunda kalan tahsisleri iptal
edilerek, 5 yıl süre ile tahsis verilmez. Ayrıca bu fabrika ve
tesislerle İdaremiz arasında protokol imzalanacak olup, bu y p
6877/1.6. “Müşteri elde ettiği ürünleri nakletmek
istediğinde; işletme şefliğince düzenlenen ölçü ve tespit
tutanakları esas alınarak ürünlerin nakliye tezkeresi ile
nakline izin verilir. Dikili ağaç satış işleminin her aşaması
idarece kontrol edilerek, tüm ölçü tespit tutanaklarının birer
nüshası son ölçü tespit tutanağı ile birlikte İşletme
Müdürlüğüne gönderilir. Yapılan ölçü tespitleri sırasında,
dikili ağaç satışından elde edilen toplam ürün miktarı; verim
yüzdesinin %10'u üzerinde gerçekleşmesi halinde durum
İşletme Müdürlüğünce Bölge Müdürlüğüne bildirilir. Bölge
Müdürlüğünce kurulacak komisyon marifetiyle müşterinin
da katılabileceği şekilde en geç bir hafta içerisinde durum
incelenerek herhangi bir usulsüzlük olup olmadığını tespitle
İşletme Müdürlüğüne bildirilecektir. Usulsüzlük olması
durumunda
mevzuat
gereğince
işlem
yapılacaktır. Usulsüzlük yoksa ürünün nakline izin verilecektir.” y
6521/14-
“Satışın
yapılmasını
müteakip,
İşletme
Müdürlüklerince satışın konusu, yeri ve mahiyeti hakkında
ilgili Sosyal Sigortalar Kurumu’na bilgi verilecektir. Ancak Turkish Journal of Forestry 2023, 24(4): 378-389 384 protokol ve şartnamelerdeki hükümlere göre işlemler
gerçekleştirilecektir.” belirtilen gün sayıları dikkate alınarak idareyle belirledikleri
iş takvimine göre iş mahallinde bulundurmak zorundadır.” 3.3. İlgi gruplarının dikili satış tamim değişikliği hakkındaki
görüşleri Değişiklik 8. 6521/ (Tamimde yok) Tomruk
piyasası
aktörlerinin
dikili
satış
yönetmeliklerinde yapılan değişiklikler hakkındaki bilgi
düzeyleri ve görüşleri arasında fark olup olmadığının
belirlenmesine yönelik oluşturulan araştırma hipotezinin test
edilmesine yönelik sorulan dokuz soruya alınan cevaplar
Kruskal-Wallis ve Man-Whitney U testleri ile analiz edilerek
grupların görüşleri arasında istatistiksel olarak anlamlı
farklılıklar olup olmadığı belirlenmeye çalışılmıştır. İlgi
gruplarının görüşleri ve analiz sonuçlarında gruplar arasında
istatistiksel olarak anlamlı farklılık bulunanlar Çizelge 4-
Çizelge 13’de gösterilmiştir. 6877/4.8.2. “Orman köylerini kalkındırma kooperatifleri
ile köylerdeki gerçek kişilere yapılacak tahsisli satışlarda;
gerçek dışı beyanda bulunulması, sahte evrak ibraz edilmesi,
dikili ağaç satışı alanların üretim işini yapmaksızın aldıkları
dikili
ağaçları
başkalarına
devretmesi
veya
satması
durumunda
tahsisleri
iptal
edilerek
teminatları
irad
kaydedilir. Bu durumdaki orman köylerini kalkındırma
kooperatifleri ile köylerdeki gerçek kişilere 5 yıl tahsisen
dikili ağaç satışı yapılmaz. Tahsis talebinde bulunduğu halde
aynı yıl içerisinde iki defa satışını yaptırmayan talep
sahiplerine bir yıl tahsisli dikili ağaç satışı yapılmaz.”
Değişiklik 9. “6875 sayılı tamimle verim yüzdesi tespiti için hacim
büyüklüğünün DKGH’nin %2 ye çıkarılması sizce verim
yüzdesi hesabının daha sağlıklı yapılmasını sağlar mı?” (S1)
sorusuna ORKOP katılımcılarının %87,5’i ve OKÜİ’lerin
%93,3’ü alanın daha iyi temsil edileceği ve daha doğru sonuç
alınacağı düşüncesi ile evet derken, ORKOP’un %14,3’ü ve
OKÜİ’nin %6,7’si ise verim yüzdesinin hali hazırda yanlış
hesaplandığını ve değişme olmayacağını belirterek hayır
demiştir. İki grubun görüşleri arasında anlamlı fark olup
olmadığına dair yapılan Mann-Whitney U testi sonuçları
Çizelge 4’te gösterilmiştir. 6521/ (Tamimde yok) 6521/ (Tamimde yok)
6877/5.1. “Dikili ağaç satışı yaptırmak isteyen gerçek ve
tüzel kişiler, Bölge Müdürlüğü dâhilinde 60 günlük üretim
periyodunda 500 m3 ve üzeri satışlar için ihalenin
kesinleşmesini müteakip satış yaptırmadan önce, 5531 sayılı
Orman Mühendisliği, Orman Endüstri Mühendisliği ve Ağaç
İşleri Endüstri Mühendisliği Hakkında Kanun uyarınca
orman ürünleri üretimi alanında yetkili orman mühendisi
veya orman yüksek mühendisi çalıştırmak zorunda olup,
müşteri oda belgesi ile birlikte; 5.1.1. Bölge Müdürlüğü
dâhilinde
60
günlük
üretim
periyodunda,
işletme
müdürlüklerinden 12.000 m³ ve katları dikili ağaç satın
alanlar; her 12.000 m³ için en az 6 ay süreli hizmet akdiyle,
orman
mühendisi
veya
orman
yüksek
mühendisi
çalıştırdığına dair noterden düzenlenmiş sözleşmeyi, beşinci
bölüm 5.1.2. Bölge Müdürlüğü dâhilinde 60 günlük üretim
periyodunda, İşletme Müdürlüklerinden 12.000 m³’e kadar
dikili ağaç satın alanlar; en az 6 ay süreli hizmet akdiyle
orman
mühendisi
veya
orman
yüksek
mühendisi
çalıştırdığına dair noterden düzenlenmiş sözleşmeyi veya
dikili ağaç satış süresini kapsayacak şekilde düzenlenmiş
olmak şartıyla, serbest ormancılık bürosu ya da ormancılık
şirketiyle yapılmış danışmanlık hizmet alımı sözleşmesini
ibraz etmek zorundadır. Bir orman mühendisi veya orman
yüksek mühendisi 2 aylık üretim periyodunda 6.000 m³’e
kadar dikili ağaç satışında danışmanlık hizmeti verebilir. Müşteri, orman mühendisi ya da orman yüksek mühendisini
yukarıda yapılan açıklama çerçevesinde Ek-7'deki örnekte Çizelge 4’te görüldüğü üzere ORKOP ve OKÜİ’nin S1
hakkındaki görüşleri açısından istatistiksel olarak anlamlı bir
farklılık olmadığı tespit edilmiştir. “2013 öncesindeki ve sonrasındaki 6521 ve 6877 sayılı
tamim dönemlerinde dikili satışa verilen alanlarda transport
sorunu yaşadınız mı ve yaşıyor musunuz? (S2) sorusuna
ORKOP’un %57,1’i, OK’nin %7,3’ü ve OKÜİ’lerin %
10,4’ü
6521
sayılı
tamimde
transportta
sorunlar
yaşadıklarını, ORKOP’un %15,7’si, OK’nin %1’i ve
OKÜİ’ninde %4,4’ü de herhangi bir transport sorunu
yaşamadıklarını belirtmişlerdir. 6877 sayılı tamimle transport
sorunlarının halledildiğini söyleyenlerin oranı ise ORKOP’ta
%58,6, OK’de 56,2 ve OKÜİ’de %14,12dir. Sorunların
tamim değişikliğine rağmen devam ettiğini söyleyenlerin
oranı ise ORKOP’ta %14,2, OK’de %2,1 ve OKÜİ’de %0,7
olarak belirtilmiştir. Gruplardaki katılımcıların geri kalanları
ise bu konu hakkında fikir beyan etmemişlerdir. Üç grubun S2 hakkındaki görüşleri arasında anlamlı fark
olup olmadığına dair yapılan Kruskal-Wallis ve Mann-
Whitney U testi sonuçları Çizelge 5’te gösterilmiştir. Çizelge 4. 6521/ (Tamimde yok) İlgi gruplarının S1 hakkındaki görüşlerine dair farklılık analizi
Mann-Whitney U Testi
Gruplar
N
Sıra ortalaması
Mann-Whitney U
Wilcoxon W
Z
Anlamlılık düzeyi (p)
S1
ORKOP
57
106,09
4042,0
15068,0
-0,921
0,357
OKÜİ
148
101,81
S1-N
ORKOP
57
106,09
4042,0
15068,0
-0,921
0,357
OKÜİ
148
101,81
Anlamlılık düzeyi: p<0,05 ise anlamlı Turkish Journal of Forestry 2023, 24(4): 378-389 385 Çizelge 5. İlgi gruplarının S2 hakkındaki görüşlerine dair farklılık analizi
Kruskal-Wallis
Gruplar
N
Sıra ortalaması
Ki-Kare
sd
Anlamlılık düzeyi (p)
S2-6521 sayılı tamim
ORKOP
51
43,88
1,582
2
0,453
OK
16
39,94
OKUİ
20
47,55
S2-6877 sayılı tamim
KOOP
51
42,07
2,108
2
0,349
OK
16
44,72
OKÜİ
20
48,35
Anlamlılık düzeyi: p<0,05 ise anlamlı Çizelge 5. İlgi gruplarının S2 hakkındaki görüşlerine dair farklılık analizi Çizelge 5’te görüldüğü üzere ORKOP, OK ve OKÜİ’nin
S2 hakkındaki görüşleri açısından istatistiksel olarak anlamlı
bir farklılık olmadığı tespit edilmiştir. “Dikili satış ödemelerinin vadeli olarak yapılması dikili
satış alımlarının artmasında rol oynamakta mıdır?” (S5)
sorusuna ORKOP’un %98,6’sı, OK’un %73’ü, OİMÇ’nin
%73,3’ü ve OKÜİ’nin %94,8’i dikili satışı artıracağı,
ORKOP’un %1,4’ü, OK’un %25,4’ü, OİMÇ’nin %10’u ve
OKÜİ’nin %5,2’si dikili satışları artırmayacağı şeklinde
görüş bildirmiştir. ğ
p
ş
“2013 yılında yapılan değişiklikle birlikte 6877 sayılı
tamimde,
60
günlük
üretim
periyodunda,
orman
işletmelerinden 6.000 m³ ve katları dikili ağaç satın alanlar
için; her 6.000 m³ için en az 6 ay süreli hizmet akdiyle, orman
mühendisi veya orman yüksek mühendisi çalıştırma
zorunluluğu getirilmiş olmasını nasıl değerlendirirsiniz ve
nedenleri nelerdir?” (S3) sorusuna ORKOP’un %88,6’sı,
OK’nin %93,8’i, OİMÇ’nin %82,8’i ve OKÜİ’nin %62,2’si
gerekli ve faydalı bulduğunu belirtirken, ORKOP’un
%11,4’ü, OK’nin %6,2’si, OİMÇ’nin %17,2’si ve OKÜİ’nin
%37,8’i ise gereksiz bulduğunu belirtmiştir. Orman
mühendisliği çalıştırma zorunluluğunu gerekli bulanlar
içerisinden ORKOP’ta %11,4, OK’de %6,2, OİMÇ’de %82,8
ve OKÜİ’de %37,8 oranında bunun nedenini teknik takip ve
iş imkanı oluşturması olarak belirtmişlerdir. Gereksiz
bulanlar içerisinde ORKOP’un %88,6’sı, OK’de %93,8’i
OİMÇ’de %17,2’si ve OKÜİ’de %62,2’si ise mühendislerin
işe gitmediğini sadece çalışma belgelerini kiraladıklarını
gereksiz bulma nedenleri olarak ifade etmişlerdir. g
İlgi gruplarının S5 hakkındaki görüşleri arasında anlamlı
fark olup olmadığına dair yapılan Kruskal-Wallis ve Mann-
Whitney U testi sonuçları Çizelge 8’de gösterilmiştir. y
ç
Ç
g
g
ş
Çizelge 8’de görüldüğü üzere ORKOP, OK, OKÜİ ve
OİMÇ’nin S5 hakkındaki görüşleri açısından istatistiksel
olarak anlamlı farklılıkların olduğu tespit edilmiştir. “2013 yılında değişen (6877) tamim ile gelen “peyderpey
satış kaldırılmıştır” maddesini destekliyor musunuz?” (S6)
sorusuna ORKOP’un %14,3’ü, OK’nin %5,2’si, OİMÇ’nin
%100’ü desteklediğini, ORKOP’un %85,2’si, OK’nin
%94,8’i ve OKÜİ’nin tamamı desteklemediğini belirtmiştir. 6521/ (Tamimde yok) İlgi gruplarının peyderpey satışın kaldırılmasını destekleme
ve desteklememe nedenleri Çizelge 9’da gösterilmektedir. Çizelge 9’da görüldüğü üzere ORKOP’un %87,1’i maddi
yetersizlikler ve ihaleye girerken gereken nakit bulma
zorluğundan dolayı desteklememektedir. OK’un %72,5’i
kooperatiflerin dikili satış alamadığı ve işçilerin sigorta
sorunundan dolayı yükleniciler ile çalışılmak zorunda
kalınmasından dolayı büyük oranda desteklememektedir. OİMÇ’lerin tamamı işin kontrolünün sağlanması ve işlerin
zamanında bitirilebilmesi nedenleriyle peyderpey satışın
kaldırılmasını desteklediğini belirtmiştir. OKÜİ’lerin tamamı
da bu uygulamanın küçük işletmeler için zararlı olduğundan
dolayı eski uygulamanın devamının gerekli olduğunu
belirtmektedirler. g
İlgi gruplarının S3 hakkındaki görüşleri arasında anlamlı
fark olup olmadığına dair yapılan Kruskal-Wallis ve Mann-
Whitney U testi sonuçları Çizelge 6’te gösterilmiştir. Çizelge 6’te görüldüğü üzere ORKOP, OK, OKÜİ ve
OİMÇ’nin S3 ve nedenleri hakkındaki görüşleri açısından
istatistiksel olarak anlamlı farklılıkların olduğu tespit
edilmiştir. 6877 sayılı tamimle birlikte iş güvenliği ve çevreye daha
duyarlı
dikili
satışın
yapılmasını
nasıl
değerlendiriyorsunuz?” (S4) sorusuna ORKOP’un %25,7’si,
OK’nin %20,2’si, OİMÇ’nin %73,3’ü ve OKÜİ’nin %59,3’ü
çevre ve işçilerin sağlığı açısından olumlu olduğunu,
ORKOP’un %47,2’si OK’nin %41,5’i, OİMÇ’nin %26,7’si
ve OKÜİ’nin %34,1’i işleri yavaşlattığını belirtmişlerdir. Buna karşın ORKOP’un %27,1’i, OK’nin %38,3’ü ve
OKÜİ’nin de %6,6’sı da değişen bir şey olmadığını ifade
etmişlerdir. İlgi gruplarının S6 hakkındaki görüşleri arasında anlamlı
fark olup olmadığına dair yapılan Kruskal-Wallis ve Mann-
Whitney U testi sonuçları Çizelge 10’de gösterilmiştir. y
g
g
Çizelge 10’da görüldüğü üzere ORKOP, OK, OKÜİ ve
OİMÇ’nin S6 hakkındaki görüşleri açısından istatistiksel
olarak anlamlı farklılıkların olduğu tespit edilmiştir. “Köylerin
çevrelerindeki
ormanlarda
yapılacak
üretimlerde yöre dışından şahısların ihaleye katılmalarını
nasıl değerlendiriyorsunuz?” (S7) sorusuna ORKOP’un
tamamı, OİMÇ’lerin ise %50’si olumsuz bir durum olarak
gördüklerini belirtmiştir. OK’nin %94,5’i ve OKÜİ’nin
%89,6’sı ise bu durumu olumlu karşıladıklarını beyan
etmişlerdir. Bu düşüncelerinin nedenleri Çizelge 11’de
gösterilmiştir. İlgi gruplarının S4 hakkındaki görüşleri arasında anlamlı
fark olup olmadığına dair yapılan Kruskal-Wallis ve Mann-
Whitney U testi sonuçları Çizelge 7’de gösterilmiştir. Çizelge 7’de görüldüğü üzere ORKOP, OK, OKÜİ ve
OİMÇ’nin S4 hakkındaki görüşleri açısından istatistiksel
olarak anlamlı farklılıkların olduğu tespit edilmiştir. Turkish Journal of Forestry 2023, 24(4): 378-389 386 Çizelge 6. İlgi gruplarının S3 ve nedenler hakkındaki görüşlerine dair farklılık analizi
Kruskal-Wallis
Mann-Whitney U testi
Gruplar
N
Sıra ort. Ki-Kare sd
Anlam. düzeyi (p)
Gruplar
Mann-
Whitney U
Wilcoxon W
Z
Anlam. 6521/ (Tamimde yok) düzeyi (p)
S3
ORKOP
70
236,77
108,161
3
0,000
ORKOP-OİMÇ
291,0
726,0
-6,871
0,000
OK
193
248,19
ORKOP-OKÜİ
3480,0
12660,0
-3,942
0,000
OİMÇ
29
85,55
OK-OİMÇ
678,0
1113,0
-9,769
0,000
OKÜİ
135
180,91
OK-OKÜİ
8925,0
18105,0
-6,905
0,000
OİMÇ-OKÜİ
1077,0
1512,0
-4,398
0,000
S3-N
ORKOP
70
236,16
116,071
3
0,000
ORKOP-OİMÇ
291,0
726,0
-6,871
0,000
OK
193
249,22
ORKOP-OKÜİ
3480,0
12660,0
-3,942
0,000
OİMÇ
29
84,22
OK-OİMÇ
639,5
1074,5
-10,481
0,000
OKÜİ
135
180,03
OK-OKÜİ
8806,5
17986,5
-7,317
0,000
OİMÇ-OKÜİ
1077,0
1512,0
-4,398
0,000
Anlamlılık düzeyi: p<0,05 ise anlamlı Çizelge 6. İlgi gruplarının S3 ve nedenler hakkındaki görüşlerine dair farklılık analizi
Kruskal-Wallis
Ma Anlamlılık düzeyi: p<0,05 ise anlamlı Çizelge 7. İlgi gruplarının S4 ve nedenleri hakkındaki görüşlerine dair farklılık analizi
Kruskal-Wallis
Mann-Whitney U testi
Gruplar
N
Sıra ort. Ki-Kare
sd
Anlam. düzeyi (p)
Gruplar
Mann-
Whitney U
Wilcoxon
W
Z
Anlam. düzeyi (p)
S4
ORKOP
70
232,92
75,603
3
0,000
ORKOP-OKÜİ
2787,5
11967,5
-5,007
0,000
OK
193
257,34
ORKOP-OİM
607,5
1042,5
-3,183
0,001
OİMÇ
29
149,88
OK-İM
1510,5
1945,5
-4,235
0,000
OKÜİ
135
153,14
OK-OKÜİ
6584,5
15764,5
-8,121
0,000
Anlamlılık düzeyi: p<0,05 ise anlamlı Çizelge 7. İlgi gruplarının S4 ve nedenleri hakkındaki görüşlerine dair farklılık analizi
Kruskal-Wallis
Ma Çizelge 8. İlgi gruplarının S5 ve nedenler hakkındaki görüşlerine dair farklılık analizi
Kruskal-Wallis
Mann-Whitney U testi
Gruplar
N
Sıra ort. Ki-Kare
sd
Anlam. düzeyi (p)
Gruplar
Mann-
Whitney U
Wilcoxon W
Z
Anlam. düzeyi (p)
S5
ORKOP
70
232,92
43,603
3
0,000
ORKOP-OK
50285,0
7513,5
-4,456
0,000
OK
193
257,34
ORKOP-OKÜİ
4547,5
7032,5
-1.314
0,000
OİMÇ
29
149,88
ORKOP-OİM
747,0
3232,0
-4,133
0,000
OKÜİ
135
153,14
OK-OKÜİ
10172,0
19352,0
-5,069
0,000
Anlamlılık düzeyi: p<0,05 ise anlamlı Çizelge 8. İlgi gruplarının S5 ve nedenler hakkındaki görüşlerine dair farklılık analizi
K
k l W lli
M Çizelge 9. İlgi gruplarının peyderpey satış usulün 6877 sayılı tamim ile kaldırılması hakkındaki düşüncelerinin nedenleri
ORKOP (%) OK (%) OİMÇ (%) OKÜİ (%)
Maddi yetersizlikler ve nakit bulma zorluğu
87,1
22,3
-
-
Alanın tamamına girilmesinden dolayı iş imkânının artması
12,9
5,2
-
-
Kooperatifler dikili satışları alamadığı için ve sigorta sorunlarından dolayı yüklenici ile birlikte
çalışılması gerekiyor
-
72,5
-
-
İşin zamanında bitmesi için
-
-
46,4
-
İşin kontrolünün sağlanması ve karışıklıkların önlenebilmesi için
-
-
53,3
-
Küçük işletmeler için zararlı olduğundan kaldırılmasını ve eski uygulamanın devam etmesi daha
uygun olacaktır
-
-
-
100 Çizelge 9. 6521/ (Tamimde yok) İlgi gruplarının peyderpey satış usulün 6877 sayılı tamim ile kaldırılması hakkındaki düşüncelerinin nedenleri
ORKOP (%) OK (%) OİMÇ (%) OKÜİ (%)
Maddi yetersizlikler ve nakit bulma zorluğu
87,1
22,3
-
-
Alanın tamamına girilmesinden dolayı iş imkânının artması
12,9
5,2
-
-
Kooperatifler dikili satışları alamadığı için ve sigorta sorunlarından dolayı yüklenici ile birlikte
çalışılması gerekiyor
-
72,5
-
-
İşin zamanında bitmesi için
-
-
46,4
-
İşin kontrolünün sağlanması ve karışıklıkların önlenebilmesi için
-
-
53,3
-
Küçük işletmeler için zararlı olduğundan kaldırılmasını ve eski uygulamanın devam etmesi daha
uygun olacaktır
-
-
-
100
Çizelge 10. İlgi gruplarının S6 ve nedenler hakkındaki görüşlerine dair farklılık analizi
Kruskal-Wallis
Mann-Whitney U testi
Gruplar
N
Sıra ort. Ki-Kare
sd
Anlam. düzeyi (p)
Gruplar
Mann-
Whitney U
Wilcoxon
W
Z
Anlam. düzeyi (p)
S6
ORKOP
70
208,50
242,208
3
0,000
ORKOP-OİMÇ
145,0
580,0
-7,903
0,000
OK
193
227,94
ORKOP-OKÜİ
4085,0
6570,0
-4,071
0,000
OİMÇ
29
25,50
OK-OİMÇ
145,0
580,0
-12,483
0,000
OKÜİ
135
2337,42
OK-OKÜİ
12449,0
31170,0
-2,195
0,028
OİMÇ-OKÜİ
14,5
449,5
-12,506
0,000
ORKOP-OK
1743,5
4228,5
-10,362
0,000
S6-N
ORKOP
70
60,97
336,770
3
0,000
ORKOP-OİMÇ
,000
2485,0
-8,953
0,000
OK
193
159,58
ORKOP-OKÜİ
39,5
2524,5
-13,975
0,000
OİMÇ
29
274,17
OK-OİMÇ
140,0
18861,0
-9,645
0,000
OKÜİ
135
358,22
OK-OKÜİ
171,5
18892,5
-16,483
0,000
OİMÇ-OKÜİ
29,0
464,0
-12,346
0,000
Anlamlılık düzeyi: p<0,05 ise anlamlı Maddi yetersizlikler ve nakit bulma zorluğu Maddi yetersizlikler ve nakit bulma zorluğu Maddi yetersizlikler ve nakit bulma zorluğu Çizelge 10. İlgi gruplarının S6 ve nedenler hakkındaki görüşlerine dair farklılık analizi
Kruskal-Wallis
Mann-Whitney U testi
Gruplar
N
Sıra ort. Ki-Kare
sd
Anlam. düzeyi (p)
Gruplar
Mann-
Whitney U
Wilcoxon
W
Z
Anlam. düzeyi (p)
S6
ORKOP
70
208,50
242,208
3
0,000
ORKOP-OİMÇ
145,0
580,0
-7,903
0,000
OK
193
227,94
ORKOP-OKÜİ
4085,0
6570,0
-4,071
0,000
OİMÇ
29
25,50
OK-OİMÇ
145,0
580,0
-12,483
0,000
OKÜİ
135
2337,42
OK-OKÜİ
12449,0
31170,0
-2,195
0,028
OİMÇ-OKÜİ
14,5
449,5
-12,506
0,000
ORKOP-OK
1743,5
4228,5
-10,362
0,000
S6-N
ORKOP
70
60,97
336,770
3
0,000
ORKOP-OİMÇ
,000
2485,0
-8,953
0,000
OK
193
159,58
ORKOP-OKÜİ
39,5
2524,5
-13,975
0,000
OİMÇ
29
274,17
OK-OİMÇ
140,0
18861,0
-9,645
0,000
OKÜİ
135
358,22
OK-OKÜİ
171,5
18892,5
-16,483
0,000
OİMÇ-OKÜİ
29,0
464,0
-12,346
0,000
Anlamlılık düzeyi: p<0,05 ise anlamlı Çizelge 10. İlgi gruplarının S6 ve nedenler hakkındaki görüşlerine dair farklılık analizi
Kruskal-Wallis
Ma Turkish Journal of Forestry 2023, 24(4): 378-389 387 şartları
beğenmeyebileceğini
ve
ihaleye
girmekten
vazgeçebileceğini belirtmişlerdir. Çizelge 11’den de görüldüğü üzere ORKOP, OK ve
OKÜİ büyük oranda aynı fikirdedir ve sadece köylülerin
kendi köy sınırları içerisinde yapılacak işlerde çalışmasının
gerekli olduğunu belirtmektedirler. İlgi gruplarının S8 hakkındaki görüşleri arasında anlamlı
fark olup olmadığına dair yapılan Kruskal-Wallis ve Mann-
Whitney U testi sonuçları Çizelge 13’te gösterilmiştir. İlgi gruplarının S7 hakkındaki görüşleri arasında anlamlı
fark olup olmadığına dair yapılan Kruskal-Wallis ve Mann-
Whitney U testi sonuçları Çizelge 12’de gösterilmiştir. Çizelge 13’de görüldüğü üzere ORKOP, OK, OKÜİ ve
OİMÇ’nin S8 hakkındaki görüşleri açısından istatistiksel
olarak anlamlı farklılıkların olduğu tespit edilmiştir. y
g
g
Çizelge 12’de görüldüğü üzere ORKOP, OK, OKÜİ ve
OİMÇ’nin S7 hakkındaki görüşleri açısından istatistiksel
olarak anlamlı farklılıkların olduğu tespit edilmiştir. “Üretim pazarlama periyodunda süre yetiyor mu? (S9)
sorusuna ORKOP’un %35,7’si,
OK’nin
%6,7’si ve
OKÜİ’nin %12,6’sı evet yanıtını vermiştir. Bazen diyenlerin
oranı ORKOP’ta %17,i, OK’de %3,1 ve OKÜİ’de %8,12dir. ORKOP’un %20’si ve OK’nin %1,6’sı ise hayır cevabını
vermişlerdir. Geri kalan katılımcılar ise konu hakkında
fikirlerinin olmadığını belirtmişlerdir. Yapılan Kruskal-
Wallis analizi sonuçlarında ilgi gruplarının üretim ve
pazarlama periyodunda sürenin yeterliliği hakkındaki
görüşleri arasında istatistiksel olarak anlamlı bir fark tespit
edilememiştir. “Aynı yıl içerisinde iki defa tahsis talebinde bulunduğu
halde satış anlaşmasını imzalamayanların yeni bir ihaleye
katılımının
iptal
edilme
yetkisini
nasıl
değerlendiriyorsunuz?” (S8) sorusuna ORKOP’un %82,92u
OK’nin %93,8’i, OİMÇ’nin tamamı ve OKÜİ’nin %98,5’i
olumlu bir değişim olduğunu beyan etmişlerdir. Bu kararların
nedeni olarakta değişikliğin suistimallerin engellenmesine
fayda sağlayacağını düşünmeleri olarak belirtmişlerdir. Olumsuz olarak değerlendirenler ise alıcının gerektiğinde Çizelge 11. 4. Tartışma ve sonuç Ormancılık mesleği iş kazalarının yoğun olarak yaşandığı
bir iş koludur. Ancak bunun dışında aşırı fiziki strese neden
olan aşırı hava koşulları, gürültü, vibrasyon, gibi nedenlerle
duyma bozukluğu vb. çeşitli meslek hastalıklarına da neden
olmaktadır. Yeni Zelanda’da yapılan bir çalışmada da on beş
yıl tomruk üretiminde çalışan orman işçilerinin yarısında
duyma sorunları göründüğü belirtilmektedir. Bu kapsamda
yeni tamimde iş güvenliği ile ilgili değişiklikler olumlu
gözükmektedir. 15 yıl odun üretim işinde çalışan işçilerin
yarısında duyma bozukluğu meydana geldiği belirlenmiştir
(Poschen, 1993). Bu çalışma tamime eklenen maddeyi
destekler
niteliktedir. Buna
karşın
özellikle
orman
köylülerinin büyük çoğunluğunun yapılan çalışmaların bir işe
yaramayacağını ve işleri yavaşlatacağını düşünmesi üretim
işinde çalışan ve tehlikelerle yüz yüze olan kişilerin bu
durumu
anlamamış
ve
içselleştirememiş
olduğunu
göstermektedir. Bu durumun özellikle eğitimsizlikten
kaynaklandığı düşünülmektedir. Buna karşın orman işçileri iş
güvenliği donanımlarının pahalı olmasından dolayı da iş
güvenliği çalışmalarına sıcak bakmadıklarını görüşmeler
esansında belirtmişlerdir. Oysaki yapılan çalışmalar başlık,
göz koruyucu, güvenlik eldiveni, pantolon ve bot gibi kişisel
koruyucu donanım kullanımının yaralanmaları yıllık bazda
%20 azalttığını göstermektedir. Finlandiya’da yapılan bu
çalışma
da
özellikle
botların
bu
yaralanmaların
azaltılmasında çok önemli olduğunu belirtmektedir (Klen ve
Vayrynen, 1984). Belirtilmesi gereken bir husus da
ergonomik olmayan iş güvenliği ekipmanlarının kaza riskini
artırmasıdır. Burada önemli olan bir diğer önemli hususta
orman idarecilerinin yaklaşık 527’sinin bu uygulamaların
kazaların önüne geçmede etkili olmadığını düşünmeleridir. Bu işleri koordine edecek ve uygulamasını yönetecek
kişilerin bu işe inanmıyor oluşu iş güvenliği çalışmalarının
henüz idareciler tarafından bile tam olarak öneminin
anlaşılamamış olduğunu göstermektedir. Araştırmada dikili satışla alakalı olan dört farklı ilgi
grubuna (orman kooperatifleri, orman köylüleri, orman
işletmesi çalışanları ve odun kökenli ürün işleyen işletmeler)
anket uygulaması yapılmıştır. Anket, gözlem ve mülakatlar
sonucunda elde edilen bulgular neticesinde elde edilen
sonuçlar aşağıda verilmiştir. ç
ş ğ
ş
Dikili satış uygulamasının en önemli aşaması olan verim
yüzdesi tespitine göre ihaleye çıkılmakta ve bölmeyi teslim
edecek ölçümler yeterince dikkatli ve hassas bir şekilde
yapılmadığında alıcı ya da orman işletmeleri maddi zarara
uğrayabilmektedir. Verim yüzdesinin hesabı ile ilgili
değişikliği odun kökenli ürün işleyen işletmelerin ve
kooperatiflerin büyük çoğunluğu olumlu karşılamıştır. Doğu
Karadeniz’de yapılan bir çalışmada çalışma sonuçlarını
destekler niteliktedir. Odun kökenli ürün işleyen işletmelerin
yaklaşık %75’i verim yüzdesi hesabından memnun
olmadıklarını belirtmişlerdir. Bunun nedeni olarak ta %44,8’i
seçilen ağaçların verim yüzdesini yansıtmadığını, %33,3’ü
ise orman işletmesinin verim yüzdesini hesaplamada
yanlışlıklar
yaptığı
şeklinde
görüş
bildirmişlerdir. 4. Tartışma ve sonuç Kooperatiflerin ise neredeyse yarıya yakını (%42,8) verim
yüzdesinden memnun olmadıklarını belirtmiş ve bu kişilerde
verim yüzdesi hesabındaki ağaç seçiminin yanlış yapıldığını
ve arazi şartlarından kaynaklanan değer kayıplarının,
bölmecikler bazında hesaplanan verim yüzdesi değerine
eklenmediğini belirtmişlerdir (İslamoğlu, 2010). Ayrıca ilgi
gruplarının %10-15’inin verim yüzdesi hesabının yanlış
yapıldığını ve değişikliklerin bunu değiştirmeyeceğini
düşünmeleri orman idaresine olan güvensizliği göstermesi
bakımından önemlidir. Üretim işlerinin daha çok kooperatifler yolu ile
yapılmasından dolayı en fazla sorununda kooperatifler
tarafından yaşadığını görmekteyiz. Odun kökenli ürün
işleyen işletmeler müşteri pozisyonunda olduklarından
onların da yüksek oranda sorun yaşadığı görülmektedir. Boylama ve bölmeden çıkarma dikili satış uygulamasının en
önemli aşamalarından biri olduğundan bu kısımda çıkan
olumsuzluklar ve sorunlar paydaşların dikili satış üzerindeki
görüşlerini olumsuz etkilemektedir. Mevcut transport
sorunları bir nebzede olsa çözülmüş görünse de halen devam
eden sorunlar bulunmaktadır. Dikili satışların vadeli yapılmasının satışları artırıcı
etkide bulunduğu ilgi grupları tarafından dile getirilmiş olsa
da ipotek olarak gösterilebilen gayrimenkullerin yerine
değerli kâğıtların geçmesi gelir durumu çok düşük olan
orman köylüsü için huzursuzluk yaratan bir durum
oluşturmaktadır. Bunun sonucunda orman köylüleri ve
kooperatifler sadece yüklenicilerin taşeronluğunu yapan,
onlar için aracı kurum gibi çalışan bir yapı haline
bürünmüşlerdir. Bunun sonucunda da elde edilecek gelir
yörede kalmayacağından kırsal kalkınmaya olan desteği de
azalmaktadır. Dikili satışlardaki usulsüzlük ve diğer aksamaların
giderilmesi
noktasında
orman
mühendisi
çalıştırma
zorunluluğunun getirilmesi her ne kadar olumlu bir adım gibi
görünse de uygulamadaki aksaklıklar kooperatiflerin ve
orman köylülerinin büyük çoğunluğunun bu durumu
istememesine yol açmaktadır. Ancak tamimde belirtildiği
üzere orman mühendislerinin danışmanlık açısından ve alıcı
ile orman idaresi arasındaki teknik takibin sağlanması
açısından önemli oldukları bir gerçektir. Buna karşın bu
değişikliğin yapılmasının nedenlerinden biri usulsüzlüklerin
önlenmesidir. Bu kapsamda çalışanların başlarında bir
denetim görevlisinin bulunmasını istememeleri ve bunu
olumsuz olarak değerlendirmeleri de anlaşılabilir bir
durumdur. Benzer şekilde Yener (2014) çalışmasında, dikili
üretiminde çalışan orman mühendislerinin her ne kadar
orman işletme şeflerinin sorumluluklarını azaltmamış olsa da
çıkabilecek sorunlarda da muhataplarının yükleniciler yerine
bölmede çalışan orman mühendislerinin olmasının teknik
elemanların sorunlar karşısında işlerini kolaylaştırdığını
belirtmektedir. Peyderpey satışın kaldırılması işletmeler haricinde pek
destek bulmamıştır. Bu durumla birlikte üretimini yapıp
sattığı malların parası ile farklı işlere de girebilirken bu
durumda alanın tamamını almak gerektiğinden orman
köylüleri ve kooperatifler düşük gelirleri nedeni ile oldukça
zorlanmaktadırlar. Sonucunda da köylü ve kooperatifleri
müteahhitlere muhtaç eden bir durum oluşmaktadır. Yeni tamimde belirtilen altmış günlük üretim süresinin
yeterli olup olmadığı tam olarak belirli değildir. Ancak, dikili
satış sürecinde kesme, boylama, sürütme, transport ve
pazarlama faaliyetleri ile birlikte bu sürenin kısa olduğu
düşünülmektedir. Maddi yetersizlikler ve nakit bulma zorluğu İlgi gruplarının S7’ye dair görüşlerinin nedenleri
ORKOP %
OK %
OİMÇ %
OKÜİ %
Kendi sınırları içerisindeki köylülerle çalışmalı
100
93.2
3.3
89.6
Çalışabilir (işgücü yetersizse)
0
6.2
13,3
10.4
Rekabet getirecektir
-
-
3.3
-
İşin yavaşlaması davaya konu olması
-
-
26.7
-
Daha düşük fiyattan çalışılacağı için devlete kazanç sağlabır
-
-
30
-
İhaleyi alan şahıs kendi işçilerini çalıştırabilir
-
-
23.5
- Çizelge 11. İlgi gruplarının S7’ye dair görüşlerinin nedenleri Çizelge 12. İlgi gruplarının S7 ve nedenler hakkındaki görüşlerine dair farklılık analizi
Kruskal-Wallis
Mann-Whitney U testi
Gruplar
N
Sıra
ort. Ki-Kare sd Anlam. düzeyi (p)
Gruplar
Mann-Whitney
U
Wilcoxon
W
Z
Anlam. düzeyi
(p)
S7
ORKOP
70
234
71,556
3
0,000
ORKOP-OİMÇ
490,0
925,0
-6,499
0,000
OK
193
221,83
ORKOP-OKÜİ
4235,0
4235.0
-2,784
0,005
OİMÇ
29
123,57
OK-OİMÇ
1510,5
1945,5
-7,171
0,000
OKÜİ
135
211,56
OİMÇ-OKÜİ
1148,0
1583
-5,280
0,000
S7-
N
ORKOP
70
212,50
242,812
3
0,000
ORKOP-OİMÇ
,000
2485,0
-9,718
0,000
OK
193
200,13
ORKOP-OKÜİ
4235
4235
-2,784
0,005
OİMÇ
29
413,00
OK-OİMÇ
,000
18721,50
-12,826
0,000
OKÜİ
135
191,86
OİMÇ-OKÜİ
,000
9180
-10,986
0,000
Anlamlılık düzeyi: p<0,05 ise anlamlı Çizelge 12. İlgi gruplarının S7 ve nedenler hakkındaki görüşlerine dair farklılık analizi
Kruskal-Wallis
Mann- Mann-Whitney U testi Çizelge 13. İlgi gruplarının S8 ve nedenler hakkındaki görüşlerine dair farklılık analizi
Kruskal-Wallis
Mann-Whitney U testi
Gruplar
N
Sıra ort. Ki-
Kare
sd Anlam. düzeyi (p) Gruplar
Mann-Whitney
U
Wilcoxon W Z
Anlam. düzeyi
(p)
S8
ORKOP 70
237,01
21,047
3
0,000 ORKOP-OİMÇ
841
1276 -2,366
0,018
OK
193
214,92
ORKOP-OKÜİ
3985
13165 -4,205
0,000
OİMÇ
29
200,50
OKÜİ
135
203,66
S8-
N
ORKOP 70
237,60
21,804
3
0,000 ORKOP-OKÜİ
6017
24738 -2,714
0,007
OK
193
214,27
ORKOP-OKÜİ
3985
13165 -4,205
0,000
OİMÇ
29
201,00
OKÜİ
135
204,16
Anlamlılık düzeyi: p<0,05 ise anlamlı Çizelge 13. İlgi gruplarının S8 ve nedenler hakkındaki görüşlerine dair farklılık analizi
Kruskal-Wallis
Mann Turkish Journal of Forestry 2023, 24(4): 378-389 388 Kaynaklar Meydan, K., Öztürk, S., 2023. Küre Dağları Milli Parkı’nın Hızlı
Kırsal Değerlendirme Yöntemi ile Ekoturizm Potansiyelinin
Belirlenmesi. İksad
Yayınevi,
Ankara
DOI:
https://dx.doi.org/10.5281/zenodo.10057177 Ayan, S., Öztürk, S., Yiğit, 2007. Karadeniz Bölgesi Milli
Parklarının PAN Parks Sertifikasyon Sistemine Uygunlukları. 38. ICANAS Uluslararası Asya ve Kuzey Afrika Çalışmaları
Kongresi, Ankara, Türkiye, 105-129. g
Miraboğlu, M., 1983. Ormancılık İşletme İktisadı. İ.Ü. Orman
Fakültesi, İstanbul. Cevahir, E., 2020. SPSS ile nicel veri analizi rehberi. İzmir, Kibele. Orman Genel müdürlüğü (OGM), 2013. Dikili ağaç satışı, T.C. Orman ve Su İşleri Bakanlığı Orman Genel Müdürlüğü İşletme
ve Pazarlama Dairesi Başkanlığı, http://www.ogm.gov.tr/
ekutuphane/Tamimler/Dikili%20A%C4%9Fa%C3%A7%20Sat
%C4%B1%C5%9F%C4%B1%20(30.04.2013%20de%C4%9F
%C5%9F.i%C5%9Fl.).pdf, Erişim: 11.05.2015. Ö
İ
İ Counsell, S., 2006. Forest governance in the Democratic Republic
of Congo. An NGO perspective. Recommendations for a
Voluntary Partnership Agreement with the EU. Moreton in
Marsh, FERN. Daşdemir, İ., 2003. Asli Orman Ürünlerinde Fiyat Analizi
(Zonguldak Orman Bölgen Müdürlüğü Örneği). ZKÜ Bartın
Orman Fakültesi Yayınları, Bartın. Özdamar, K., 2002. Paket Programlar İle İstatistiksel Veri Analizi-2
(Çok Değişkenli Analizler) SPSS-MİNİTAB, 4. Baskı. Kaan
Kitabevi, Eskişehir. Ö
İ y
Daşdemir, İ., 2011. Ormancılık İşletme Ekonomisi. BÜ Orman
Fakültesi Yayınları, Bartın. Özdönmez, M., İstanbullu, T., Akesen, A., 1989. Ormancılık
Politikası. İÜ Orman Fakültesi Yayın No: 401, İstanbul. Demirel, E., 2006. Açık arttırmalı tomruk satışlarında fiyat oluşum
sürecinin
incelenmesi
(Kahramanmaraş
Orman
Bölge
Müdürlüğü örneği). Yüksek Lisans, ZKÜ Fen Bilimleri
Enstitüsü, Zonguldak. Poschen, P., 1993. Forestry, A Safe and Healthy Profession,
Unasylva 44 (l), 172-185 Puwein, W., 2007. Wood market in Poland towards stabilisation
Price Setting Systems in Austria. International and interindustry
conference, 4 December, Poznan. pp. 25-36 Department of Environmental Conservtion (DEC), 2023. Timber
sales on State Forests. https://www.dec.ny.gov/nature/forests-
trees/state-forests/timber-sales, Erişim: 09.09.2023 Schenker, S., 1995. Private forest companies in Austrıa: Conflicts
and compensation. https://www.fao.org/3/w3722E/w3722e12. htm#TopOfPage, Erişim: 12.10.2023. Diker, M., 1947. Türkiye’de Ormancılık, Dün- Bugün- Yarın. Tarım
Bakanlığı, Orman Genel Müdürlüğü Yayınları, Sayı: 61, Akın
Matbaası, Ankara. Şen, G., Akbulut, S., Karaköse, M., 2022. Ethnopharmacological
study of medicinal plants in Kastamonu province (Türkiye). Open Chemistry, 20(1): 873-911. Elyakime, B., Cabanettes, A., 2009. How to improve the marketing
of timber in France?.Forest Policy and Economics, 11(3): 169-
173. Teagasc, 2023. Watch: Timber categories and sales methods - conifer
forest
management,
https://www.teagasc.ie/news--events/
daily/forestry/watch-timber-categories-and-sales-methods---
conifer-forest-management-.php, Erişim. 12.09.2023. Gaizutis, A., 2005. Gaining a Position For Lithuanian Small-Scale
Forestry Through Creation Of Marketing, Network For Wood
Trade
And
Services. Unasylva,
228
(58)
48-52. https://www.fao.org/3/a1346e/a1346e13.pdf
Erişim:
06.08.2023. Toppinen, A, Viitanen, J., Leskinen, P., Toivonen, R. 2005. 4. Tartışma ve sonuç Özellikle Doğu Karadeniz Bölgesi gibi
coğrafi şartlarının ve hava koşullarının zor olabildiği
alanlarda her gün arazide çalışma yapılamaması gibi
durumlar üretim süresinin az olduğunun düşünülmesini haklı
çıkarmaktadır. Turkish Journal of Forestry 2023, 24(4): 378-389 389 Açıklama Kurnaz, S., Güneş Şen, S., 2023. İbreli ve yapraklı meşcere
türlerinde toprak özellikleri. 3rd International Conference on
Engineering and Applied Natural Sciences, 14-17 Ocak, Konya,
Türkiye, pp.507-513. Bu çalışma, “Ormancılıkta Dikili Satış Yöntemindeki Değişim
ve Uygulamadaki Sorunların Tüm Paydaşlar Açısından İncelenmesi
(Kastamonu Orman Bölge Müdürlüğü Örneği)” başlıklı yüksek
lisans tezinden üretilmiş olup, çalışmanın geniş özeti IV. Ormancılıkta Sosyo-Ekonomik Sorunlar Kongresi’nde sunulmuştur. Melkas, R., Oy, M. 2022. Wood measuring methods used in Finland
2021. https://www.metsateho.fi/wp-content/uploads/
Tuloskalvosarja-2022-7-EN-Wood-measuring-methods-used-
in-Finland-2021.pdf, Erişim: 06.08.2023. Kaynaklar Dynamics of roundwood prices in Estonia, Finland and
Lithuania. Baltic Forestry, 11(1), 88-96. Gümüş, C., 2004. Ormancılık Politikası, Cilt 1. KTÜ Orman
Fakültesi Yayın No:34.444S, Trabzon. Türker, M.F., Yazıcı, K., 1998. Üçüncü Sınıf Normal Boy Ladin
Tomruk Açık Artırmalı Satışlarında Fiyat Oluşumu Sürecinin
Araştırılması (Trabzon Orman Bölge Müdürlüğü Örneği). KTÜ
Orman Fakültesi Seminer Kitabı, Trabzon. Güngör, E., Şen, G., 2018. Selecting suitable forest areas for honey
production using the AHP: A case study in Turkey. CERNE,
24(1): 67-79. TÜİK,
2023. Nüfus
ve
Demografi
2020,
https://data. tuik.gov.tr/Kategori/GetKategori?p=Nufus-ve-Demografi-109
Erişim: 15.09.2023 ( )
İslamoğlu, N., 2010. Devlet ormanlarında yapılan dikili ağaç
satışının paydaşlar açısından irdelenmesi (Trabzon Orman
Bölge Müdürlüğü Örneği). Yüksek Lisans Tezi, KTÜ Fen
Bilimleri Enstitüsü, Trabzon. Forestry England (FE), 2023. Timber Sales. https://www. forestryengland.uk/timber/timbersales Erişim: 13.10.2023 Kaplan, E., 2005. Orman Ürünlerinin Değerlendirilmesinde Dikili
Ağaç Satışı. 1.Çevre ve Ormancılık Şurası, Cilt 3, Antalya. TOB, 2023. Kastamonu Coğrafi Yapısı, https://kastamonu. tarimorman.gov.tr/Menu/46/Cografi-Yapisi, Erişim: 01.09.2023
Ü Kastamonu Orman Bölge Müdürlüğü (KOBM), 2023. Orman
varlığı. https://www.ogm.gov.tr/kastamonuobm/ormanlarimiz/
orman-varligi, Erişim: 22.08.2023. Ünal, S., Karakaya, A., 2002. Orman ürünlerinin üretiminde dikili
ağaç satışı ve uygulamada kooperatiflerin yeri. I.Ulusal
Ormancılık Kooperatifleri Sempozyumu Bildiriler Kitabı, 22-23
Mart, Kastamonu, Türkiye, pp. 51-58. Klen, T., Vayrynen, S., 1984. The role of personal protection in the
prevention of accidental injuries in logging work. Journal of
Occupational Accidents, 6(4): 263-275. Yener, A., 2014. Kahramanmaraş Orman İşletme Müdürlüğü’nde
üretim işlerinde vahidi fiyat ve dikili satış yöntemlerinin
karşılaştırılması üzerine araştırmalar. Yüksek Lisans Tezi,
Kahramanmaraş Sütçü İmam Üniversitesi, Fen Bilimleri
Enstitüsü, Kahramanmaraş. Kravkaz-Kuscu, I.S., Sariyildiz, T., Cetin, M., Yigit, N., Sevik, H.,
Savaci, G., 2018. Evaluation of the soil properties and primary
forest tree species in Taskopru (Kastamonu) district. Fresenius
Environmental Bulletin, 27(3): 1613-1617. Yılmaz, E., 2006. R’WOT Technique: an Example Application with
Participatory Approach in Beekeeping Industry. Doğu Akdeniz
Ormancılık Araştırma Enstitüsü Yayını, Doğu Akdeniz
Ormancılık Araştırma Müdürlüğü Matbaası, Tarsus.
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https://zenodo.org/records/4270089/files/DEVELOPMENT%20OF%20METHODS%20FOR%20ASSESSMENT%20AND%20SELECTION%20OF%20UNMANNED%20AERIAL%20VEHICLE%20FOR%20MINE%20RECONNAISSANCE.pdf
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DEVELOPMENT OF METHODS FOR ASSESSMENT AND SELECTION OF UNMANNED AERIAL VEHICLE FOR MINE RECONNAISSANCE
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ScienceRise
| 2,020
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cc-by
| 4,919
|
Vadym Neroba1 y
1Department of General Military Disciplines, National Academy of the State Border Guard Service of Ukraine named after Bohdan
Khmelnytskyi, Khmelnytskyi, Ukraine
vadym.rostislavovich@gmail.com
ORCID: http://orcid.org/0000-0002-7232-3285 AB ST R AC T
Object of research: comparative assessment and selection of an unmanned aerial vehicle for
mine reconnaissance sample. Investigated problem: substantiation of the methodological apparatus for comparative as-
sessment and selection of an unmanned aerial vehicle for mine reconnaissance sample, tak-
ing into consideration the presence of both quantitative and qualitative indicators. Main scientific results: the methods of comparative assessment and selection of an unmanned
aerial vehicle for mine reconnaissance sample is developed. The technique is based on an ex-
pert method, which allows a drone sample to be evaluated and selected objectively, taking into
consideration the presence of both quantitative and qualitative indicators. At the same time,
group interaction and discussion of experts are realized. When the judgments do not coincide,
an artificial consensus is not imposed. The number of experts is not limited. The experts are
not linked in any way. The need to ensure transitive consistency (10–12 %) makes it possible
to record attempts by an expert (experts) to artificially overestimate the indicators of one of the
drone samples (or the one being evaluated), therefore, the indicators of another sample will au-
tomatically deteriorate. The principle of impartiality and fairness is maintained. A well-trained
objective coordinator is not required, and the reality is that the absence of the disrupting the
problem solution possibility is due to a change in the psychological situation among the experts. Area of practical use of research results: humanitarian demining in the interests of ensur-
ing the detection of mines for various purposes by sappers from a safe distance. At the same
time, an increase within the probability of mines detecting is ensured due to special equip-
ment installed onboard the drone. Innovative technological product: a technique has been developed that allows not only
assessing the drone samples for mine reconnaissance objectively, but making an objective
choice of a sample for specific requirements also. Scope of application of the innovative technological product: clearance of the terrain re-
maining after the end of hostilities. With the help of unmanned aerial vehicles, a significant
acceleration of the demining process is possible, especially in those territories where mines
are installed and being for a sufficiently long time. (2020). Development of methods for assessment and selection of unmanned aerial vehicle for mine reconnaissance.
ScienceRise, 5, 44–50. doi: http://doi.org/10.21303/2313-8416.2020.001496 Object of research: comparative assessment and selection of an unmanned aerial vehicle for
mine reconnaissance sample. Investigated problem: substantiation of the methodological apparatus for comparative as-
sessment and selection of an unmanned aerial vehicle for mine reconnaissance sample, tak-
ing into consideration the presence of both quantitative and qualitative indicators. Main scientific results: the methods of comparative assessment and selection of an unmanned
aerial vehicle for mine reconnaissance sample is developed. The technique is based on an ex-
pert method, which allows a drone sample to be evaluated and selected objectively, taking into
consideration the presence of both quantitative and qualitative indicators. At the same time,
group interaction and discussion of experts are realized. When the judgments do not coincide,
an artificial consensus is not imposed. The number of experts is not limited. The experts are
not linked in any way. The need to ensure transitive consistency (10–12 %) makes it possible
to record attempts by an expert (experts) to artificially overestimate the indicators of one of the
drone samples (or the one being evaluated), therefore, the indicators of another sample will au-
tomatically deteriorate. The principle of impartiality and fairness is maintained. A well-trained
objective coordinator is not required, and the reality is that the absence of the disrupting the
problem solution possibility is due to a change in the psychological situation among the experts. Area of practical use of research results: humanitarian demining in the interests of ensur-
ing the detection of mines for various purposes by sappers from a safe distance. At the same
time, an increase within the probability of mines detecting is ensured due to special equip-
ment installed onboard the drone. Innovative technological product: a technique has been developed that allows not only
assessing the drone samples for mine reconnaissance objectively, but making an objective
choice of a sample for specific requirements also. Scope of application of the innovative technological product: clearance of the terrain re-
maining after the end of hostilities. With the help of unmanned aerial vehicles, a significant
acceleration of the demining process is possible, especially in those territories where mines
are installed and being for a sufficiently long time. © The Author(s) 2020. This is an open access article under the CC BY license http://creativecommons.org/licenses/by/4.0). 1. 1. The object of research 1. 1. The object of research The object of research is a comparative assessment and selection of an unmanned aerial
vehicle for mine reconnaissance sample while using quantitative and qualitative indicators. No. 5 (70), 2020 No. 5 (70), 2020 ScienceRise. Special issue ISSN 2313-8416 Vadym Neroba1 A RT I C L E I N F O
Article history:
Received date 14.09.2020
Accepted date 22.10.2020
Published date 30.10.2020
Section:
Control systems
D O I
10.21303/2313-8416.2020.001496
K E Y W O R D S
mines reconnaissance
unmanned aerial vehicle
methods of system analysis
expert review
technique for a drone assessing and
sampling
ScienceRise, 5, 44 50. doi: http://doi.org/10.21303/2313 8416.2020.001496
© The Author(s) 2020. This is an open access article under the CC BY license http://creativecommons.org/licenses/by/4.0). AB ST R AC T AB ST R AC
A RT I C L E I N F O A RT I C L E I N F O DEVELOPMENT OF METHODS FOR ASSESSMENT AND
SELECTION OF UNMANNED AERIAL VEHICLE FOR MINE
RECONNAISSANCE Vadym Neroba1
1Department of General Military Disciplines, National Academy of the State Border Guard Service of Ukraine named after Bohdan
Khmelnytskyi, Khmelnytskyi, Ukraine
vadym.rostislavovich@gmail.com
ORCID: http://orcid.org/0000-0002-7232-3285 1. 3. Suggested solution to the problem A number of Ukrainian and foreign specialists and scientists were involved in various issues
related to research on the use of unmanned aircraft for solving the problems of military facilities
reconnaissance: A. Ananin, L. Artiushin, M. Dougherty, V. Kirilenko, V. Kolesnikov, M. Mitrak-
hovich, S. Mosov, S. Saliy, A. Samkov, A. Selyukov, V. Silkov, A. Feschenko and others. S. Mosov and V. Kolesnikov in their work [1] gave the system requirements for the selection
of unmanned aerial systems for performing reconnaissance and surveillance tasks. At the same
time, these requirements are of a general nature, although they can be used to determine quantita-
tive indicators for evaluating UAV samples. A team of authors in [2] presented the results of research related to unmanned aerial systems
from the standpoint of a comparative assessment of their combat capabilities. The methodological
approaches proposed in [2] do not consider the issue of assessment and sampling of UAVs with
specified equipment for reconnaissance of mines. A team of authors in [3] analyzed the use of unmanned aircraft in military conflicts of our
time, highlighting the features of the use of unmanned aircraft systems of various purposes. At the
same time, the issues of using UAVs for detecting mines are not covered in the monograph. A team of authors in [3] analyzed the use of unmanned aircraft in military conflicts of our
time, highlighting the features of the use of unmanned aircraft systems of various purposes. At the
same time, the issues of using UAVs for detecting mines are not covered in the monograph. A feature of work [4] is the mathematical apparatus of multi-criteria choice of reconnais-
sance unmanned aircraft systems. At the same time, the given mathematical apparatus uses only
quantitative indicators. A. Ananiev in [5] proposed a set of tactical and technical requirements for unmanned air-
craft systems and defined their tasks in the system of protecting the state border. Among the tasks,
the detection of mines is not defined, which today is important for the protection of the state border
on the south-eastern border of Ukraine. A. Ananiev in [5] proposed a set of tactical and technical requirements for unmanned air-
craft systems and defined their tasks in the system of protecting the state border. Among the tasks,
the detection of mines is not defined, which today is important for the protection of the state border
on the south-eastern border of Ukraine. 1. 2. Problem description The territory of Ukraine, which is considered to be contaminated with explosives, is ap-
proximately 16,000 km2. By 2019 almost 300 military and more than 2,500 civilians died since the
beginning of hostilities due to the explosion of mines and explosive objects. The threat of a mine situation to troops, border guards and the civilians indicates the exis-
tence of a discrepancy. On the one hand, these are requirements for the relevant level of technical
means for area engineering reconnaissance conducting for the mines presence, taking into consid-
eration the achievements of modern scientific and technological progress. On the other hand – the 44 Innovative technologies in industry moral obsolescence of ground-based technical means of mines searching left over from the times
of the Soviet Union. moral obsolescence of ground-based technical means of mines searching left over from the times
of the Soviet Union. Elimination of this discrepancy is possible through the development of more effective tech-
nical means of engineering reconnaissance of mines. New approaches are needed for timely and
safe remote detection of mines, taking into consideration the latest achievements of scientific and
technological progress. One of these innovative approaches was the idea of using an unmanned aerial vehicle
(hereinafter – UAV) for mine reconnaissance. The payload of the drone is a view (with image
formation) technical means of mine reconnaissance. With the help of such reconnaissance drones, it is possible to accelerate the demining
process significantly, especially in those territories where mines have been installed and be-
ing for a sufficiently long time. Such studies are currently being carried out by the scientists
of a number of leading countries of the world: the USA, Great Britain, Israel, Russia, China,
Switzerland, etc. The use of specific technical means of UAVs for mine reconnaissance is an urgent di-
mension of scientific research. This requires the development of the methods for comparative
assessment and selection of UAVs based on the results of the study of system analysis. 3. Research results One of the specific features of the task of comparative assessment and selection of a specific
UAV sample from a certain set of existing is a significant number of possible alternative solutions
based on the indicators of their technical improvement. To compare several samples, methods of expert assessments are most often used, as well as
theoretical methods associated with solving single-criterion or multi-criteria problems. Objectification of the selection of UAVs for mine reconnaissance is aimed, firstly, the ap-
plication of a systematic approach in the process of assessing various UAVs, and, secondly, the
introduction or development of new effective methods for comparing and selecting UAVs. To ensure objectivity in the assessment and selection of UAVs for mine reconnaissance, it is
advisable to develop a method of comparative assessment and selection based on the results of the
study of system analysis methods. One of these methods is the rather well-known Delphi method, which is a method of estab-
lishing expert judgments on the basis of the anonymity of experts and their physical separation, as
well as the presence of controlled feedback [9]. To maintain anonymity and physical separation,
this method aims to avoid some of the potential pitfalls of group decision making, and the purpose
of feedback is to allow each expert to read the average opinion of other experts. If there are sufficient positive aspects, the method has corresponding disadvantages. The first
drawback should be attributed to the fact that the separation of experts practically excludes the possi-
bility of the emergence of new approaches to solving the problem, which can be developed and tested
in the course of discussions. Another disadvantage lies in the way of constructing the questionnaire,
according to which the survey of experts is carried out. If the questionnaires are not constructed suf-
ficiently, then the conclusions of the experts who answer the question will also be not objective due to
the fact that the conclusions are actually determined by the questions that are asked. Another well-known systemic method of assessment and selection is the Strategic Assumption
Surfacing and Testing (hereinafter – SAST) method, which is based on preliminary “polishing” of as-
sumptions (elimination of inconsistency), which are subsequently used to solve the problem, as well as
their ranking [10]. The method is well adapted to the analysis of poorly structured problems, in which
the development of a solution is based on acutely conflicting assumptions. 1. 3. Suggested solution to the problem In collective work [6] the authors provide approximate calculations of the main parameters
and characteristics of unmanned aerial vehicles, which do not take into consideration the tasks of
conducting aerial reconnaissance (observation). V. Neroba in [7] systematized the conditions and factors that would affect the specific tech-
nical means of UAV in terms of mines reconnaissance, allowed to determine the quantitative pa-
rameters for assessing the samples of UAVs. V. Neroba in [7] systematized the conditions and factors that would affect the specific tech-
nical means of UAV in terms of mines reconnaissance, allowed to determine the quantitative pa-
rameters for assessing the samples of UAVs. M. Dougherty [8] cited the features of the use of unmanned aircraft in local wars and con-
flicts in the civilian sphere, without parameters systematizing, which can be used to assess and
select samples of unmanned aerial vehicles. M. Dougherty [8] cited the features of the use of unmanned aircraft in local wars and con-
flicts in the civilian sphere, without parameters systematizing, which can be used to assess and
select samples of unmanned aerial vehicles. The analysis results show that the issue of using the specific technical means of reconnais-
sance (hereinafter – STMR) installed on UAVs to solve the tasks of mines reconnaissance as objects
of aerial reconnaissance remains in the topical arsenal. Only quantitative indicators are used to
assess the UAV. The issues of assessment and selection of UAVs for mine reconnaissance require
research. This requires the development of a comparative assessment methods and the choice of a 45 ScienceRise. Special issue ISSN 2313-8416 No. 5 (70), 2020 UAV model, on which specific technical means of mine reconnaissance are installed, while taking
into consideration quantitative and qualitative indicators. UAV model, on which specific technical means of mine reconnaissance are installed, while taking
into consideration quantitative and qualitative indicators. The aim of the article. To develop a methods for comparative assessment and selection of
an UAV sample, on which STMR are installed for mine reconnaissance, while taking into consid-
eration quantitative and qualitative indicators. 2. Materials and methods The development of the methods for the comparative assessment and selection of an UAV
sample for mine reconnaissance based on the results of the study of system analysis methods. 3. Research results However, it requires such
an objective arbiter, proficient in the art of interpersonal dialogue and experienced in group dynamics
theory. Otherwise, the application of the SAST method is doomed to failure. Moreover, the use of
the method requires the involvement of a sufficiently large number of experienced qualified experts. Another systematic procedure is the hierarchy analysis method (hereinafter – HAM) [11]. In
contrast to the Delphi method, HAM supports group interaction and discussions. Thus, during explo-
ration of the assumptions underlying individual decisions, new and important knowledge emerges. The expediency of this approach is confirmed by the experience of conducting business
games during the USSR period [12]. In case of disagreement, the HAM does not impose an artifi-
cial consensus, since it does not withdraw, but takes into consideration the opinions that fall out of
the general channel in the calculations (the value of the discrepancy is allowed). Comparison of HAM with SAST allows to conclude that they are similar at the stage of the
problematic task structuring. At the same time, a well-trained objective coordinator is not required,
and it is real that there is no possibility of disrupting the solution of the problem due to a change in
the psychological situation among experts. Thus, according to the results of the analysis, it can be concluded that the HAM is the most
well-known of the acceptable system methods, which is advisable to use in terms of methods for 46 Innovative technologies in industry assessing and sampling of UAVs for mines reconnaissance developing. The theoretical work [2]
confirms this conclusion. The software implementation of the HAM is available online on the
Internet resource [13]. The first step of the HAM is to decompose the problem of assessing and selecting a UAV
model for mine reconnaissance and presenting it in the form of a hierarchy (Fig. 1). At the first
(highest) level, there is a common target z – the best UAV model for mine reconnaissance. At the
second level, there are indicators K={ki}, which clarify the goal, and at the third (lower) level, there
is a set of applicants A={dj} from among the UAV samples that must be evaluated. Level 1
Level 2
Level 3
Fig. 1. Scheme of decomposition of tasks, estimation and visualization of UAVs Fig. 1. 3. Research results Special issue ISSN 2313-8416 No. 5 (70), 2020 No. 5 (70), 2020 Table 2
The scale for assessing the relative importance w
w
1
3
5
7
9
2, 4, 6, 8
Advantage degree
Equal importance
Moderate impor-
tance
Essential
Significant
Very strong
Intermediate
Table 3
Pairwise comparison matrices
k1
d1d2...dm
Priority vector
k2
d1d2...dm
Priority vector
d1
1
1
2
d
d
w
w
... 1
m
d
d
w
w
1 1
k d
u
d1
1
1
2
d
d
w
w
... 1
m
d
d
w
w
2 1
k d
u
d2
2
1
d
d
w
w
1 ... 2
m
d
d
w
w
1
2
k d
u
d2
2
1
d
d
w
w
1 ... 2
m
d
d
w
w
2
2
k d
u
... ... ... ... dm
1
m
d
d
w
w
2
m
d
d
w
w
... 1
1
m
k d
u
dm
1
m
d
d
w
w
2
m
d
d
w
w
... 1
2
m
k d
u
... d1d2...dm
Priority vector
kn
d1d2...dm
Priority vector
d1
1 ... ... ... ... d1
1
1
2
d
d
w
w
... 1
m
d
d
w
w
1
n
k d
u
d2
... 1 ... ... ... d2
2
1
d
d
w
w
1 ... 2
m
d
d
w
w
2
n
k d
u
... ... ... 1
... ... ... ... dm
... dm
1
m
d
d
w
w
2
m
d
d
w
w
... 1
n
m
k d
u Pairwise comparison matrices In the process of pairwise comparisons, to identify and eliminate possible violations of
transitive consistency, the value of the consistency index r should be determined to calculate the
consistency ratio χ: max
,
1
λ
−
=
−
s
r
s
0
100 %,
χ = r
r
(2) (2) r – the index of random consistency [14]: 1.0808
0
1.67
,
−
=
s
r
e
(3) (3) λmax – the maximum eigenvalue of the matrix: λmax – the maximum eigenvalue of the matrix: λmax – the maximum eigenvalue of the matrix: max
1
1
. 3. Research results Scheme of decomposition of tasks, estimation and visualization of UAVs At the second step, it is necessary to establish the local priorities of the kj indicators by con-
cluding square matrices of i pairwise comparisons (Table 1) and conducting expert comparisons
using the scale for assessing the relative importance w (Table 2). Square matrices are inversely
symmetric, that is, the matrix element bji=wj/wi=1/bij. Similar matrices should be constructed for
paired comparisons of each dj sample of the UAV at the third level in relation to the indicators ki of
the second level (Table 3). Sets of local priorities are formed from the groups of matrices of pairwise comparisons,
indicating the relative influence of a set of elements on an element of the level adjacent from above. The process of forming local priorities is based on calculating eigenvectors for each matrix and
normalizing the result to one: 1
1
1
1
,
s
i
s
i
s
s
j
j
i
s
j
i
j
w
u
w
w
w
=
=
=
=
∏
∑∏
(1) (1) ui – the local priority of the i-th element of the column of the matrix of pairwise comparisons; s – the
number of elements of the matrix of pairwise comparisons in the column. ui – the local priority of the i-th element of the column of the matrix of pairwise comparisons; s – the
number of elements of the matrix of pairwise comparisons in the column. Table 1
Setting local priorities for indicators kj
Indicators
k1
k2
. . . k(n–1)
kn
Local priority
k1
1
1k
w
2
k
w
. . . 1k
w
(
1)
−
n
k
w
1k
w
n
k
w
1ku
k2
2
k
w
1k
w
1
. . . 2
k
w
(
1)
−
n
k
w
2
k
w
n
k
w
2
ku
. . . . . . . . . . . . . . . . . . . . . k(n–1)
(
1)
−
n
k
w
1k
w
(
1)
−
n
k
w
2
k
w
. . . 1
(
1)
−
n
k
w
n
k
w
(
1)
−
n
ku
kn
n
k
w
1k
w
n
k
w
2
k
w
. . . n
k
w
(
1)
−
n
k
w
1
n
ku 47 ScienceRise. 4. Discussion of research results The proposed technique allows, firstly, not only to evaluate the UAV samples for mine
reconnaissance objectively, but to carry out an objective selection of the UAV sample also. Secondly, the need to ensure transitive consistency makes it possible to record attempts to
artificially overestimate the indicators of one of the UAV samples (or the one being evaluated),
therefore, the indicators of another UAV sample will automatically deteriorate and the consis-
tency ratio will go beyond the acceptable limits. This allows the principle of impartiality and
fairness to be maintained. When applying the methods, it is proposed to add the following to the composition of
indicators K={ki}, which clarify the goal – the choice of a UAV model for mines reconnais-
sance: the presence of a stabilized platform on board the UAV sample for placing a STMR;
maximum payload weight of the UAV sample; operating temperature (winter, summer) of the
UAV sample; time spent in the air of the UAV sample; controllability of the UAV sample by the
external pilot (pilot-operator); purchase cost of the UAV sample; the cost of operating a UAV
sample; the maneuverability of the UAV sample; resistance of the UAV sample to wind gusts;
maintainability of the UAV sample; operational reliability of the UAV sample; diagnostic abil-
ity to the state of a UAV sample. y
p
The composition of indicators K={ki} may vary depending on the goal being achieved. The technique can be applied in the context of comparative assessment and selection of
UAVs for mines reconnaissance, which are equipped with STMR: RGB– cameras, infrared camer-
as, multi- and hyper spectral cameras. To ensure the objectivity of obtaining the results of applying the methods, the value of the
agreement ratio should not exceed 10–12 %. The technique can be used in conditions when it is necessary to evaluate each (or one) UAV
sample. In this case, a “normative” UAV model should be used as an additional UAV sample. Af-
ter determining the global priority of the “normative” UAV sample, the level of the UAV sample
is compared, evaluated relative to the normative by calculating the difference between the global
priorities of the “normative” UAV sample and the UAV sample estimated, or calculating the per-
centage between them. The direction of further research should be considered the development of recommenda-
tions for organizing the training of external pilots and UAV operators for mine reconnaissance. 3. Research results =
=
λ
= ∑∑
s
s
j
i
i
j
i
w
u
w
(4) (4) The value χ should not exceed 10–12 %, otherwise it will be necessary to additionally check
the judgments of experts [11]. In the case where experts do not have sufficient experience in making judgments or a suf-
ficient level of professional training to carry out the process of setting local priorities, the criteria
should be pre-ranked. The penultimate step in determining z is the implementation of the principle of synthesis. The priorities are synthesized starting from the second level down. Local priorities
i
j
k d
u
are multi-
plied by the priority
iku of the corresponding indicator at the highest level and summed up for each
element in accordance with the criteria that this element affects: 48 Innovative technologies in industry (
)
(
)
1
1 1
1
2 1
2 2
2
1
2
1
2
1
2
... ... ,
,... ,
,... . ... ... ... ...
=
d
k d
k dm
k d
k d
k dm
d
d
dm
k
k
kn
knd
knd
kndm
u
u
u
u
u
u
l
l
l
u
u
u
u
u
u
(5) (5) The procedure for synthesizing local priorities is carried out to the lowest level, as a result of
which the global priorities lj of each UAV sample – dj are determined. The ranking of the obtained
priorities allows to determine the UAV sample with the highest priority value, which will be the
most pleasant among the UAV samples that are evaluated. 4. Discussion of research results Another promising area may be the development of recommendations for technical operation of an
unmanned aircraft complex designed organizing to carry out mine reconnaissance tasks. References [1] Mosov, S. P., Kolesnikov, V. O. (2016). Vymohy do vyboru bezpilotnykh aviatsiinykh kompleksiv dlia vykonannia zavdan roz-
vidky ta sposterezhennia. Zbirnyk naukovykh prats Tsentru voienno-stratehichnykh doslidzhen Natsionalnoho universytetu
oborony Ukrainy imeni Ivana Cherniakhovskoho, 1 (56), 24–28. [2] Mitrakhovich, M. M., Silkov, V. I., Samkov, A. V., Burshtynskaia, Kh. V. et. al.; Silkov, V. I. (Ed.) (2012). Bespilotnye aviat-
sionnye kompleksy: metodika sravnitelnoi otsenki boevykh vozmozhnostei. Kyiv: TSNII VVT VS Ukrainy, 288. [3] Mosov, S. P., Pohoretskyi, M. V., Salii, S. M., Sieliukov, O. V., Feshchenko, A. L.; Mosov, S. P. (Ed.) (2019). Bezpilotna aviatsiia
u viiskovii spravi. Kyiv: Interservis, 324. [4] Kyrylenko, V., Artiushyn, L., Steshenko, P. (2018). Matematychnyi aparat bahatokryterialnoho vyboru rozviduvalnykh bezpi-
lotnykh aviatsiinykh kompleksiv. Zbirnyk naukovykh prats Natsionalnoi akademii Derzhavnoi prykordonnoi Ukrainy. Seriia:
viiskovi ta tekhnichni nauky, 1 (75), 115–133. [5] Ananin, O. (2016). Taktyko-tekhnichni vymohy do bezpilotnykh aviatsiinykh kompleksiv ta yikh zavdannia v systemi ok-
horony derzhavnoho kordonu. Zbirnyk naukovykh prats Natsionalnoi akademii Derzhavnoi prykordonnoi Ukrainy. Seriia:
viiskovi ta tekhnichni nauky, 2 (68), 181–194. [6] Iliushko, V. M., Mitrakhovich, M. M., Samkov, A. V., Silkov, V. I. et. al. Silkov, V. I. (Ed.) (2009). Bespilotnye letatelnye
apparaty: metodiki priblizhennykh raschetov osnovnykh parametrov i kharakteristik. Kyiv: TSNII VVT VS Ukrainy, 302. [7] Neroba, V. R. (2019). Systematyzatsiia umov i faktoriv, shcho vplyvatymut na vydovi tekhnichni zasoby rozvidky min bezpi-
lotnoho litalnoho aparatu. Zbirnyk naukovykh prats Viiskovoi akademii, 2 (12 (1)), 48–54. [8] Dogerti, M. (2017). Drony: pervii illiustrirovannii putevoditel po BPLA. Moscow: Izd-vo «E», 224. [9] Pill, J. (1971). The Delphi method: Substance, context, a critique and an annotated bibliography. Socio-Economic Planning
Sciences, 5 (1), 57–71. doi: http://doi.org/10.1016/0038-0121(71)90041-3 [10] Mitroff, I. I., Sagasti, F. (1973). Epistemology as General Systems Theory: An Approach to the Design of Complex Deci-
sion-Making Experiments. Philosophy of the Social Sciences, 3 (2), 117–134. doi: http://doi.org/10.1177/004839317300300202
[11] Saati, T. (1993). Priniatie reshenii: metod analiza ierarkhii. Moscow: Radio i sviaz, 320. sion-Making Experiments. Philosophy of the Social Sciences, 3 (2), 117–134. doi: http://doi.org/10.1177/004839317300300202
[11]
Saati T (1993) Priniatie reshenii: metod analiza ierarkhii Moscow: Radio i sviaz 320 [11] Saati, T. (1993). Priniatie reshenii: metod analiza ierarkhii. Moscow: Radio i sviaz, 320. [12] Schedrovitskii, G. P. (1995). Izbrannye trudy. Moscow: Shk. Kult. Polit., 800. [13] Metod analiza ierarkhii onlain. Available at: https://axd.semestr.ru/upr/hierarchies.php Last accessed: 20.09.2019 [14] Zahorka, O. M., Mosov, S. P., Sbitniev, A. I., Stuzhuk, P. I. (2005). 5. Conclusions A methods of comparative assessment and selection of a UAV sample for mine reconnais-
sance have been developed, based on the well-known expert method for analyzing hierarchies. The
methods have several advantages. The first is to support group interaction and discussion. Second,
if the judgments do not coincide, an artificial consensus is not imposed, because opinions that
fall out of the general channel in the calculations are not withdrawn, but taken into consideration. Third, a well-trained objective coordinator is not required, and the reality is that the lack of the
possibility of disrupting the solution of the problem is due to a change in the psychological environ-
ment among experts. Fourth, both quantitative and qualitative indicators are compared simultane-
ously through quantitative formalization. 49 ScienceRise. Special issue ISSN 2313-8416 No. 5 (70), 2020 References Elementy doslidzhennia skladnykh system viiskovoho
pryznachennia. Kyiv: NAOU, 100. 50
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https://openalex.org/W2464953261
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https://www.aclweb.org/anthology/N16-1163.pdf
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English
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Retrofitting Sense-Specific Word Vectors Using Parallel Text
| null | 2,016
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cc-by
| 3,700
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Proceedings of NAACL-HLT 2016, pages 1378–1383,
San Diego, California, June 12-17, 2016. c⃝2016 Association for Computational Linguistics Abstract create a graph structure comprising two classes of
relations: form-based relations between each word
form and its respective senses, and meaning-based
relations between word senses with similar mean-
ings. This graph structure is then used to transform a
traditional VSM into an enriched VSM, where each
point in the space represents a word sense, rather
than a word form. This approach is appealing as, un-
like with prior sense-aware representations, senses
are defined categories in a semantic lexicon, rather
than clusters induced from raw text (Reisinger and
Mooney, 2010; Huang et al., 2012; Neelakantan et
al., 2015; Tian et al., 2014), and the method does
not require performing word sense disambiguation
(Guo et al., 2014). Jauhar et al. (2015) recently proposed to
learn sense-specific word representations by
“retrofitting” standard distributional word rep-
resentations to an existing ontology. We ob-
serve that this approach does not require an
ontology, and can be generalized to any graph
defining word senses and relations between
them. We create such a graph using transla-
tions learned from parallel corpora. On a set of
lexical semantic tasks, representations learned
using parallel text perform roughly as well as
those derived from WordNet, and combining
the two representation types significantly im-
proves performance. In this paper, we observe that the crucial mean-
ing relationships in the Jauhar et al. retrofitting
process—the word sense graph—can be inferred
based on another widely available resource: bilin-
gual parallel text. This observation is grounded in
a well-established tradition of using cross-language
correspondences as a form of sense annotation (Gale
et al., 1992; Diab and Resnik, 2002; Ng et al., 2003;
Carpuat and Wu, 2007; Lefever and Hoste, 2010,
and others). Using parallel text to define sense dis-
tinctions sidesteps the persistent difficulty of identi-
fying a single correct sense partitioning based on hu-
man intuition, and avoids large investments in man-
ual curation or annotation. 1
Introduction Vector space models (VSMs) provide a powerful
tool for representing word meanings and modeling
the relations between them. While these models
have demonstrated impressive success in capturing
some aspects of word meaning (Landauer and Du-
mais, 1997; Turney et al., 2010; Mikolov et al.,
2013; Baroni et al., 2014; Levy et al., 2014), they
generally fail to capture the fact that single word
forms often have multiple meanings. This can lead
to counterintuitive results—for example, it should be
possible for the nearest word to rock to be stone in
everyday usage, punk in discussions of music, and
crack (cocaine) in discussions about drugs. We use parallel text and word alignment to in-
fer both word sense identities and inter-sense rela-
tions required for the sense graph, and apply the
approach of Jauhar et al. to retrofit existing word
vector representations and create a sense-based vec- In a recent paper, Jauhar et al. (2015) introduce
a method for “retrofitting” generic word vectors to
create sense-specific vectors using the WordNet se-
mantic lexicon (Miller, 1995). From WordNet, they 1378 1378 types of relations between senses: tor space, using bilingual correspondences to define
word senses. When evaluated on semantic judgment
tasks, the vector spaces derived from this graph per-
form comparably to and sometimes better than the
WordNet-based space of Jauhar et al., indicating that
parallel text is a viable alternative to WordNet for
defining graph structure. Combining the output of
parallel-data-based and WordNet-based retrofitted
VSMs consistently improves performance, suggest-
ing that the different sense graph methods make
complementary contributions to this sense-specific
retrofitting process. C(V ) = arg min
V
X
i−ij
α∥ˆui −vij∥2
+
X
ij−i′j′
βr∥vij −vi′j′∥2
(1) (1) The objective encourages similarity between a
word’s vector and its senses’ vectors (first term), as
well as similarity between the vectors for senses that
are related in the sense graph (second term). Defining a sense graph from parallel text. Our
key observation is that, although Jauhar et al. (2015)
assume their sense graph to be an ontology, this
graph can be based on any inventory of word-sense
and sense-sense relationships. In particular, given
a parallel corpus, we can follow the tradition of
translation-as-sense-annotation: the senses of an En-
glish word type can be defined by different possible
translations of that word in another language. 2
Model Retrofitting. The technique introduced by Jauhar
et al. (2015) is based on what we will call a sense
graph, which we formulate as follows. Nodes in
the sense graph comprise the words wi in a vocabu-
lary W together with the senses sij for those words. Labeled, undirected edges include word-sense edges
⟨wi, si,j⟩, which connect each word to all of its pos-
sible senses, and sense-sense edges ⟨sij, si′j′⟩la-
beled with a meaning relationship r that holds be-
tween the two senses. Operationalizing this observation is straightfor-
ward, given a word-aligned parallel corpus. If En-
glish word form ei is aligned with Chinese word
form cj, then ei(cj) is a sense of ei in the sense
graph, and there is a word-sense edge ⟨ei, ei(cj)⟩. Edges signifying a meaning relation are drawn be-
tween sense nodes if those senses are defined by the
same translation word. For instance, English senses
swear(发誓) and vow(发誓) both arise via align-
ment to 发誓(fashi), so a sense-sense edge will be
drawn between these two sense nodes. See Figure 2
for illustration. Jauhar et al. use WordNet to define their sense
graph. Synsets in the WordNet ontology define the
sense nodes, a word-sense edge exists between any
word and every synset to which it belongs, and
WordNet’s synset-to-synset relations of synonymy,
hypernymy, and hyponymy define the sense-sense
edges. Figure 1 illustrates a fragment of a WordNet-
based sense graph, suppressing edge labels. 3
Evaluation Tasks. We evaluate on both the synonym selection
and word similarity rating tasks used by Jauhar et al. Synonym selection nicely demonstrates the advan-
tages afforded by sense partitioning: if we believe
that spin means “make up a story”, then we are not
likely to perform well on a question in which the
correct synonym is twirl. Word similarity rating, on
the other hand, is a classic test of the extent to which
vector representations simulate human intuitions of
word relations in general. Adopting Jauhar et al.’s notation, the original vec-
tor space to be retrofitted is defined by the original
word-form vectors ˆui for each wi ∈W, and the goal
is to infer a set V of sense-specific vectors vij cor-
responding to each sense sij. Jauhar et al. use the
sense graph to define a Markov network with vari-
ables for all word vectors and sense vectors, within
which each word’s vector ˆui is connected to all of
its sense vectors vij, and the variables for sense vec-
tors vij and vi′j′ are connected iff the corresponding
senses are connected in the sense graph. For synonym selection, we follow Jauhar et al. in
testing with ESL-50 (Turney, 2001), RD-300 (Jar-
masz and Szpakowicz, 2004), and TOEFL-80 (Lan-
dauer and Dumais, 1997), using maxSim for multi- Retrofitting then consists in optimizing the fol-
lowing objective, where α is a sense-agnostic
weight, and βr are relation-specific weights for 1379 Figure 2: Illustration of parallel-text-based sense graph. Figure 1: Illustration of WordNet-based sense graph. Figure 2: Illustration of parallel-text-based sense graph. Figure 1: Illustration of WordNet-based sense graph. sense models (Jauhar et al., 2015, eq. 9) to select the
most similar word.1 For similarity rating, we again
mirror Jauhar et al., testing with WS-353 (Finkel-
stein et al., 2001), RG-65 (Rubenstein and Good-
enough, 1965), MC-30 (Miller and Charles, 1991),
and the designated test subset (1000 items) of MEN-
3k (Bruni et al., 2014), using avgSim (Jauhar et al.,
2015, eq. 8) as the similarity rating, and evaluating
model ratings against human similarity ratings via
Spearman’s rank correlation coefficient (ρ).2 ∼5.8M lines of segmented Chinese-English paral-
lel text from the DARPA BOLT project and the
Broadcast Conversation subset of the segmented
Chinese-English parallel data in the OntoNotes cor-
pus (Weischedel et al., 2013).4 We perform word
alignment with the Berkeley aligner (Liang et al.,
2006). 4English was lemmatized post-alignment via lookup in the
XTAG morphological database (XTAG Research Group, 2001). 3
Evaluation We filter out noisy alignments using the G-
test statistic (Dunning, 1993), with a threshold se-
lected during tuning on a development set. We set α (see Equation 1) to 1.0. Each sense-
sense edge ⟨ei(cj), ei′(cj)⟩has individual weight
0 < βr ≤1, computed by obtaining the G-test
statistic for the alignment of ei with cj and for
the alignment of ei′ with cj, running these values
through a logistic function, and averaging. Param-
eters for these computations, as well as the G-test
statistic threshold below which we filtered out noisy
alignments, were selected during tuning on the de-
velopment set. Initial
word
representations. We
use
the
word2vec (Mikolov et al., 2013) skip-gram archi-
tecture to train 80-dimensional word vectors (in
keeping with Jauhar et al.), based on evidence that
this model shows consistently strong performance
on a wide array of tasks (Baroni et al., 2014; Levy
et al., 2015). Training is on ukWaC (Ferraresi et al.,
2008), a diverse 2B-word web corpus.3 Note that we have not currently incorporated spe-
cial treatment for alignments of a single word to a
multi-word phrase. This does create the possibil-
ity of noisy or uninformative sense annotations (e.g.,
sense annotations corresponding to parts of aligned
Chinese phrases) when such alignments are not fil-
tered out by the G-test thresholding. 1Because it is not clear how multi-word phrases should best
be treated (and this is not a question being investigated here),
we filter out any questions containing multi-word phrases for
any of the relevant items (probe or possible response), and any
questions for which any of the relevant items is completely out
of vocabulary (no vectors available) for any of the evaluated
models. This leaves 48 items in ESL, 87 items in RD, and 77
items in TOEFL. 2The designated development set of MEN-3k (2000 items)
was used for tuning.
3 1Because it is not clear how multi-word phrases should best
be treated (and this is not a question being investigated here),
we filter out any questions containing multi-word phrases for
any of the relevant items (probe or possible response), and any
questions for which any of the relevant items is completely out
of vocabulary (no vectors available) for any of the evaluated
models. This leaves 48 items in ESL, 87 items in RD, and 77
items in TOEFL.
2The designated development set of MEN-3k (2000 items)
was used for tuning.
3To alleviate sparsity we lemmatized the ukWaC corpus.
Runs without lemmatization produced weaker results. 3To alleviate sparsity we lemmatized the ukWaC corpus.
Runs without lemmatization produced weaker results. Sense-graph construction from parallel text.
To
construct the sense graph per Section 2, we use Sense-graph construction from parallel text. To
construct the sense graph per Section 2, we use 1Because it is not clear how multi-word phrases should best
be treated (and this is not a question being investigated here),
we filter out any questions containing multi-word phrases for
any of the relevant items (probe or possible response), and any
questions for which any of the relevant items is completely out
of vocabulary (no vectors available) for any of the evaluated
models. This leaves 48 items in ESL, 87 items in RD, and 77
items in TOEFL. Experimental conditions. We evaluate the fol-
lowing experimental conditions: Skip-gram (SG)
uses the un-retrofitted word2vec vectors, Word- 2The designated development set of MEN-3k (2000 items)
was used for tuning. 3To alleviate sparsity we lemmatized the ukWaC corpus. Runs without lemmatization produced weaker results. 1380 Synonym Selection SYMM (%)
ESL-48
RD-87
TOEFL-77
SG
58.3
58.6
71.4
WN
66.7
74.7
81.8
PD
68.8
62.1
80.5
PD-WN
70.8
79.3
84.4
Table 1: Synonym selection task results: accuracy Net (WN) retrofits using the WordNet-based sense
graph, and Parallel Data (PD) retrofits using the
sense graph built from parallel text. We also com-
bine the two retrofitting approaches (PD-WN). For
synonym selection, we compute maxSim over all
sense pairs for WN and PD separately, and select
the sense pair with the overall maximum cosine sim-
ilarity across the two. For similarity rating, we
explore two PD-WN combination approaches: for
each word pair, we take the avgSim from each sep-
arate model, and then we (a) take the average of the
values given by the two models (avg), or (b) take the
maximum value between the two models (max). Word similarity: avgSim SYMM (ρ)
WS-353
RG-65
MC-30
MEN-1k
SG
.708
.729
.722
.763
WN
.610
.725
.750
.739
PD
.636
.777
.715
.769
PD-WN (avg)
.666
.777
.742
.773
PD-WN (max)
.630
.731
.758
.756
Table 2: Similarity rating task results Table 2: Similarity rating task results 4
Results raters were explicitly told to rate relatedness, rather
than similarity, while the retrofitting process is in-
tended to encourage similarity per se. If we exclude
this set from consideration, we can observe that SG
is outperformed by at least one sense-specific model
in all cases.5 Table 1 shows that combining our new method
with Jauhar et al.’s WN retrofitting performs best
on synonym selection across all datasets, and both
retrofitted models consistently outperform the no-
retrofitting model (SG). Error analysis on RD-87,
the only set on which WN substantially outperforms
PD, suggests that PD’s errors are driven by the large
number of lower frequency items that characterize
this dataset. Given that WordNet is a hand-curated
lexicon while the parallel data mirrors actual us-
age, it is not surprising that the latter suffers when
it comes to low frequency items. Note that as expected, the amount of training data
has an impact on the quality of the alignments and of
the sense graph. Retrofitting sense-specific embed-
dings using only 300k sentence pairs, which repre-
sent about 5% of the total training data, does not give
clear benefit over word-form embeddings. Error analysis also indicates that PD performs
particularly well on the synonym task precisely
when one would expect: when the probe and the
correct answer have an alignment to the same Chi-
nese word form, so that the corresponding sense vec-
tors are extremely close in vector space. Occasion-
ally, PD yields “the wrong answer for the right rea-
son”, choosing an option for which there is indeed a
correct alignment that matches an alignment of the
probe word. For instance, though the probe passage
is intended to have the answer hallway, PD chooses
ticket because both passage and ticket have a sense
defined by alignment to the Chinese word 机票(jip-
iao), meaning “air ticket”. Though this is a less fre-
quent sense of passage, it is a reasonable one. 5We also explored using maxSim for similarity ratings, on
the intuition that when human annotators give similarity judg-
ments, they are likely to judge based on senses of the given
words that are biased toward the words with which they are
paired. However, top performance is similarly scattered when
using maxSim for similarity scores and fails to improve over the
SG baseline for two of the datasets.
6Sample sense-specific vectors and code for generat-
ing a sense graph from parallel data can be accessed at
http://ling.umd.edu/~aetting/retropd.html. 5We also explored using maxSim for similarity ratings, on
the intuition that when human annotators give similarity judg-
ments, they are likely to judge based on senses of the given
words that are biased toward the words with which they are
paired. However, top performance is similarly scattered when
using maxSim for similarity scores and fails to improve over the
SG baseline for two of the datasets. 6Sample sense-specific vectors and code for generat-
ing a sense graph from parallel data can be accessed at
http://ling.umd.edu/~aetting/retropd.html. Acknowledgments The authors would like to thank Sujay Kumar Jauhar
for sharing software and data and for helpful dis-
cussion. Thanks also to Manaal Faruqui and Pe-
ter Turney for help in acquiring evaluation datasets,
to Amittai Axelrod for his assistance with data, and
to the anonymous reviewers for valuable comments
and suggestions. This work was supported in part
by an NSF Graduate Research Fellowship under
Grant No. DGE 1322106. Any opinions, findings,
and conclusions or recommendations expressed are
those of the authors and do not necessarily reflect the
views of the NSF. Eric H Huang, Richard Socher, Christopher D Manning,
and Andrew Y Ng. 2012. Improving word representa-
tions via global context and multiple word prototypes. In Proceedings of the 50th Annual Meeting of the Asso-
ciation for Computational Linguistics: Long Papers-
Volume 1, pages 873–882. Mario Jarmasz and Stan Szpakowicz. 2004. Roget’s
thesaurus and semantic similarity. Recent Advances
in Natural Language Processing III: Selected Papers
from RANLP, 2003:111. Sujay Kumar Jauhar, Chris Dyer, and Eduard Hovy. 2015. Ontologically grounded multi-sense represen-
tation learning for semantic vector space models. In
Proceedings of NAACL, pages 683–693. Thomas K Landauer and Susan T Dumais. 1997. A so-
lution to plato’s problem: The latent semantic analysis
theory of acquisition, induction, and representation of
knowledge. Psychological review, 104(2):211. 5
Conclusions and future work Building on Jauhar et al. (2015), we have presented
an alternative means of deriving information about
senses and sense relations to build sense-specific
vector space representations of words, making use
of parallel text rather than a manually constructed
ontology. We show that this is a viable alterna-
tive, producing representations that perform on par
with those retrofitted to sense graphs based on Word-
Net.6 Results on the similarity rating task (presented in
Table 2) are less clearly interpretable, top perfor-
mance being divided between the PD model and the
combined models—with the exception of WS-353. We note that WS-353 is a test set for which human 1381 Based on these results, it would be interesting
to evaluate further refinements of the sense graph:
alignment-based senses could be clustered, or fur-
ther filtered to reduce the impact of alignment noise;
new edges could be added using other multilingual
resources. Finally, it will be important to evaluate
the effectiveness of the retrofitted word embeddings
on extrinsic tasks that require disambiguating word
meaning in context. Lev Finkelstein, Evgeniy Gabrilovich, Yossi Matias,
Ehud Rivlin, Zach Solan, Gadi Wolfman, and Eytan
Ruppin. 2001. Placing search in context: The con-
cept revisited. In Proceedings of the 10th international
conference on World Wide Web, pages 406–414. William A Gale,
Kenneth W Church,
and David
Yarowsky. 1992. A method for disambiguating word
senses in a large corpus. Computers and the Humani-
ties, 26(5-6):415–439. Jiang Guo, Wanxiang Che, Haifeng Wang, and Ting
Liu. 2014. Learning sense-specific word embeddings
by exploiting bilingual resources. In Proceedings of
COLING, pages 497–507. References Marco Baroni, Georgiana Dinu, and Germ´an Kruszewski. 2014. Don’t count, predict! A systematic compari-
son of context-counting vs. context-predicting seman-
tic vectors. In Proceedings of the 52nd Annual Meet-
ing of the Association for Computational Linguistics,
volume 1, pages 238–247. Els Lefever and Veronique Hoste. 2010. Semeval-2010
task 3: Cross-lingual word sense disambiguation. In
Proceedings of the 5th International Workshop on Se-
mantic Evaluation, pages 15–20. Elia Bruni, Nam-Khanh Tran, and Marco Baroni. 2014. Multimodal distributional semantics. Journal of Arti-
ficial Intelligence Research, 49:1–47. Omer Levy, Yoav Goldberg, and Israel Ramat-Gan. 2014. Linguistic regularities in sparse and explicit
word representations. In Proceedings of the Confer-
ence on Natural Language Learning (CoNLLL), pages
171–180. Marine Carpuat and Dekai Wu. 2007. Improving statisti-
cal machine translation using word sense disambigua-
tion. In EMNLP-CoNLL, volume 7, pages 61–72. Omer Levy, Yoav Goldberg, and Ido Dagan. 2015. Im-
proving distributional similarity with lessons learned
from word embeddings. Transactions of the Associa-
tion for Computational Linguistics, 3:211–225. Mona Diab and Philip Resnik. 2002. An unsupervised
method for word sense tagging using parallel corpora. In Proceedings of the 40th Annual Meeting on Associ-
ation for Computational Linguistics, pages 255–262. Percy Liang, Ben Taskar, and Dan Klein. 2006. Align-
ment by agreement. In Proceedings of the main con-
ference on Human Language Technology Conference
of the North American Chapter of the Association of
Computational Linguistics, pages 104–111. Ted Dunning. 1993. Accurate methods for the statistics
of surprise and coincidence. Computational linguis-
tics, 19(1):61–74. Adriano Ferraresi, Eros Zanchetta, Marco Baroni, and
Silvia Bernardini. 2008. Introducing and evaluating
ukWaC, a very large web-derived corpus of English. In Proceedings of the 4th Web as Corpus Workshop
(WAC-4) Can we beat Google, pages 47–54. Tomas Mikolov, Kai Chen, Greg Corrado, and Jeffrey
Dean. 2013. Efficient estimation of word representa-
tions in vector space. arXiv preprint arXiv:1301.3781. 1382 George A Miller and Walter G Charles. 1991. Contex-
tual correlates of semantic similarity. Language and
Cognitive Processes, 6(1):1–28. George A Miller. 1995. Wordnet: a lexical database for
English. Communications of the ACM, 38(11):39–41. Arvind Neelakantan, Jeevan Shankar, Alexandre Pas-
sos,
and
Andrew
McCallum. 2015. Effi-
cient non-parametric estimation of multiple embed-
dings per word in vector space. arXiv preprint
arXiv:1504.06654. Hwee Tou Ng, Bin Wang, and Yee Seng Chan. 2003. Ex-
ploiting parallel texts for word sense disambiguation:
An empirical study. References In Proceedings of the 41st An-
nual Meeting on Association for Computational Lin-
guistics, pages 455–462. Joseph Reisinger and Raymond J Mooney. 2010. Multi-
prototype vector-space models of word meaning. In
Human Language Technologies:
The 2010 Annual
Conference of the North American Chapter of the As-
sociation for Computational Linguistics, pages 109–
117. Herbert Rubenstein and John B Goodenough. 1965. Contextual correlates of synonymy. Communications
of the ACM, 8(10):627–633. Fei Tian, Hanjun Dai, Jiang Bian, Bin Gao, Rui Zhang,
Enhong Chen, and Tie-Yan Liu. 2014. A probabilistic
model for learning multi-prototype word embeddings. In Proceedings of COLING, pages 151–160. Peter D Turney, Patrick Pantel, et al. 2010. From fre-
quency to meaning: Vector space models of semantics. Journal of Artificial Intelligence Research, 37(1):141–
188. Peter Turney. 2001. Mining the web for synonyms: PMI-
IR versus LSA on TOEFL. In Proceedings of the 12th
European Conference on Machine Learning. Ralph Weischedel, Martha Palmer, Mitchell Marcus, Ed-
uard Hovy, Sameer Pradhan, Lance Ramshaw, Nian-
wen Xue, Ann Taylor, Jeff Kaufman, Michelle Fran-
chini, et al. 2013. Ontonotes release 5.0 ldc2013t19. Linguistic Data Consortium, Philadelphia, PA. XTAG Research Group. 2001. A lexicalized tree adjoin-
ing grammar for English. Technical Report IRCS-01-
03, IRCS, University of Pennsylvania. 1383
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Variant detection sensitivity and biases in whole genome and exome sequencing
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© 2014 Meynert 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/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. RESEARCH ARTICLE Open Access Abstract Background: Less than two percent of the human genome is protein coding, yet that small fraction harbours the
majority of known disease causing mutations. Despite rapidly falling whole genome sequencing (WGS) costs, much
research and increasingly the clinical use of sequence data is likely to remain focused on the protein coding exome. We set out to quantify and understand how WGS compares with the targeted capture and sequencing of the exome
(exome-seq), for the specific purpose of identifying single nucleotide polymorphisms (SNPs) in exome targeted
regions. Results: We have compared polymorphism detection sensitivity and systematic biases using a set of tissue samples
that have been subject to both deep exome and whole genome sequencing. The scoring of detection sensitivity was
based on sequence down sampling and reference to a set of gold-standard SNP calls for each sample. Despite
evidence of incremental improvements in exome capture technology over time, whole genome sequencing has
greater uniformity of sequence read coverage and reduced biases in the detection of non-reference alleles than
exome-seq. Exome-seq achieves 95% SNP detection sensitivity at a mean on-target depth of 40 reads, whereas WGS
only requires a mean of 14 reads. Known disease causing mutations are not biased towards easy or hard to sequence
areas of the genome for either exome-seq or WGS. Conclusions: From an economic perspective, WGS is at parity with exome-seq for variant detection in the targeted
coding regions. WGS offers benefits in uniformity of read coverage and more balanced allele ratio calls, both of which
can in most cases be offset by deeper exome-seq, with the caveat that some exome-seq targets will never achieve
sufficient mapped read depth for variant detection due to technical difficulties or probe failures. As WGS is intrinsically
richer data that can provide insight into polymorphisms outside coding regions and reveal genomic rearrangements,
it is likely to progressively replace exome-seq for many applications. Keywords: SNP, Sensitivity, Protein-coding genes, Next-generation sequencing, Whole genome sequencing, Exome
sequencing Variant detection sensitivity and biases in
whole genome and exome sequencing Alison M Meynert*, Morad Ansari, David R FitzPatrick and Martin S Taylor Meynert et al. BMC Bioinformatics 2014, 15:247
http://www.biomedcentral.com/1471-2105/15/247 Meynert et al. BMC Bioinformatics 2014, 15:247
http://www.biomedcentral.com/1471-2105/15/247 *Correspondence: alison.meynert@igmm.ed.ac.uk
MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine,
University of Edinburgh, Western General Hospital, Crewe Road,
EH4 2XU Edinburgh, UK Background causal disease variants identified to date [2]. Addition-
ally, experimental approaches to determine the function
of candidate disease variants at protein coding or tran-
script splice sites are well developed and accepted by the
research community. The cost of sequencing DNA has decreased steeply since
the introduction of next-generation short read technolo-
gies [1]. It is now at the point where cohorts of whole
human genomes are sequenced for study. However, inves-
tigations of disease-causing variation continue to focus
on the protein-coding exome, which is a small fraction
of the whole genome. It contains fewer repetitive ele-
ments than non-coding regions and contains most of the For these reasons, exome centric analysis will remain
common in research and is increasingly used in clini-
cal genetic settings [3]. The targeted capture followed by
sequencing of specific regions, such as the 30 Mb human
exome (exome-seq), has proven to be a cost-effective
and productive strategy for the identification of single
nucleotide polymorphisms (SNPs) and small insertions
and deletions in this rich vein of the genome. However, *Correspondence: alison.meynert@igmm.ed.ac.uk
MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine,
University of Edinburgh, Western General Hospital, Crewe Road,
EH4 2XU Edinburgh, UK Page 2 of 11 Page 2 of 11 Meynert et al. BMC Bioinformatics 2014, 15:247
http://www.biomedcentral.com/1471-2105/15/247 target capture kits estimated between 20X and 46X mean
on-target depth was required to successfully genotype
95% of heterozygous SNPs, with the commercially avail-
able kits at the higher end of that range. as sequencing technology rapidly improves and cost per
sequenced nucleotide falls, there is likely to come a point
where it is more economic to sequence a whole genome
rather than target-capture and sequence, even if analysis is
confined to just the exome. Where that point lies depends
both on the costs of the technologies but also on the uni-
formity of coverage and biases inherent in the data. In
this work we set out to compare exome-seq with whole
genome sequencing (WGS) in terms of their sensitivity to
correctly detect known variants over the whole exome. g
g
We examine previously established measures of SNP
detection sensitivity [9] in coding regions from exome-
seq and WGS samples. Background SNP detection sensitivity can be
measured both at a site level, considering the number
of reads mapped over a given position in the reference
genome, or as an overall estimate based on the mapped
read depth across a region or regions (Figure 1). We com-
puted the per-site measure for different sequencing tech-
nologies and compared them directly when the per-site
mapped depth is identical (point A, Figure 1). Because of
the allele distribution bias in exome-seq, we expected that
WGS would require fewer reads to successfully genotype
heterozygous SNPs. The greater variability in coverage
from exome-seq means that greater mean on-target depth
should be required to identify the same proportion of
SNPs in exome-seq as compared to WGS [10] (points B
and C, Figure 1). We measured the estimated overall SNP
detection sensitivity across a given set of target regions
by using the per-site SNP detection sensitivity for the
sequencing method combined with the coverage distri-
bution for samples sequenced by the same method. This
relates the overall sensitivity of a method to the mean on-
target depth in the sample, which can be used to calculate
the cost of sequencing to a given sensitivity. The process of exome-seq has known issues that impact
negatively on SNP detection sensitivity. These include
PCR amplification, which tends towards lower cover-
age in GC-rich regions due to annealing during ampli-
fication [4-6], and the preferential capture of reference
sequence alleles, which biases the allele distribution away
from alternate alleles at heterozygous SNP sites [7-9]. Exome-seq produces a relatively heterogeneous profile of
read coverage over target regions when compared to the
more homogeneous WGS [10]. Better uniformity of cov-
erage yields improved SNP detection sensitivity across the
regions of interest [9-11]. Previous estimates of the amount of sequencing
required to accurately identify SNPs in WGS and exome-
seq are variable. Bentley et al. estimated that 15X
mapped read depth of WGS samples would be suffi-
cient to detect almost all homozygous SNPs and 33X
for almost all heterozygous SNPs [12]. 50X was esti-
mated by Ajay et al. for all SNPs and small indels
[13]. Depending on the capture kit, Clark et al. cal-
culated that exome-seq required 80X mean on-target
depth to reach the common threshold of 10X per-site
depth in 90% or more of all targeted regions [10]. Our previous work on some of the original exome-seq Results and discussion
Site level SNP detection sensitivity Results and discussion
Site level SNP detection sensitivity Site level SNP detection sensitivity Site level SNP detection sensitivity Site level SNP detection sensitivity is the mapped
read
depth
directly
over
a
polymorphic
site
that
is required to reliably call that polymorphism [9]. Figure 1 SNP detection sensitivity in exome and whole genome sequencing. Exome and whole genome sequencing mapped read depth
across the exons of an example gene. The grey vertical lines denote exon boundaries. At point A, the depth is equal and we can compare the
per-site SNP detection sensitivity. Points B and C are examples of unequal depth, where per-site sensitivity cannot be directly compared, but the
overall estimated sensitivity of the region can be calculated to account for the variability in coverage. Figure 1 SNP detection sensitivity in exome and whole genome sequencing. Exome and whole genome sequencing mapped read depth
across the exons of an example gene. The grey vertical lines denote exon boundaries. At point A, the depth is equal and we can compare the
per-site SNP detection sensitivity. Points B and C are examples of unequal depth, where per-site sensitivity cannot be directly compared, but the
overall estimated sensitivity of the region can be calculated to account for the variability in coverage. Page 3 of 11 Meynert et al. BMC Bioinformatics 2014, 15:247
http://www.biomedcentral.com/1471-2105/15/247 Meynert et al. BMC Bioinformatics 2014, 15:247
http://www.biomedcentral.com/1471-2105/15/247 Ten human whole genome sequences (TCGA-WGS)
and matched whole exome sequences from the same
patients, plus ten additional exome samples (all TCGA-
WXS), were obtained from The Cancer Genome Atlas
(TCGA; only non-tumour samples were used). A fur-
ther six human whole genome samples (1KG-WGS)
were obtained from the 1000 Genomes Project [14],
all aligned to the reference genome. An additional 13
exome samples were captured, sequenced, and aligned
in house as part of two ongoing disease studies (HGU-
WXS) (Methods, Additional file 1: Table S1, Additional
file 2: Tables S2–S4, Additional file 1: Figures S1 and S2). We randomly downsampled all 49 alignments to simu-
late shallower sequencing and called SNPs in the coding
regions of the alignments as in our previous work [9]. gold standard (Additional file 1: Figure S4). We measured
sensitivity as a function of the per-site depth for heterozy-
gous (Figure 2) and homozygous (not shown) SNPs. We
focused on heterozygous SNPs as the more challenging
problem: only 2-3X per-site depth was required to accu-
rately detect at least 95% of homozygous SNPs in all four
data sets. Direct comparison of matched samples The matched WGS and exome-seq samples from TCGA
were compared directly. Considering only SNPs in the
regions targeted by the exome capture kit, 98.3 ± 0.007%
of variant sites were called as polymorphic by both meth-
ods with matched genotypes, and 0.3 ± 0.3% with mis-
matched genotypes. A further 1.3 ± 0.4% were called
as polymorphic by whole genome sequencing only, and
0.1 ± 0.1% by exome sequencing only. 93% of the sites
called as polymorphic only by whole genome sequencing
had greater mapped per-site depth in the WGS sam-
ple than in the exome-seq sample (Additional file 1:
Figure S7). Of these, 34% are at sites with no reads in the
exome-seq sample, which could be due to probe failure or
other technical problems. The majority of mismatched genotypes were cases
where the whole genome sample was genotyped as het-
erozygous and the exome sample was genotyped as
homozygous (Additional file 1: Figure 8a and Table S7). Mismatches generally occurred at sites where the whole
genome sample had higher per-site mapped depth than
the exome sample (Additional file 1: Figure S8b). Some
sites with very high mapped depth in the exome sample
also had mismatched genotypes with the whole genome
sample. This could be caused by random accumulation
of the same sequencing error at a given position if that
position is sequenced to very high mapped depth. Both of
these results imply that improving uniformity of coverage
will improve SNP detection sensitivity. The number of variants called from the TCGA-WGS
data set did not improve greatly with grouped sample
calling (mean of 37 heterozygous and 11 homozygous
additional sites), though there were a large number of
mismatched genotypes between the group calling and
the single-sample calling. This was observed in only 6 of
the samples; the other 4 all had ≤2 mismatched geno-
types. The TCGA-WGS samples had both excellent mean
on-target depth and uniformity of coverage, which made
them easy to accurately genotype using single-sample
variant calling. Grouped variant calling would therefore
not provide the same boost as with the other data sets. We took the subset of coding SNPs where the alle-
les and genotypes were identical in the full alignments
between the TCGA-WGS and TCGA-WXS samples for
the same individual, and compared the mapped depth of
sequencing required to correctly identify the genotypes
of both heterozygous and homozygous SNPs (Figure 3). Effect of grouped and single sample variant calling same generation of machine, this difference was likely
caused by reference bias from the capture step. Low-depth
exome sequencing projects may need to take this into
consideration. Effect of grouped and single sample variant calling The results in this paper are derived from calling variants
for one sample at a time; however, it is standard prac-
tice to call variants in groups of samples (pooled calling)
as this improves accuracy by allowing the use of reads
across all samples at a position to determine the presence
of a polymorphism. To investigate the relative benefits of
pooled calling, we grouped our samples by data source,
called variants on the full alignments for each group,
and compared the results to the variants called on the
full alignments by single sample calling (Additional file 2:
Table S8a). All the data sets converged at 99% sensitivity for sites
with between 15 and 20X mapped depth. The recall
curves were virtually identical for variants drawn from
the Ensembl 72 coding regions and each of the sets
of targeted regions from the two exome sequencing
data sets (Additional file 1: Figure S5, Additional file 1:
Table S6). Specificity (1 - false positive rate) reached 99%
at 9X for the 1KG-WGS data set and 8X for the other three
(Additional file 1: Figure S6). For sites in HapMap 3.3, there were very few cases of
mismatched genotypes between the two calling methods;
the main difference was in additional sites called as poly-
morphic when the samples were grouped. For all data sets,
of the sites with mismatched genotypes or where only
one method called the site as polymorphic, the mapped
read depth was on average lower than for sites where
genotypes were matched (Additional file 1: Figure S9). The two exome capture data sets benefited significantly
from grouped sample calling, with a mean of 186 (594)
heterozygous and 100 (326) homozygous additional sites
genotyped for the HGU-WXS (TCGA-WXS) data set. These data sets had the most samples, which may have
been the major cause of the improvement, or possibly
the uneven coverage of the exomes was smoothed by the
inclusion of multiple samples. Effect of grouped and single sample variant calling The 1KG-WGS data set
also benefited to the same degree as the exome capture
data sets for heterozygous sites (mean 317 additional),
but not for homozygous sites (mean 52 additional), per-
haps because the 1KG-WGS data set comprises two family
trios, which would help to resolve heterozygous positions. Site level SNP detection sensitivity BMC Bioinformatics 2014, 15:247
http://www.biomedcentral.com/1471-2105/15/247 Page 4 of 11 Effect of grouped and single sample variant calling Site level SNP detection sensitivity The oldest data set (1KG-WGS) had the worst per-
formance for heterozygous SNPs, requiring at least 13X
to reach 95% sensitivity. This could be due to shorter
read lengths or higher sequencing error rates on older
technologies. The difference in per-site SNP detection
sensitivity between this data set and the newer three data
sets indicates that analysis of older data sets requires more
stringent thresholds. We defined a gold-standard set of SNP calls for each
sample, based on the full alignments (using all avail-
able reads for the sample, i.e. not down sampled) and
confined to known HapMap 3.3 variants (Additional
file 2: Table S5, Additional file 1: Figure S3, Additional
file 3). We validated the use of HapMap 3.3 variants
as the gold standard in sample NA12878 by compar-
ing results to those obtained using the Genome in a
Bottle 2.18 highly confident variant call set [15] as the All of the newer three data sets, which are contempo-
rary with each other, performed similarly. The TCGA-
WXS samples reached 95% sensitivity at 10X, while the
HGU-WXS and TCGA-WGS samples had a slight edge
at 9X. There was a slight advantage in sensitivity for the
newer TCGA-WGS data set as opposed to the two exome-
seq data sets, though this equalized at 12-13X per-site
depth. Given that read lengths are the same between these
three data sets and most samples were sequenced on the Depth at SNV position
Heterozygous SNP sensitivity
0.0
0.2
0.4
0.6
0.8
1.0
5
10
15
20
1KG−WGS
HGU−WXS
TCGA−WGS
TCGA−WXS
Figure 2 Site level heterozygous SNP detection sensitivity for exome and whole genome sequencing samples. Sensitivity is calculated from
heterozygous HapMap 3.3 positions [16] located within coding sequence as determined by Ensembl 72 [17]. 95% sensitivity is reached at per-site
mapped depths of 9X for the TCGA-WGS samples, 10X for the TCGA-WXS and HGU-WXS samples, and 13X for the 1KG-WGS samples. Figure 2 Site level heterozygous SNP detection sensitivity for exome and whole genome sequencing samples. Sensitivity is calculated from
heterozygous HapMap 3.3 positions [16] located within coding sequence as determined by Ensembl 72 [17]. 95% sensitivity is reached at per-site
mapped depths of 9X for the TCGA-WGS samples, 10X for the TCGA-WXS and HGU-WXS samples, and 13X for the 1KG-WGS samples. Page 4 of 11 Meynert et al. BMC Bioinformatics 2014, 15:247
http://www.biomedcentral.com/1471-2105/15/247 Meynert et al. Direct comparison of matched samples To accurately genotype 95% of heterozygous SNPs, the
TCGA-WGS data set required a minimum per-site depth
of 12X and the TCGA-WXS data set required 34X. For
homozygous sites, the minimum per-site depths were 8X
and 33X respectively. We also examined rare variants, as defined by absence
from HapMap 3.3, presence in the Exome Variant Server
ESP6500 (http://evs.gs.washington.edu/EVS) set at less
than 0.01 minor allele frequency, and minimum geno-
type quality at least 60 in each of the grouped and single
sample call sets (Additional file 2: Table S8b). The 1KG-
WGS, HGU-WXS, TCGA-WGS, and TCGA-WXS data
sets gained a mean of an additional 0.28%, 0.25%, 1.23%,
and 4.30% respectively of these rare variants by grouped Meynert et al. BMC Bioinformatics 2014, 15:247
http://www.biomedcentral.com/1471-2105/15/247 Page 5 of 11 (a)
Minimum depth for correct genotype call
Cumulative density
0.0
0.2
0.4
0.6
0.8
1.0
0
10
20
30
40
Heterozygous
0.0
0.2
0.4
0.6
0.8
1.0
Homozygous
WGS
WXS
(b)
Difference (exome − genome) in minimum depth for correct genotype call
Density
0.00
0.02
0.04
0.06
0
20
40
Heterozygous
Homozygous
re 3 Minimum per-site mapped depth required to correctly genotype a site in matched TCGA exome and genome samples. Coding
at HapMap 3.3 positions [16] with identical genotypes and alleles between matched TCGA exome and genome samples in the full alignments. e minimum per-site mapped depth required for a correct genotype call in TCGA-WXS and TCGA-WGS matchd samples. b) The number of extra
required to correctly identify a SNP in the TCGA-WXS sample. (a)
Minimum depth for correct genotype call
Cumulative density
0.0
0.2
0.4
0.6
0.8
1.0
0
10
20
30
40
Heterozygous
0.0
0.2
0.4
0.6
0.8
1.0
Homozygous
WGS
WXS (a) Cumulative density (b)
Difference (exome
genome) in minimum depth for correct genotype call
Density
0.00
0.02
0.04
0.06
0
20
40
Heterozygous
Homozygous (b) Density Difference (exome − genome) in minimum depth for correct genotype call Figure 3 Minimum per-site mapped depth required to correctly genotype a site in matched TCGA exome and genome samples. Coding
SNPs at HapMap 3.3 positions [16] with identical genotypes and alleles between matched TCGA exome and genome samples in the full alignments. a) The minimum per-site mapped depth required for a correct genotype call in TCGA-WXS and TCGA-WGS matchd samples. b) The number of extra
reads required to correctly identify a SNP in the TCGA-WXS sample. Characteristics of difficult target regions As has previously been noted for both whole genome
and exome sequencing, regions of high G+C content
and regions containing repetitive elements are gener-
ally harder to sequence to high depth [19]. We define
difficult regions based on poor coverage (see Methods)
in at least half the samples from a given data set, and
easy regions based on excellent coverage (see Methods)
in all the samples from that set. Our samples show the
expected characteristics, with the bulk of difficult regions
occurring at G+C content above 60% (Additional file 1:
Figure S11), and with a significantly higher proportion
of difficult regions overlapping repetitive elements com-
pared to relatively easy target regions (Additional file 1:
Figure S12). The HGU-WXS data set also had a large
number of difficult target regions that were of low G+C
content. Because the classification of a region as difficult is
based on at least half the samples in a data set, this was not
caused by capture failure of one or a few samples; however,
a larger scale failure could be implicated. Very few target
regions were classed as difficult for the TCGA-WGS data
set for either of the two exome capture target region sets. However, approximately one third of all regions identified
as difficult in any of the four data sets were classed that
way for both the TCGA-WXS and 1KG-WGS data set, and
15% for both the HGU-WXS and the 1KG-WGS data set
(Additional file 1: Figure S13). Our estimates for WGS required mapped depth are
lower than those from Bentley et al. (33X) [12] and Ajay
et al. (50X) [13], though both were attempting to quan-
tify detection of all or almost all SNPs rather than to a
given percentage as here. It is unsurprising that the harder
to sequence variants will require proportionally greater
additional numbers of reads to accurately genotype. Addi-
tionally, we are analysing only coding sequence variants,
which are in the least repetitive portion of the human
genome. The higher figures reported by the other WGS
analyses will be influenced by the different qualities of
non-coding sequence, especially repetitive regions. The
TCGA-WXS and HGU-WXS exome-seq data sets used
in this analysis can update the figures provided by Clark
et al. Characteristics of difficult target regions of 80X mean on-target depth required for 10X
mapped read depth in 90% of targeted regions [10]: a
median of 59X mean on-target depth is needed for the
same coverage in both of our more recent exome-seq data
sets. The equivalent figure was 18X for the TCGA-WGS
data set and 20X for the 1KG-WGS data set. To quantify the contributions of repetitive sequence
and nucleotide composition to target difficulty, we iden-
tified targets meeting our criteria for difficult (see
Methods) in any of the samples for a data set. The num-
ber of samples in which that target was defined as difficult
was multiply regressed against target G+C content, pres-
ence of annotated repeats and alignability [20]. All factors
were significant to p < 0.001; however, their predic-
tive power was slight (Additional file 1: Table S8). The
adjusted R-squareds were 0.265, 0.086, 0.150, and 0.171
for 1KG-WGS, HGU-WXS, TCGA-WGS, and TCGA-
WXS respectively. As the analysis was run on the intersec-
tion of the target capture regions for the two exome-seq Direct comparison of matched samples Figure 3 Minimum per-site mapped depth required to correctly genotype a site in matched TCGA exome and genome samples. Coding
SNPs at HapMap 3.3 positions [16] with identical genotypes and alleles between matched TCGA exome and genome samples in the full alignments. a) The minimum per-site mapped depth required for a correct genotype call in TCGA-WXS and TCGA-WGS matchd samples. b) The number of extra
reads required to correctly identify a SNP in the TCGA-WXS sample. Page 6 of 11 Meynert et al. BMC Bioinformatics 2014, 15:247
http://www.biomedcentral.com/1471-2105/15/247 calling, while losing only 0.31%, 0.04%, 0.19%, and 0.35%
that were only called in the single sample method. The
TCGA-WXS data set gained by far the most rare variants
by use of the grouped calling, similar to the results for
known common HapMap 3.3 sites. given a particular sequencing strategy. To demonstrate
and at the same time discover if known disease causing
mutations are preferentially located in easy or difficult to
sequence regions of the genome: we obtained the loca-
tions of such coding and splice variants from HGMD [18]. From these we generated the coverage distributions for
disease-causing and disease-associated SNVs separately
to compare their overall estimated sensitivity with cod-
ing regions in general for both whole genome and exome
sequencing. For 87,663 disease-causing and 2,241 disease-
associated sites, we found no difference in the measure
across all four of our sample sets (Additional file 1:
Figure S10). Overall estimated sensitivity Using the depth of coverage distributions for every down-
sampled and full alignment on the regions targeted by
each of the two exome capture kits, and the per-site SNP
detection sensitivity for each data set, we calculated the
overall estimated sensitivity for each of the four data sets. We compared this measure to the mean on-target read
depth across the targeted regions and found that the two
whole genome data sets performed considerably better
than each of the exome data sets (Figure 4). In order to
reach an overall estimated 95% sensitivity for heterozy-
gous SNPs in the targeted regions, the 1KG-WGS samples
required at least 18X and the TCGA-WGS 14X mean on-
target depth. The HGU-WXS samples required 41X mean
on-target depth, and the TCGA-WXS samples 39X. This
effect is almost entirely due to the lack of uniformity in
coverage for the exome samples: The difference in per-site
sensitivity is relatively slight between the two exome data
sets and the TCGA-WGS data set (Figure 2), and both of
them perform better than the 1KG-WGS data set on that
measure. Cost of sequencing to a given level of sensitivity We compared exome and whole genome sequencing costs
on current standard technology (Illumina HiSeq) with an
exome capture kit of the same size as the Nimblegen
SeqCap EZ Exome v3 (65Mbp) used for the HGU-WXS
samples, assuming 60% of exome reads on target (Table 1)
and holding the per sample cost of the exome capture
kit constant. To achieve 93–94% overall estimated het-
erozygous sensitivity in the coding regions of the genome,
exome sequencing is 4.2X cheaper than whole genome
sequencing (12 exome samples/lane vs. 1 lane/sample
whole genome). Likewise, for 98–99% sensitivity, exome
sequencing is 5.4X cheaper (4 exome samples/lane vs. 2
lanes/sample whole genome). The mean on-target depth needed for 95% SNP detec-
tion sensitivity shown by our analysis of WGS data from
multiple sources is also lower than previous estimates
[12,13]. The earlier of these two estimates describes reads
from the first next-generation sequencing experiments,
which were shorter than the reads used for our WGS
samples, and additionally contained no paired-end reads. The second estimate is more comparable in terms of
data, and we conclude that improvement in variant calling
algorithms is likely to be a factor in the difference here. Uniformity of coverage is clearly still a major issue
for exome sequencing in terms of capturing a reason-
able number of reads across all of the targeted regions. PCR amplification-free library preparation can mitigate
the issue somewhat for WGS samples [4,5] but it is still
required to provide a sufficiently large library for exome-
seq samples. Allele distribution biases introduced by the
reference bias of exome-seq target probes could be min-
imised by the use of alternate probes containing common
haplotypes, but the problem will remain for rare variants. The additional allele distribution bias introduced by treat-
ing the reference genome as truth during computational
analysis affects both WGS and again exome-seq and is not
easily fixed for rare variants. We estimate that the cost per lane of sequence would
have to be 15–20% of the current cost for the two meth-
ods to reach cost parity, holding the cost of exome capture
constant (Additional file 1: Figure S14). The projected
$1000 genome at 30X depth enabled by the Illumina
HiSeq X Ten (X10) system reaches this cost point for 93–
94% overall estimated heterozygous sensitivity in the cod-
ing regions of the genome. Sensitivity at sites in Human Gene Mutation Database
(HGMD) Overall estimated sensitivity is a useful measure that can
be applied to more specific subsets of target regions. For instance, estimating how many known disease caus-
ing or disease associated SNV sites can be recovered Meynert et al. BMC Bioinformatics 2014, 15:247
http://www.biomedcentral.com/1471-2105/15/247 Page 7 of 11 Figure 4 Overall estimated sensitivity for targeted regions. Calculated from the per-site sensitivity for each data set combined with the depth
of coverage distributions for samples across the regions targeted by each of the two exome capture kits. a) HGU-WXS (Nimblegen SeqCap EZ
Exome v3). b) TCGA-WXS (Whole exome Agilent 1.1 plus boosters). Figure 4 Overall estimated sensitivity for targeted regions Calculated from the per-site sensitivity for each data set combin Figure 4 Overall estimated sensitivity for targeted regions. Calculated from the per-site sensitivity for each data set combined with the depth
of coverage distributions for samples across the regions targeted by each of the two exome capture kits. a) HGU-WXS (Nimblegen SeqCap EZ
Exome v3). b) TCGA-WXS (Whole exome Agilent 1.1 plus boosters). Page 8 of 11 Meynert et al. BMC Bioinformatics 2014, 15:247
http://www.biomedcentral.com/1471-2105/15/247 Meynert et al. BMC Bioinformatics 2014, 15:247
http://www.biomedcentral.com/1471-2105/15/247 for heterozygous SNPs [9]. The two data sets in this
analysis from more recent capture kits (HGU-WXS and
TCGA-WXS) show 40X is required for the same level of
sensitivity. This progressive improvement in technology
could partially explain the difference between our results
and the higher mean on-target depth of 80X suggested by
other previous analyses such as Clark et al. [10]. data sets; the particularly low R-squared for the HGU-
WXS data set may be due to differences in probe design
but is not due to a different set of targets. Cost of sequencing to a given level of sensitivity Holding the per sample exome
kit cost constant, the X10 system claims to sequence
genomes to 12X depth at 77% the cost of sequencing
exomes to 29X depth, with roughly equivalent sensitiv-
ity. However, for higher sensitivity of 98–99%, we estimate
that WGS on the X10 system will still be 31% more expen-
sive than exome-seq, and decreases in exome capture kit
costs will likely keep the two methods at close to cost
parity. The amount of raw sequencing is the main cost driver
for both WGS and exome-seq, and the drop in cost to the
$1000 human genome at 30X depth has brought the two
methods roughly into parity. However, smaller sequenc-
ing centres relying on the previous generation of machines
will continue to charge three to four times exome-seq
costs for the same level of SNP detection sensitivity across
coding regions using WGS. When taking into account the
considerably higher data storage requirements of WGS All costs have been normalised against the cheapest exome sequencing (16 samples per lane). Estimated costs include library preparation, exome capture and
multiplexing where applicable, and paired-end sequencing on Illumina HiSeq. Alignments Reads for the 13 HGU-WXS exomes were aligned to
the hg19/GRCh37 assembly of the human genome ref-
erence sequence with BWA 0.5.9 [21]. Duplicate reads
were removed using the MarkDuplicates function of
Picard 1.79 (http://picard.sourceforge.net). Reads were re-
aligned around indels and quality scores re-calibrated
using the Genome Analysis Toolkit (GATK) 2.2-8-
gec077cd [22]. Full parameters are given in the Additional
file 1: Supplementary Information and Additional file 4. We randomly down-sampled reads from exome align-
ments using Picard DownsampleSam, which maintains
read pair information. The probability of sampling each
read varied from 0.1 to 0.9 at intervals of 0.1. Ethical approval and consent The samples used for in-house exome sequencing were
collected under approval by the UK Multiregional Ethics
Committee (References: 06/MRE00/76 and 04/MRE00/
19). Conclusions Exome-seq target capture technology is clearly improv-
ing. Our previous results from a solution-based target
capture kit suggested a mean on-target depth of 46X
was needed to obtain 95% overall estimated sensitivity Table 1 Cost of sequencing to achieve a given level of heterozygous SNV detection sensitivity
Method
Lane usage
Mean on-target depth
Sensitivity
Cost
Whole genome
1 lane/sample
11X
94.0%
4.60
2 lanes/sample
22X
98.5%
8.79
Exome
16 samples/lane
22X
91.1%
1.00
12 samples/lane
29X
93.4%
1.09
8 samples/lane
44X
95.9%
1.28
6 samples/lane
58X
96.9%
1.46
4 samples/lane
88X
98.1%
1.63
All costs have been normalised against the cheapest exome sequencing (16 samples per lane). Estimated costs include library preparation, exome capture and
multiplexing where applicable, and paired-end sequencing on Illumina HiSeq. Table 1 Cost of sequencing to achieve a given level of heterozygous SNV detection sensitivity All costs have been normalised against the cheapest exome sequencing (16 samples per lane). Estimated costs include library preparation, exome capture and
multiplexing where applicable, and paired-end sequencing on Illumina HiSeq. All costs have been normalised against the cheapest exome sequencing (16 samples per lane). Estimated costs include library preparation, exome capture and
multiplexing where applicable, and paired-end sequencing on Illumina HiSeq. Meynert et al. BMC Bioinformatics 2014, 15:247
http://www.biomedcentral.com/1471-2105/15/247 Meynert et al. BMC Bioinformatics 2014, 15:247
http://www.biomedcentral.com/1471-2105/15/247 Page 9 of 11 Page 9 of 11 Page 9 of 11 Page 9 of 11 Target regions and the extra compute time required to perform align-
ment and subsequent bioinformatic analyses on WGS
samples, the cost difference will be further amplified. We defined a common set of target regions using the cod-
ing regions of exons from Ensembl 72 [17]. The coding
regions were merged so that every position was repre-
sented only once, 50bp of flanking sequence added to each
resulting region, and the regions merged again. We also
used the provided targets for the Nimblegen SeqCap EZ
Exome v3 kit, a set of targets labelled ‘Whole exome Agi-
lent 1.1 RefSeq plus 3 boosters’ obtained directly from
TCGA, and a merged set of these two targets. WGS provides a much richer data set, capturing
information on polymorphisms over whole genome and
potentially capturing genomic rearrangements. The dra-
matically improved uniformity of read coverage and
reduced bias of allele ratios in WGS, both lend themselves
to improved detection of copy number changes and mea-
surement of sample heterogeneity. These are likely to be
extremely useful measures in some settings, such as for
the sequencing of primary tumours whose analysis, even
when focused on the exome, is confounded by copy num-
ber change, sample heterogeneity and a desire to detect de
novo mutations. Mapped read depth across all the target regions was
calculated using the DepthOfCoverage tool from GATK
2.6-5-gba531bd [22]. The target regions were split into
maximally 100bp non-overlapping tiles for further anal-
ysis, with small tiles at target region edges. We defined
difficult target region tiles as those with fewer than 50%
of their bases covered at least 15X in the full align-
ments for at least half of the samples in a given data
set, and easy target region tiles as those with all their
bases covered at least 15X in the full alignments for all
the samples in a given data set. G+C content for tar-
get regions was obtained using the GCContentByInterval
tool from GATK GenomeAnalysisTK-2.5-2-gf57256b, and
repeat element occurrences were mapped from Ensembl
73. HGMD disease causing mutations were obtained from
the HGMD Professional database (March 2013 release). Exome capture and sequencing The 13 HGU-WXS exomes were captured using a Nim-
blegen SeqCap EZ Exome v3 kit. Paired-end reads of 98
bp were generated on the Illumina HiSeq platform. Six
whole genome samples were downloaded from the 1000
Genomes Project [14] Pilot 2 (high coverage family trios). Individuals with both whole genome and exome samples
from TCGA were filtered for cases with similar num-
bers of reads to the 1KG-WGS and HGU-WXS exome
samples. From these, 20 individuals with exome samples
labelled with the same set of target capture regions were
randomly selected. Exome alignments for all 20 individ-
uals and whole genome alignments for a random subset
of 10 individuals were downloaded from TCGA Data Por-
tal. Additional file 1: Table S1 summarises the technology
used to generate each of the four data sets. Individuals with both whole genome and exome samples
from TCGA were filtered for cases with similar num-
bers of reads to the 1KG-WGS and HGU-WXS exome
samples. From these, 20 individuals with exome samples
labelled with the same set of target capture regions were
randomly selected. Exome alignments for all 20 individ-
uals and whole genome alignments for a random subset
of 10 individuals were downloaded from TCGA Data Por-
tal. Additional file 1: Table S1 summarises the technology
used to generate each of the four data sets. Multiple linear regression of number of samples where
a target region had less than 50% of bases covered at
least 15X in the full alignment was performed using R
lm on factors G+C content, presence of repeats, and
alignability [20] with no interactions, where at least one
sample met the criteria for a given data set. Alignabil-
ity tracks were downloaded from the UCSC Genome
Browser [23]. 36mer alignability was used for 1KG-WGS
samples, 75mer for TCGA-WXS, and 100mer for HGU-
WXS and TCGA-WGS, to best match the read lengths for
each data set (Additional file 1: Table S1). Variants Variants were called on the full and down-sampled align-
ments using the GATK 2.6-5-gba531bd UnifiedGeno-
typer tool [24], one sample at a time (full parameters
in Additional file 1: Supplementary Information). We
obtained HapMap Phase III sites and genotypes from the
project FTP site [16]. Variants from this set were mapped
by position and alleles to called variants in the full and
down-sampled alignments. Additional variant calls were
made by grouping samples by data source and running the
UnifiedGenotyper tool on the full alignments for samples
within each group. Sensitivity Sensitivity for per-site mapped read depth and estimated
overall sensitivity were calculated as in [9], using sites Page 10 of 11 Meynert et al. BMC Bioinformatics 2014, 15:247
http://www.biomedcentral.com/1471-2105/15/247 Meynert et al. BMC Bioinformatics 2014, 15:247
http://www.biomedcentral.com/1471-2105/15/247 facilitates improved mapping and assembly of (G+C)-biased
genomes. Nat Methods 2009, 6(4):291–295. 5. Aird D, Ross MG, Chen W-S, Danielsson M, Fennell T, Russ C, Jaffe DB,
Nusbaum C, Gnirke A: Analyzing and minimizing PCR amplification
bias in Illumina sequencing libraries. Genome Biol 2011, 12(2):18. 6. Veal CD, Freeman PJ, Jacobs K, Lancaster O, Jamain S, Leboyer M, Albanes
D, Vaghela RR, Gut I, Chanock SJ, Brookes AJ: A mechanistic basis for
amplification differences between samples and between genome
regions. BMC Genomics 2012, 13:455. 7. Quail MA, Kozarewa I, Smith F, Scally A, Stephens PJ, Durbin R, Swerdlow
H, Turner DJ: A large genome center’s improvements to the Illumina
sequencing system. Nat Methods 2008, 5(12):1005–1010. 8. Heinrich V, Stange J, Dickhaus T, Imkeller P, Krüger U, Bauer S, Mundlos S,
Robinson PN, Hecht J, Krawitz PM: The allele distribution in
next-generation sequencing data sets is accurately described as the
result of a stochastic branching process. Nucleic Acids Res 2012,
40(6):2426–2431. 9
Meynert AM Bicknell LS Hurles ME Jackson AP Taylor MS: Quantifying in HapMap 3.3 and the Ensembl 72 coding regions with
minimum genotype quality of 60 in the full alignments. in HapMap 3.3 and the Ensembl 72 coding regions with
minimum genotype quality of 60 in the full alignments. facilitates improved mapping and assembly of (G+C)-biased
genomes. Nat Methods 2009, 6(4):291–295. 5. Aird D, Ross MG, Chen W-S, Danielsson M, Fennell T, Russ C, Jaffe DB,
Nusbaum C, Gnirke A: Analyzing and minimizing PCR amplification
bias in Illumina sequencing libraries. Genome Biol 2011, 12(2):18. Acknowledgements We thank Edinburgh Genomics for providing information regarding the
relative costs of whole genome sequencing and targeted exome capture
followed by sequencing. This work was supported by the UK Medical Research
Council and the Medical Research Foundation. 17. Flicek P, Ahmed I, Amode MR, Barrell D, Beal K, Brent S, Carvalho-Silva D,
Clapham P, Coates G, Fairley S, Fitzgerald S, Gil L, García-Girón C, Gordon
L, Hourlier T, Hunt S, Juettemann T, Kähäri AK, Keenan S, Komorowska M,
Kulesha E, Longden I, Maurel T, McLaren WM, Muffato M, Nag R, Overduin
B, Pignatelli M, Pritchard B, Pritchard E, et al: Ensembl 2013. Nucleic Acids
Res 2013, 41(Database issue):48–55. Authors’ contributions
AMM
d MST d
i
d 15. Zook JM, Chapman B, Wang J, Mittelman D, Hofmann O, Hide W, Salit M:
Integrating human sequence data sets provides a resource of
benchmark SNP and indel genotype calls. Nat Biotechnol 2014,
32(3):246–251. AMM and MST designed the analysis and wrote the manuscript. AMM
performed the analysis. MA and DRF provided and sequenced the HGU-WXS
exomes. All authors read and approved the final manuscript. AMM and MST designed the analysis and wrote the manuscript. AMM
performed the analysis. MA and DRF provided and sequenced the HGU-WXS
exomes. All authors read and approved the final manuscript. 16. The International HapMap 3 Consortium: Integrating common and rare
genetic variation in diverse human populations. Nature 2010,
467(7311):52–58. Availability of supporting data The TCGA-WXS and TCGA-WGS data are available
through dbGaP and the Cancer Genome Hub (http://
cghub.ucsc.edu/) and 1KG-WGS data from the 1000
Genomes Project (http://www.1000genomes.org/data),
see Additional file 1: Table S2 for accession identifiers. Reads for the HGU-WXS exome sequence data are avail-
able upon request. VCF files containing the HapMap 3.3
SNP sites and associated genotype calls and read depths
in the downsampled and full alignments used to generate
the main results in this article are included as additional
files. The command lines for producing the downsampled
alignments and calling variants are included as a plain text
additional file. 6. Veal CD, Freeman PJ, Jacobs K, Lancaster O, Jamain S, Leboyer M, Albanes
D, Vaghela RR, Gut I, Chanock SJ, Brookes AJ: A mechanistic basis for
amplification differences between samples and between genome
regions. BMC Genomics 2012, 13:455. 7. Quail MA, Kozarewa I, Smith F, Scally A, Stephens PJ, Durbin R, Swerdlow
H, Turner DJ: A large genome center’s improvements to the Illumina
sequencing system. Nat Methods 2008, 5(12):1005–1010. 8. Heinrich V, Stange J, Dickhaus T, Imkeller P, Krüger U, Bauer S, Mundlos S,
Robinson PN, Hecht J, Krawitz PM: The allele distribution in
next-generation sequencing data sets is accurately described as the
result of a stochastic branching process. Nucleic Acids Res 2012,
40(6):2426–2431. 9. Meynert AM, Bicknell LS, Hurles ME, Jackson AP, Taylor MS: Quantifying
single nucleotide variant detection sensitivity in exome
sequencing. BMC Bioinformatics 2013, 14:195. 10. Clark MJ, Chen R, Lam HYK, Karczewski KJ, Chen R, Euskirchen G, Butte AJ,
Snyder M: Performance comparison of exome DNA sequencing
technologies. Nature Biotechnologyotech 2011, 29(10):908–914. 11. Sulonen A-M, Ellonen P, Almusa H, Lepisto M, Eldfors S, Hannula S,
Miettinen T, Tyynismaa H, Salo P, Heckman C, Joensuu H, Raivio T,
Suomalainen A, Saarela J: Comparison of solution-based exome
capture methods for next generation sequencing. Genome Biol 2011,
12(9):94. Received: 7 February 2014 Accepted: 7 July 2014
Published: 19 July 2014 18. Stenson PD, Ball EV, Mort M, Phillips AD, Shiel JA, Thomas NST,
Abeysinghe S, Krawczak M, Cooper DN: Human Gene Mutation
Database (HGMD): 2003 update. Hum Mutat 2003, 21(6):577–581. Additional files Additional file 1: Supplementary Information. Supplementary
methods, figures, and small tables. Additional file 2: Supplementary Tables. Large supplementary tables. Additional file 3: VCF files of HapMap 3.3 coding sites and genotypes. Additional file 4: Command lines. 12. Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, Brown
CG, Hall KP, Evers DJ, Barnes CL, Bignell HR, Boutell JM, Bryant J, Carter RJ,
Keira Cheetham R, Cox AJ, Ellis DJ, Flatbush MR, Gormley NA, Humphray
SJ, Irving LJ, Karbelashvili MS, Kirk SM, Li H, Liu X, Maisinger KS, Murray LJ,
Obradovic B, Ost T, Parkinson ML, Pratt MR, et al: Accurate whole human
genome sequencing using reversible terminator chemistry. Nature
2008, 456(7218):53–59. Abbreviations 1KG: 1000 Genomes Project; HGU: Human Genetics Unit; GATK: Genome
Analysis ToolKit; TCGA: The Cancer Genome Atlas; TCGA-WXS: TCGA Whole
eXome Sequence; TCGA-WGS: TCGA Whole genome sequence. 13. Ajay SS, Parker SC, Ozel Abaan H, Fuentes Fajardo KV, Margulies EH:
Accurate and comprehensive sequencing of personal genomes. Genome Res 2011, 21(9):1498–1505. 14. 1000 Genomes Project Consortium, Durbin RM, Abecasis GR, Altshuler DL,
Auton A, Brooks LD, Durbin RM, Gibbs RA, Hurles ME, McVean GA: A map
of human genome variation from population-scale sequencing. Nature 2010, 467(7319):1061–1073. References 1. Wetterstrand KA: DNA Sequencing Costs: Data from the NHGRI
Genome Sequencing Program. 2013. [http://www.genome.gov/
sequencingcosts/] 1. Wetterstrand KA: DNA Sequencing Costs: Data from the NHGRI
Genome Sequencing Program. 2013. [http://www.genome.gov/
sequencingcosts/] 19. Sims D, Sudbery I, Ilott NE, Heger A, Ponting CP: Sequencing depth and
coverage: key considerations in genomic analyses. Nat Rev Genet
2014, 15(2):121–132. coverage: key considerations in genomic analyses. Nat Rev Genet
2014, 15(2):121–132. 20. Derrien T, Estellé J, Marco Sola S, Knowles DG, Raineri E, Guigó R, Ribeca P:
Fast computation and applications of genome mappability. PloS One
2012, 7(1):30377. 21. Li H, Durbin R: Fast and accurate short read alignment with
Burrows-Wheeler transform. Bioinformatics 2009, 25(14):1754–1760. 22. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A,
Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA: The Genome
Analysis Toolkit: a MapReduce framework for analyzing
next-generation DNA sequencing data. Genome Res 2010,
20(9):1297–1303. 2. Cooper DN, Krawczak M, Antonarakis S. In The Metabolic and Molecular
Bases of Inherited Disease. 7th edn. Edited by Scriver C, Beaudet A, Sly W,
Valle D, Childs B, Kinzler K, Vogelstein B. New York: McGraw Hill;
1995:259–291. 2. Cooper DN, Krawczak M, Antonarakis S. In The Metabolic and Molecular
Bases of Inherited Disease. 7th edn. Edited by Scriver C, Beaudet A, Sly W,
Valle D, Childs B, Kinzler K, Vogelstein B. New York: McGraw Hill;
1995:259–291. 20. Derrien T, Estellé J, Marco Sola S, Knowles DG, Raineri E, Guigó R, Ribeca P:
Fast computation and applications of genome mappability. PloS One
2012, 7(1):30377. 21. Li H, Durbin R: Fast and accurate short read alignment with
Burrows-Wheeler transform. Bioinformatics 2009, 25(14):1754–1760. 3. Yang Y, Muzny DM, Reid JG, Bainbridge MN, Willis A, Ward PA, Braxton A,
Beuten J, Xia F, Niu Z, Hardison M, Person R, Bekheirnia MR, Leduc MS,
Kirby A, Pham P, Scull J, Wang M, Ding Y, Plon SE, Lupski JR, Beaudet AL,
Gibbs RA, Eng CM: Clinical whole-exome sequencing for the diagnosis 3. Yang Y, Muzny DM, Reid JG, Bainbridge MN, Willis A, Ward PA, Braxton A,
Beuten J, Xia F, Niu Z, Hardison M, Person R, Bekheirnia MR, Leduc MS,
Kirby A, Pham P, Scull J, Wang M, Ding Y, Plon SE, Lupski JR, Beaudet AL,
Gibbs RA, Eng CM: Clinical whole-exome sequencing for the diagnosis
f
d li
di
d
N E
l J M d 2013 369(16) 1502 1511 3. Additional file 1: Supplementary Information. Supplementary
methods, figures, and small tables.
Additional file 2: Supplementary Tables. Large supplementary tables.
Additional file 3: VCF files of HapMap 3.3 coding sites and genotypes.
Additional file 4: Command lines. References Yang Y, Muzny DM, Reid JG, Bainbridge MN, Willis A, Ward PA, Braxton A,
Beuten J, Xia F, Niu Z, Hardison M, Person R, Bekheirnia MR, Leduc MS,
Kirby A, Pham P, Scull J, Wang M, Ding Y, Plon SE, Lupski JR, Beaudet AL,
Gibbs RA, Eng CM: Clinical whole-exome sequencing for the diagnosis
of mendelian disorders. N Engl J Med 2013, 369(16):1502–1511. 4. Kozarewa I, Ning Z, Quail MA, Sanders MJ, Berriman M, Turner DJ:
Amplification-free Illumina sequencing-library preparation 3. Yang Y, Muzny DM, Reid JG, Bainbridge MN, Willis A, Ward PA, Braxton A,
Beuten J, Xia F, Niu Z, Hardison M, Person R, Bekheirnia MR, Leduc MS,
Kirby A, Pham P, Scull J, Wang M, Ding Y, Plon SE, Lupski JR, Beaudet AL,
Gibbs RA, Eng CM: Clinical whole-exome sequencing for the diagnosis
of mendelian disorders. N Engl J Med 2013, 369(16):1502–1511. 4. Kozarewa I, Ning Z, Quail MA, Sanders MJ, Berriman M, Turner DJ:
Amplification-free Illumina sequencing-library preparation 22. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A,
Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA: The Genome
Analysis Toolkit: a MapReduce framework for analyzing
next-generation DNA sequencing data. Genome Res 2010,
20(9):1297–1303. Gibbs RA, Eng CM: Clinical whole-exome sequencing for the diagnosis
of mendelian disorders. N Engl J Med 2013, 369(16):1502–1511. 4. Kozarewa I, Ning Z, Quail MA, Sanders MJ, Berriman M, Turner DJ:
Amplification-free Illumina sequencing-library preparation Gibbs RA, Eng CM: Clinical whole-exome sequencing for the diagnosis
of mendelian disorders. N Engl J Med 2013, 369(16):1502–1511. 4. Kozarewa I, Ning Z, Quail MA, Sanders MJ, Berriman M, Turner DJ:
Amplification-free Illumina sequencing-library preparation Page 11 of 11 Page 11 of 11 Meynert et al. BMC Bioinformatics 2014, 15:247
http://www.biomedcentral.com/1471-2105/15/247 23. Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler
D: The human genome browser at UCSC. Genome Res 2002,
12(6):996–1006. 24. Depristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C,
Philippakis AA, Del Angel G, Rivas MA, Hanna M, McKenna A, Fennell TJ,
Kernytsky AM, Sivachenko AY, Cibulskis K, Gabriel SB, Altshuler D, Daly MJ:
A framework for variation discovery and genotyping using
next-generation DNA sequencing data. Nat Genet 2011, 43(5):491–498. doi:10.1186/1471-2105-15-247
Cite this article as: Meynert et al.: Variant detection sensitivity and biases
in whole genome and exome sequencing. BMC Bioinformatics 2014 15:247. 23. References Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler
D: The human genome browser at UCSC. Genome Res 2002,
12(6):996–1006. 24. Depristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C,
Philippakis AA, Del Angel G, Rivas MA, Hanna M, McKenna A, Fennell TJ,
Kernytsky AM, Sivachenko AY, Cibulskis K, Gabriel SB, Altshuler D, Daly MJ:
A framework for variation discovery and genotyping using
next-generation DNA sequencing data. Nat Genet 2011, 43(5):491–498. doi:10.1186/1471-2105-15-247
Cite this article as: Meynert et al.: Variant detection sensitivity and biases
in whole genome and exome sequencing. BMC Bioinformatics 2014 15:247. doi:10.1186/1471-2105-15-247
Cite this article as: Meynert et al.: Variant detection sensitivity and biases
in whole genome and exome sequencing. BMC Bioinformatics 2014 15:247. Submit your next manuscript to BioMed Central
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http://www.fortunejournals.com/articles/im-not-sure-we-had-a-choice-decision-quality-and-the-use-of-cardiac-implantable-electronic-devices-in-older-adults-with-cognitive.pdf
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Im Not Sure We Had A Choice?: Decision Quality and The Use of Cardiac Implantable Electronic Devices In Older Adults With Cognitive Impairment
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Cardiology and cardiovascular medicine
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Research Article Research Article Cardiol Cardiovascmed 2018; 2 (1): 010-026 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 “I’m Not Sure We Had A Choice”: Decision Quality and The Use
of Cardiac Implantable Electronic Devices In Older Adults With
Cognitive Impairment Nicole R. Fowler1,2,3*, C. Elizabeth Shaaban4, Alexia M. Torke1,2,3, Kathleen A. Lane5, Samir Saba6,
and Amber E. Barnato7 1Indiana University Center for Aging Research, Indianapolis, IN, USA
2Regenstrief Institute, Inc., Indianapolis, IN, USA
3Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA
4Department of Epidemiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, USA
5Department of Biostatistics, Indiana University School of Medicine, Indianapolis, IN, USA
6Division of Cardiology, Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, PA,
USA
7The Dartmouth Institute of Health Policy and Clinical Practice, Geisel School of Medicine at Dartmouth, Lebanon,
NH, USA *Corresponding Author: Nicole R. Fowler, Indiana University School of Medicine, Indiana University Center
for Aging Research, 1101 West 10th Street, Indianapolis, IN 46202, USA, Telephone: (317) 274-9021; E-mail:
fowlern@iupui.edu Received: 30 January 2018; Accepted: 08 February 2018; Published: 12 February 2018 Keywords: Alzheimer’s Disease; Cardiac Implantable Electronic Device; Decision Making; Dementia Keywords: Alzheimer’s Disease; Cardiac Implantable Electronic Device; Decision Making; Dementia Abstract Background: The decision to implant a cardiac device in a person with Alzheimer’s disease or related dementia
requires considering the possible trade-offs of quality of life (QOL) and quantity of life. This study measured the
decision-making experience of patients with and without cognitive impairment (CI) who received a cardiac device
and their family members who were involved in the decision. Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292]
10
Methods and Results: Semi-structured interviews and questionnaires were administered with 15 patient-family
member dyads. Interviews revealed few conversations between physicians, patients and family members about the
patient’s cognitive status or about the benefits, risks, and long-term implications of the device for someone with CI. Participants largely stated that the decision to get the device was based on the patient’s functional status at the time
of the implant, and not on expectations about future functioning. Patients with CI had more regret, measured with Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292]
10 10 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 the Decision Regret Scale (DRS), (p=0.037) and family members of patients without CI reported more decisional
conflict, measured with the Decisional Conflict Scale (p=0.057). the Decision Regret Scale (DRS), (p=0.037) and family members of patients without CI reported more decisional
conflict, measured with the Decisional Conflict Scale (p=0.057). Conclusions: Although CI impacts life expectancy and QOL, cognitive status was largely not discussed prior to
device implant. Few differences were found between the experiences of dyads that included patients with or without
CI. ogy and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-929 1. Introduction In the U.S., there are 5.5 million people with Alzheimer’s disease or a related dementia (AD) [1] and 6 million
people with mild cognitive impairment (MCI) [2, 3]. The presence of AD and MCI are independent predictors of a
shortened life expectancy [4-6]. Recent meta-analyses and large cohort studies indicate that people with AD have an
average life expectancy of 4.5 years from diagnosis [4-8]. That is impacted by dementia subtype, patient age, and
severity of impairment at diagnosis [4-8]. Family caregivers are frequently involved in medial decision-making for
patients with AD regarding their medical care and treatment [9-12]. Most adults 65 and over have multimorbidity—the presence of two or more medical conditions, and this prevalence
increases with age. Cardiac co-morbidities, such as coronary heart disease are common in older adults, including
those with AD [13-15]. Moreover, some studies have found an association between atrial fibrillation and incident
dementia, beyond its effect on stroke [16]. Among Medicare beneficiaries with AD, 29.7% have coronary heart
disease, 27.8% have congestive heart failure (CHF), and 24.7% have cardiac dysrhythmias [17]. A potential
treatment option for patients with these cardiac co-morbidities is the implantation of a cardiac implantable electronic
device, such as a pacemaker or implantable cardioverter-defibrillator (ICD). Age and functional status at the time of device implant impact patient’s mortality post-device [18-21]. Despite
having shorter life expectancies and other co-morbidities, many older adults with cognitive impairment (CI) receive
cardiac devices, some beyond the time that it is clinically beneficial [12]. This raises questions about the risks of
device placement in patients with CI as they are twice as likely to be admitted to a nursing home immediately and
require long-term use of nursing homes after receiving a cardiac device [22]. The decision to implant a cardiac
device in a person with CI involves unique considerations about the possible trade-offs of quality of life (QOL) and
quantity of life [23]. Following device implantation, death from AD, which has been characterized by some as a
state worse than death, may be prolonged [24-26]. We are unaware of any prior studies addressing decision-making
by patients with CI, or their family members, regarding cardiac devices. The goals of this study are to compare the
decision-making experience of a cohort of patients with and without CI who received a cardiac device as well as a
family member who was involved in the decision to get a cardiac device. 2. Methods Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292] Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032
We recruited participants from the University of Pittsburgh Alzheimer Disease Research Center (ADRC). All study
procedures were approved by the Institutional Review Board (IRB) at the University of Pittsburgh. Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 We recruited participants from the University of Pittsburgh Alzheimer Disease Research Center (ADRC). All study
procedures were approved by the Institutional Review Board (IRB) at the University of Pittsburgh. We recruited participants from the University of Pittsburgh Alzheimer Disease Research Center (ADRC). All study
procedures were approved by the Institutional Review Board (IRB) at the University of Pittsburgh. Patients in this study were active ADRC participants with or without CI, ≥50 years of age, able to read and speak
English, and had received a single or dual chamber pacemaker and/or ICD after their baseline ADRC visit between
2002 and 2013. Inclusion criteria for family members included those who were named as the primary caregiver for
an ADRC patient with MCI or AD or named by the non-cognitively impaired patient as the person who helped them
make a decision about the device implantation. Patients were excluded from the study if they were too cognitively
impaired to participate in an interview. Family members were excluded if they reported not being involved in the
decision about the device implantation. We used a maximum variation sampling strategy for ADRC participants without CI and with varying levels of
severity of cognitive impairment at the time of device implant [27]. Sampling for family members included someone
for each living patient participant and for every deceased patient who met study criteria. Interviews were conducted with all family members and the subset of patients who were still alive and cognitively
able to participate to obtain socio-demographic information, questionnaire, and semi-structured interview data. Additional research and clinical data were collected from the ADRC research database and from the patients’
medical records. 2.2 Measures The range of scores for the DRS is 0 to 100, where higher scores indicate more regret (and low satisfaction with the
decision) [32-34]. Scores >25 are considered to reflect significant ambivalence about a decision and scores between
10-25 are defined as mild ambivalence [33]. Semi-structured interview data was collected from all enrolled family members and the seven patients who were still
alive and cognitively able to participate, including five patients without CI and two patients with CI. Interviews were
conducted in-person and over the phone and separately from the other dyad member. Interview questions were
developed by the research team and piloted with the first two interviews. Two study team members (NRF and CES)
conducted all in-depth interviews with questions that were broad and open-ended. We invited participants to relate
their personal narratives regarding their experiences with decision-making about cardiac device implantation. The
audio recordings were examined and transcribed, and no systematic differences were discerned between
interviewers. Participants responded in an equally open and forthcoming way to both interviewers. To maximize the
trustworthiness of our data collection, we continued recruiting participants until no new major themes emerged [35,
36]. 2.2 Measures Socio-demographic data collected from patient participants included age, sex, race, and ethnicity. For patients who
had died, date of death was obtained from the ADRC research database and confirmed with the family member. For
family members, we collected sex, ethnicity, race, and their relationship to the patient. Cognitive, functional, and clinical data collected included the Mini-Mental State Examination (MMSE) score [28]
and the Functional Activities Questionnaire (FAQ), [29] a measure of activities of daily living closest to the date of
device implant. Additionally, we reviewed all of the patient’s medical records at any point during the encounter for
when the device was implanted to verify the date of device implant, type of device, device vendor, indication for the
device, ACC/AHA Classification noted by the implanting physician, and to determine if any diagnosis of CI (AD,
MCI or other) was noted. Questionnaire data focused on measuring known constructs of medical decision-making quality. To measure role in
decision making, we used the Control Preferences Scale (CPS), which consists of one item with a five-point
scale.[30] Patient and family participants were categorized as having an active, shared, or passive role in the
decision regarding receipt of the cardiac device. We used the Decisional Conflict Scale (DCS) [31] to measure patients’ and family members’ level of uncertainty
about getting a cardiac device. The DCS contains three sub-scales: (1) uncertainty, (2) factors contributing to the 12 Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 uncertainty, and (3) perceptions of effective decision-making. The DCS includes 16 questions regarding a medical
decision the participant has made or is about to make using a five-point Likert scale [31]. The range of scores for the
DCS is 0 to100, where higher scores indicate more conflict. uncertainty, and (3) perceptions of effective decision-making. The DCS includes 16 questions regarding a medical
decision the participant has made or is about to make using a five-point Likert scale [31]. The range of scores for the
DCS is 0 to100, where higher scores indicate more conflict. To measure regret regarding the decision to get a cardiac device, we used the Decision Regret Scale (DRS), [32]
which measures “distress or remorse after a health care decision”. 2.3 Analysis We conducted qualitative analyses of the interview data guided by the methods of grounded theory [36]. The steps
included open coding, assigning topical codes to the text of the interviews to form a codebook, organizing themes,
comparing the content of each new interview to the existing codebook categories and modifying the codebook
accordingly and developing categories and defining the relationships among them and their possible implications
[37]. To ensure reliability between the coders in identifying and assigning codes, each coder reviewed a random 10%
sample of the transcripts coded by the other research team member. The crosschecking coder could either endorse or
contest the existing coding. Following a consensus meeting of the coders, the contested codes were deleted,
replaced, modified, or added to the codebook. Statistical analyses for the questionnaire data included descriptive
analysis of the population, including means, standard deviations for continuous data and counts and percentages for
categorical data. T-tests were used to compare the DRS and DCS scores of both patients and family members by the
type of device and patient cognitive status at the time of implant. 3. Results 13 Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292] Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292] 13 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 Twenty-five patients received a pacemaker or ICD during the observation period; 10 patients or family members
declined to participate. Of the15 patient-family member dyads who participated, all family members participated in
an interview and completed questionnaires. Seven patients from the dyads were alive and cognitively able to
participate in an interview and completed questionnaires. More patients with CI at the time of device implant were
deceased or too impaired to participate at study enrollment than patients who were not cognitively impaired. The mean age (SD) of the patients at the time of device implant was 80.9 (SD 5.1). Sixty percent were male, and
100% were white. At the time of device implant, eight patients had a diagnosis of AD, two had MCI, and five had
no CI. Nine patients received a pacemaker and six received an ICD. Twelve patients received a device for an AHA/ACA
Class I indication defined as evidence and/or general agreement that the device would be beneficial, useful, and
effective for the patient [12, 38, 39]. 2.3 Analysis Six had sinus node dysfunction, four received a device for the primary
prevention of sudden cardiac death (SCD), three for atrioventricular block, and two for secondary prevention
of SCD. Family members who participated in this study were mostly female (80%); eight were the spouse of the patient
(53%), and seven (47%) were an adult child. Table 1 shows the descriptive data of the patient-family dyads. Family members who participated in this study were mostly female (80%); eight were the spouse of the patient
(53%), and seven (47%) were an adult child. Table 1 shows the descriptive data of the patient-family dyads. Patient
n=15
Caregiver
n=15
Age*, years mean (SD)
80.9 (5.1)
Sex, n (%)
Male
9 (60)
3 (20)
Female
6(40)
12 (80)
Race, n (%)
White
15 (100)
15 (100)
Relationship to the patient, n(%)
Spouse
8 (53.3)
Adult child or child-in-law
7 (46.7)
Cognitive status of patient,* n (%)
AD
8 (53.3)
Mild cognitive impairment
2 (13.3)
Not impaired
5 (33.3)
MMSE Score,* mean (SD)
24.7 (5.1)
FAQ score,* mean (SD)
6.4 (7.9)
Device Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292] 14 DOI: 10.26502/fccm.92920032 Pacemaker, n (%)
9 (60)
Right ventricular only
2
Right atrial and right ventricular
7
ICD, n(%)
6 (40)
Right ventricular only
2
Right atrial and right ventricular
3
Right atrial, right ventricular and left
ventricular
1
Indications for the device
Sinus node dysfunction
6 (40)
Primary prevention of Sudden Cardiac Death
4 (26.7)
Atrioventricular block
3 (20)
Secondary prevention of Sudden Cardiac Death
2 (13.3)
Indications for device therapy expressed in the standard ACC/AHA
Classification format±
I
12 (80)
-
II
0
-
IIa
0
-
IIb
3 (20)
-
III
0
-
Role in the decision to get a cardiac device‡, n
(%)
Active or Shared
5 (71.4)
11 (73.3)
Passive
2 (28.6)
4 (26.7)
*Values at the time of device implant
†Class I: Conditions for which there is evidence and/or general agreement that a given
procedure or treatment is beneficial, useful, and effective. Class II: Conditions for which there is conflicting evidence and/or a divergence of
opinion about the usefulness/efficacy of a procedure or treatment. Class IIa: Weight of evidence/opinion is in favor of usefulness/efficacy. Class IIb: Usefulness/efficacy is less well established by evidence/opinion. 2.3 Analysis Class III: Conditions for which there is evidence and/or general agreement that a
procedure/treatment is not useful/effective and in some cases may be harmful. T bl 1 Ch
t i ti
f
ti
t
d
i 3.2 Qualitative Findings As shown in Table 2, three main themes emerged from the interviews: (1) the limited influence of the patient’s
cognitive status in medical decision-making about cardiac co-morbidities and cardiac devices, (2) circumstances
around the decision to get the device, and (3) discussions about the risks, benefits, and long-term implications of
getting a cardiac device. Representative quotations for each of these themes from the patients and caregivers are
shown in Table 2. Theme
Quotation
number
Respondent information
Representative Quotation
Limited influence of
the patient’s
cognitive status in
medical decision-
making
1
Spouse of a patient with
AD who received ICD
“Oh yeah, [telling the doctor she has AD] that’s
one of the first things I mention to all the doctors. In fact, they had a sitter several days with her
[while in the hospital]. That’s one of the first
things I mention to medical people. No one
mentioned her AD when we discussed the
pacemaker.”
2
Family member of a patient
who had MCI at the time of
their implant that
progressed to AD at the
time of the interview who
received ICD
“Once the [device] is in there, if you have other
kinds of medical problems, it could keep you
going kind of artificially rather than letting your
life end in a natural kind of way. Just think about
that. Is that what you want?”
3
Patient without CI who
received pacemaker
“Unless I am so debilitated, so sick, I don’t want
to continue, but I can’t envision that at the
moment.”
Circumstances
around the decision
to get the device
4
Daughter of a patient with
AD who received a
pacemaker
“I believed it was urgent. It was a priority
according to the doctors. It wasn’t something that
you could debate.”
5
Spouse of a patient with
MCI who received an ICD
for primary prevention of
sudden cardiac death
“Well, the doctor strongly advised it because he
was [in] atrial fib, and so he advised us then to put
it in. He just presented to us what difference it
would make in him and, you know, that he’d have
to feel better afterwards because it [the atrial fib]
was weakening his heart. He really highly
recommended that we do it.”
6
Family member of a patient
without CI who received a
pacemaker
“My role was to see what he [the patient] decides
and to discuss with it him. Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 Table 1: Characteristics of patients and caregivers 15 Cardiol Cardiovascmed 2018; 2 (1): 010-026 3.2 Qualitative Findings Discuss the options,
and let him make the decision.” Representative Quotation “Oh yeah, [telling the doctor she has AD] that’s
one of the first things I mention to all the doctors. In fact, they had a sitter several days with her
[while in the hospital]. That’s one of the first
things I mention to medical people. No one
mentioned her AD when we discussed the
pacemaker.” “Well, the doctor strongly advised it because he
was [in] atrial fib, and so he advised us then to put
it in. He just presented to us what difference it
would make in him and, you know, that he’d have
to feel better afterwards because it [the atrial fib]
was weakening his heart. He really highly
recommended that we do it.” “My role was to see what he [the patient] decides
and to discuss with it him. Discuss the options,
and let him make the decision.” 16 16 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 Ca diol Ca diovascmed 0 8;
( ): 0 0 0 6
O : 0. 650 /fccm.9 9 003
7
Patient without CI at the
time of ICD implant but
who had progressed to MCI
at the time of the interview
“I didn’t want it then, and I don’t want it now. I
think the whole thing was ridiculous.”
8
Spouse of a patient without
CI who received an ICD
“On that [ICD] decision that was made – I don’t
worry about it anymore. I think I made the right
one. Even if it wasn’t the right one, it seems to be
working out.”
9
Patient without CI and an
ICD
“I’m not sure I had a choice. My thing is, now that
I look back, I felt that, if this is necessary, let’s do
it.”
10
Patient without CI who
received a pacemaker
“They [the doctors] just suggested. It was almost
like that is the way they thought it should be done. I mean, that I should have the pacemaker, and I
went along with it, because I thought that is
probably what I needed.”
11
Adult child of a patient
with AD who received
pacemaker
“Obviously if it would prolong her life and
preserve her QOL by doing it, then I would go for
it. Cardiol Cardiovascmed 2018; 2 (1): 010-026 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 3.3 CI was Not a Factor in Decision Making Few patients with CI or their family members reported having a discussion about the patient’s cognitive status with
the physician. In all cases but one, it was unclear from the interviews and medical record reviews if the physician
was even aware of the patient’s MCI or AD or if they believed that it was an important factor to be discussed. Only
one out of ten patients with CI had a diagnosis or notation in the medical record. Because the patients and family
members largely did not bring up the patient’s CI with the physician, there was no discussion about the impact of the
device on prolonging life, improving QOL, or risks post-implantation in the setting of also having MCI or AD. Even
in the one case where it was made explicit to the medical team about the patient’s AD, it was not discussed in the
context of the cardiac device (Quote 1). None of the patients with cognitive impairment or the family members of patients with cognitive impairment,
reported discussing their cognitive status with the physician implanting the device or its role in the decision to get a
device. As previously mentioned, the medical records largely lacked information on the patients’ cognitive status; it
was noted in only one case. Although family members acknowledged the patient’s MCI or AD during the
interviews, opinions were mixed as to whether the CI should influence the decision-making process for getting a
cardiac device. Most had a difficult time articulating the impact of AD on life expectancy and its role in medical
decision-making about cardiac co-morbidities, and many were unaware of the trajectory and prognosis of AD. Patients and family members largely stated that the decision to get the device was based on the patient’s functional
status at the time of the implant; however, a majority of them could imagine a time when the patient’s CI would be
too severe to get a device and acknowledged that they would focus on QOL only (Quote 2). Regarding the future health status and the need for re-implantation or a new battery or device, all seven patients
interviewed stated that they would make the same decision. However, half of them stated that they might reconsider
based on their functional status at the time (Quote 3). 3.4.1 Urgent Need Both patients and family members perceived the decision to get the device as urgent. All patients received a device
during an inpatient stay; no devices were placed electively, although many had a history of abnormal heart rhythm
or cardiac disease (Quote 4). 3.2 Qualitative Findings But if all the surgery would do is maybe
prolong her life, but if she no longer had any
QOL, then I would be reluctant to do it.”
Risks, benefits and
long-term
implications of
getting a cardiac
device
12
Adult child of a patient
with MCI that received an
ICD
“When something like this happens, I think you
don’t know what to ask. You’re just not informed
enough as a layperson. We have no experience
with this kind of thing, so you don’t even know
what kind of questions you should ask, and if you
should do something differently… Little things
like how is that going to affect him, or how much
does it cost, or can it wait... I don’t think we knew
enough about what to ask.”
13
Family member of a patient
with AD who received ICD
“I would want to know how does it work; what are
some of the side effects; what are the long-range
aspects; can it keep someone alive; if they
happened to go into a vegetative state, is it
something that would cause someone to live
longer than what they would normally want to live
in a bad situation?”
Table 2: Themes and Quotations “When something like this happens, I think you
don’t know what to ask. You’re just not informed
enough as a layperson. We have no experience
with this kind of thing, so you don’t even know
what kind of questions you should ask, and if you
should do something differently… Little things
like how is that going to affect him, or how much
does it cost, or can it wait... I don’t think we knew
enough about what to ask.” 17 3.4.6 Risks, benefits and long-term implications of getting a cardiac device Few family members and patients reported having a conversation about the benefits, risks, and long-term
implications of getting the device. Many reported that they were unsure what types of questions to ask because they
did not have any experience with cardiac devices (Quote 12). Many family members reported wishing they would have discussed more about the risks and alternatives to getting a
cardiac device (Quote 13). Family members also expressed interest in learning more information regarding the
benefits of the device and if it would increase the patient’s life expectancy and what specific symptoms should
improve after the device is implanted. 3.4.4 Not a Decision An overwhelming majority of patients and family members did not acknowledge that getting the device was a
decision and were strongly influenced by the presentation of information by the physician, especially regarding how
getting it would impact symptom relief (Quote 9). In some cases, patients and family members reported that the
physician described the implant of a cardiac device as “necessary” and would help the patient “feel better”. In other
cases, patients and family members did not perceive getting the cardiac device as a choice, rather a decision that was
made by their doctor and simply presented to them (Quote 10). 3.4.5 QOL Discussions with the patient’s physician tended to focus on the “necessity” of the device rather than the benefits and
risks. Topics focused more on the benefits of getting the device, including enhanced QOL and symptom relief. Some
family members thought or were told that that receipt of a device might have a positive impact on the patients’
cognitive function. Family members largely stated that the biggest factor was to improve the QOL for the patient
(Quote 11). 3.4.2 The Physician’s Recommendation Many family members and patients believed that if the physician says the patient needs it, they must need it and
there are no other options. Family members reported that the physician strongly advised that the patient get the
device (Quote 5). 6). Regarding the role of the family member in decision-making, five stated that they went along with what the
physician recommended without discussion, and ten reported that they were involved in the decision-making (Quote 18 ogy and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-929 Cardiol Cardiovascmed 2018; 2 (1): 010-026 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 DOI: 10.26502/fccm.92920032 DOI: 10.26502/fccm.92920032 3.4.3 Decision Regret When asked about whether the decision to get a cardiac device was a “good” or “bad” decision, or if they had regret,
some patients reflected that it was not a “good decision” (Quote 7). Some family members of patients with CI also
reported reflecting on whether getting the device was a “good decision” (Quote 8). 4. Conclusion The present study was undertaken to describe and compare the decision-making experience of patients with and
without CI and their family members regarding cardiac devices. While a variety of circumstances precipitated the
decision to implant a cardiac device, most felt that there was no decision to make and were greatly influenced by
their physician’s recommendations. All patients in this study met the indications for the device; yet, few of the
indications were described as clinically urgent in the medical record, despite what some dyads reported. The state of uncertainty about future events related to a decision is a key element of all decision-making, and
decision makers often experience regret when decision outcomes, particularly poor outcomes, are compared with
alternative outcomes had another option been chosen [38]. Even the most informed patients may have regret and
psychological consequences if the outcomes of their treatment are not as expected or if treatment-related side-effects
compromise their QOL [40]. However, this belief has not been assessed in adults who receive a cardiac device while
already having CI or among their family members. Cognitive function and progression of AD affect prognosis and
QOL of the patient and, therefore, should be incorporated into medical decision making. Circumstances in which CI
co-occurs with other medical conditions will be increasingly common in our age of multimorbidity. The results of
the present study suggest that CI is rarely incorporated despite the fact that functional status of the patient at the time
of implant was a strong influence on decision-making. Indeed, most participants exhibited decision myopia [22] and
had a difficult time articulating the relationship between having MCI or AD and its role in medical decision making
about cardiac co-morbidities. Thus, further studies are required to analyze particular decision needs among patients
with CI and their family caregivers. Additionally, patients with no CI were more likely to report that they made the decision to get the device either
alone or with their doctor. In many cases, the patient’s description of the process was congruent with that of the
family member. Family members of patients with CI at the time of device implant were more likely to report their
role in the decision as either active or shared, both with and without the patient’s involvement. Many patients and
family members described the physician’s role as active. 3.4.7 Decision Quality Measures Most participants described having an active role in the decision and levels of decisional regret and decisional
conflict were low to moderate. While statistical tests should be interpreted cautiously in this small sample, there
were some statistically significant differences between patients and families by patient cognitive status. Overall,
patients had an average DRS score of 16.4 (SD 24.4, range 0-65) while the average DRS score for family members
was 14.9 (SD 19.1, range 0-50). The average DRS score for patients with CI was 45 (SD 28.3), and for patients
without CI, the average DRS score was 5 (SD 11.2) (p=0.03). There was no difference in DRS scores for patients
who received a pacemaker vs. an ICD (p=0.12) or their family members (p=0.28). Patients had an average DCS
score of 38.5 (SD 36.1, range, 3.1-100) while family members reported slightly lower DCS, with a mean DCS score 19 Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292]
19 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 of 38.2 (SD 24.9, range 1.6-100). Family members of patients without CI reported higher levels of decisional
conflict at the time of the decision to implant a device compared to family members of patients with CI (DCS scores
57.8 vs. 30.3, p=0.06) although differences did not meet statistical significance in this small sample. Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292]
20 4. Conclusion Although patients and family members did not
overwhelmingly report feelings of regret about getting a device in the questionnaires, expressions from the sample
were more conflicted in the interviews. Some patients and family members had regret or questioned whether the
decision was “the right one.” However, few patients endorsed a sense of regret, which may be related to the fact that
few patients and family members reported any adverse events as a result of getting the device, which has been
correlated with regret [41, 42]. If faced with the decision to replace the battery, most patients and family members
felt they would make the same decision again; however, many said that they might reconsider based on their
functional status at that time. 20 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 Regarding medical decision-making, there are two main sources of conflict. The first is the inherent difficulty that
arises from making decisions that have both positive and negative effects. The second source is from aspects of the
decision that are modifiable (e.g., lack of knowledge about options and outcomes, unrealistic expectations, unclear
values, unclear perceptions of those presenting information, social pressures, lack of support or mismatch between
the preferred role in decision-making and the actual role, lack of skills and self-confidence to make the decision, and
lack of other resources) [38]. In the present study, patients and family members reported limited discussion
regarding their decision to get a cardiac device. There was little evidence that the physicians who were implanting
the cardiac devices had knowledge of or engaged in discussions about the patient’s cognitive status; an indication of
dementia was identified in only one medical record. This is consistent with previous research showing providers’
lack of awareness of patient’s cognitive status and a reluctance to have discussions about both the risks and benefits
of device implantation and deactivation [41, 42]. Many noted that their physicians concentrated on the “necessity”
of the device, and few family members felt that there was a real choice, deferring to the physician’s
recommendations. This is consistent with previous studies about other cardiac devices, including left ventricular
assist devices (LVADs) [43]. Some family members thought or were told that that receipt of a device might have an
impact on the patients’ cognitive function; however, evidence is mixed to support this claim [45, 46]. 4. Conclusion The family
members’ lack of informed thinking about the implications of the device on longer-term quantity and QOL in their
loved one with CI, as well as a lack of competing death risks is similar to that reported in other studies of cardiac
devices [22, 44, 47]. However, many family members reflected that they would have liked to have more discussion
regarding the risks and alternatives to getting a cardiac device. Further studies will evaluate the experiences of dyads
that included patients with CI who were eligible for cardiac devices, but chose not to receive them. Patients with AD and MCI endorsed more regret on the DRS, although the sample size limits our confidence in
statistical comparisons. DRS scores for family members in the present study are comparable to what has been
published in the literature for family caregivers who are involved in a decision for a family member with AD and
who do not receive any support in the decision [48]. Whereas no differences in decisional conflict were observed in
the patients irrespective of cognitive status, differences were noted in the family members. Among the family
members of patients without CI who reported higher decisional conflict, they also reported being less certain about
the risks and benefits at the time of implant compared to those family members of patients with CI. These findings
are likely driven by the differences among the role of the caregiver in their role in medical decision-making (i.e.,
active vs. shared/passive). Although these results are counter to what we found regarding decisional regret and to
what we hypothesized, they are similar to what has been reported in the literature for surrogate decision makers who
do not receive any support for decision-making [49]. In the present study, patients received a pacemaker or ICD for a variety of reasons, including sinus node
dysfunction, primary or secondary prevention of sudden cardiac death, and atrioventricular block. It is, therefore,
possible that the decisions surrounding receipt of these devices may differ by device or indication. For example,
primary prevention ICD is used to prolong life without improving QOL whereas a pacemaker for sinus node
dysfunction could not only prolong life, but also improve QOL if the patient was experiencing syncope, fatigue, or
palpitations. Moreover, the decision to receive a pacemaker for symptomatic bradycardia may be relatively Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. 4. Conclusion [ISSN 2572-9292]
21 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 straightforward, particularly in a patient with MCI, as compared with the decision to implant a primary prevention
ICD without cardiac resynchronization therapy (CRT) in an asymptomatic patient, which poses considerable trade-
offs. Thus, further studies with larger cohorts are necessary to explore the decision-making experiences of patient
and caregiver dyads by cardiac device type. In addition to the small sample size, the present study is limited by the single site data and lack of racial or ethnic
diversity in the sample, which have been shown by others to impact receipt of device [50]. In addition, for those
patients who died following the procedure, the cause of death was unknown. Furthermore, the time since receiving
the cardiac implant was considerable for some dyads (up to 11 years); thus, there may have been recall bias as well
as survival bias, particularly for those who received a dual chamber pacemaker or ICD. Finally, there was no
medical record data regarding the incidences of cardioversion or adverse events. In conclusion, although the decision to receive a cardiac device in a person with AD requires unique considerations
about the possible trade-offs of quality versus quantity of life, the patients’ cognitive status was largely not
discussed with their family or physician. Most patients and family members described the procedure as “necessary”
to improve the patient’s QOL, but some patients with CI had expressed regret or questioned whether the decision
was “right.” Both patients and family members expressed a wish to have more information regarding the risks,
benefits, and alternatives, and a majority of family members could envision a time when the patient’s cognitive and
functional impairment would be too severe to get a device and acknowledge that they would focus on QOL. Multimorbidity is the new normal in our aging society, and thus medical decision-making will increasingly involve
trade-offs between various lifespan and QOL-increasing interventions, which have implications for how we prefer to
live and die. A better understanding of medical decision-making in these circumstances is critical to assist patients in
making decisions that are right for them. Acknowledgements We would like to thank MaryAnn Oakley, MA of the University of Pittsburgh ADRC for assistance with
recruitment and Jeff Lee, MD with his assistance reviewing the medical records of patient participants. Sources of Funding This study was supported by the National Institutes of Health, National Institute on Aging grant P50 AG005133. References References 1. Association. As. 2017 Alzheimer's disease facts and figures. Alzheimers Dement 13 (2017): 325-373. 1. Association. As. 2017 Alzheimer's disease facts and figures. Alzheimers Dement 13 (2017): 325-373. 2. De Bruijn RF, Akoudad S, Cremers LG, Hofman A, Niessen WJ, et al. Determinants, MRI correlates, and
prognosis of mild cognitive impairment: the Rotterdam Study. J Alzheimers Dis 42 (2014): S239-S249. 2. De Bruijn RF, Akoudad S, Cremers LG, Hofman A, Niessen WJ, et al. Determinants, MRI correlates, and
prognosis of mild cognitive impairment: the Rotterdam Study. J Alzheimers Dis 42 (2014): S239-S249. 3. Roberts R and Knopman DS. Classification and epidemiology of MCI. Clin Geriatr Med 29 (2013): 753-
772. 3. Roberts R and Knopman DS. Classification and epidemiology of MCI. Clin Geriatr Med 29 (2013): 753-
772. 4. Brodaty H, Seeher K and Gibson L. Dementia time to death: a systematic literature review on survival time
and years of life lost in people with dementia. Int Psychogeriatr 24 (2012): 1034-1045. 5. Connors MH, Ames D, Boundy K, Clarnette R, Kurrle S, Mander A, Ward J, Woodward M and Brodaty H. Predictors of Mortality in Dementia: The PRIME Study. J Alzheimers Dis 52 (2016): 967-974. 6. Sachs GA, Carter R, Holtz, Smith F, Stump TE, Tu W, Callahann. Cognitive Impairment: An independent
predictor of excess mortality: A Cohort Study. Ann Inten Med 155 (2011): 300-308. 7. Tom SE, Hubbard RA, Crane PK, Haneuse SJ, Bowen J, McCormick WC, McCurry S and Larson EB. Characterization of dementia and Alzheimer's disease in an older population: updated incidence and life
expectancy with and without dementia. Am J Public Health 105 (2015): 408-413. 8. Xie J, Brayne C, Matthews FE. Survival times in people with dementia: analysis from population based
cohort study with 14 year follow-up. BMJ 336 (2008): 258-262. 9. Hirschman KB, Xie SX, Feudtner C and Karlawish JH. How does an Alzheimer's disease patient's role in
medical decision making change over time? J Geriatr Psychiatry Neurol 17 (2004): 55-60. 10. Karlawish JH, Casarett D, Propert KJ, James BD and Clark CM. Relationship between Alzheimer's disease
severity and patient participation in decisions about their medical care. J Geriatr Psychiatry Neurol 15
(2002): 68-72. 11. Fowler NR, Hansen AS, Barnato AE, Garad L. Association between anticipatory grief and problem solving
among family caregivers of persons with cognitive impairment. J Aging Health 25 (2013): 493-509. 12. Conflicts of Interest Conflicts of Interest The authors Fowler, Shaaban, Torke, Lane, and Barnato have no conflict of interest regarding the publication of this
article. The authors Fowler, Shaaban, Torke, Lane, and Barnato have no conflict of interest regarding the publication of this
article. Saba has received research funding from the National Heart, Lung, and Blood Institute and significant research grant
support from Boston Scientific. Saba has received research funding from the National Heart, Lung, and Blood Institute and significant research grant
support from Boston Scientific. Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292]
22 Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2 Cardiol Cardiovascmed 2018; 2 (1): 010-026 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 References Fowler NR, Johnson KG, Li J, Moore CG, Saba S, Lopez OL and Barnato AE. Use of cardiac implantable
electronic devices in older adults with cognitive impairment. JAMA Intern Med 174 (2014): 1514-1516. 13. DeCarli C, Massaro J, Harvey D, Hald J, Tullberg M, et al. Measures of brain morphology and infarction in
the framingham heart study: establishing what is normal. Neurobiol Aging 26 (2005): 491-510. 14. Launer LJ, Ross GW, Petrovitch H, Masaki K, Foley D, et al. Midlife blood pressure and dementia: the
Honolulu-Asia aging study. Neurobiol Aging 21 (2000): 49-55. 15. Vermeer SE, Prins ND, den Heijer T, Hofman A, Koudstaal PJ and Breteler MM. Silent brain infarcts and
the risk of dementia and cognitive decline. N Engl J Med 348 (2003): 1215-1222. 16. Dublin S, Anderson ML, Haneuse SJ, Heckbert SR, Crane PK, et al. Atrial fibrillation and risk of dementia:
a prospective cohort study. J Am Geriatr Soc 59 (2011): 1369-1375. 17. Maslow K. Dementia and serious coexisting medical conditions: a double whammy. Nurs Clin North Am
39 (2004): 561-579. 18. Jahangir A, Shen WK, Neubauer SA, Ballard DJ, Hammill SC, et al. Relation between mode of pacing and
long-term survival in the very elderly. J Am Coll Cardiol 33 (1999): 1208-1216. 23 Cardiol Cardiovascmed 2018; 2 (1): 010-026 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 DOI: 10.26502/fccm.92920032 19. Kramer DB, Reynolds MR, Normand SL, Parzynski CS, Spertus JA, Mor V and Mitchell SL. Hospice Use
Following Implantable Cardioverter-Defibrillator Implantation in Older Patients: Results From the National
Cardiovascular Data Registry. Circulation 133 (2016): 2030-2037. 20. Pyatt JR, Somauroo JD, Jackson M, Grayson AD, Osula S, et al. Long-term survival after permanent
pacemaker implantation: analysis of predictors for increased mortality. Europace 4 (2002): 113-119. 21. Rickard J, Cheng A, Spragg D, Green A, Leff B, et al. Survival in octogenarians undergoing cardiac
resynchronization therapy compared to the general population. Pacing Clin Electrophysiol 37 (2014): 740-
744. 22. Kramer DB, Buxton AE and Zimetbaum PJ. Time for a change--a new approach to ICD replacement. N
Engl J Med 366 (2012): 291-293. 23. Patrick DL, Starks HE, Cain KC, Uhlmann RF and Pearlman RA. Measuring preferences for health states
worse than death. Med Decis Making 14 (1994): 9-18. 24. Butler K. What broke my father’s heart? New York Times Magazine (2010) 18. 25. Ditto PH, Druley JA, Moore KA, Danks JH and Smucker WD. References A report of the American College of Cardiology/American Heart Association
Task Force on Practice Guidelines (ACC/AHA/NASPE Committee to Update the 1998 Pacemaker
Guidelines). J Cardiovasc Electrophysiol 13 (2002): 1183-1199. 39. Mitty E and Post LF. Healthcare Decision Making. In: E. C. M. Boltz, T. Fulmer, & D. Zwicker ed. Evidence-based geriatric nursing protocols for best practice 4th ed. New York: Springer Publishing
Company 2012: 562-578. 40. Davison BJ and Goldenberg SL. Decisional regret and quality of life after participating in medical
decision-making for early-stage prostate cancer. BJU Int 91 (2003): 14-17. 41. Goldstein NE, Lampert R, Bradley E, Lynn J and Krumholz HM. Management of implantable cardioverter
defibrillators in end-of-life care. Ann Intern Med 141 (2004): 835-838. 42. Russo JE. Original research: deactivation of ICDs at the end of life: a systematic review of clinical
practices and provider and patient attitudes. Am J Nurs 111 (2011): 26-35. 43. Blumenthal-Barby JS, Kostick KM, Delgado ED, Volk RJ, Kaplan HM, et al. Assessment of patients' and
caregivers' informational and decisional needs for left ventricular assist device placement: Implications for
informed consent and shared decision-making. J Heart Lung Transplant 34 (2015): 1182-1189. 44. Green AR, Boyd CM, Rickard J, Gomon R and Leff B. Attitudes of older adults with serious competing
health risks toward their implantable cardioverter-defibrillators: a pilot study. BMC Geriatr 15 (2015): 173. 45. Gribbin GM, Gallagher P, Young AH, McComb JM, McCue P, et al. The effect of pacemaker mode on
cognitive function. Heart 91 (2005): 1209-1210. 46. Lamas GA, Orav EJ, Stambler BS, Ellenbogen KA, Sgarbossa EB, et al. Quality of life and clinical
outcomes in elderly patients treated with ventricular pacing as compared with dual-chamber pacing. Pacemaker Selection in the Elderly Investigators. N Engl J Med 338 (1998): 1097-1104. 47. Goldstein NE, Mehta D, Siddiqui S, Teitelbaum E, Zeidman J, et al. "That's like an act of suicide" patients'
attitudes toward deactivation of implantable defibrillators. J Gen Intern Med 23 (2008): 7-12. 48. Epstein AE, DiMarco JP, Ellenbogen KA, Estes NA, Freedman RA, et al. American College of
Cardiology/American Heart Association Task Force on Practice G, American Association for Thoracic S
and Society of Thoracic S. References Fates worse than death: the role of valued
life activities in health-state evaluations. Health Psychol 15 (1996): 332-343. 26. Patrick DL, Pearlman RA, Starks HE, Cain KC, Cole WG and Uhlmann RF. Validation of preferences for
life-sustaining treatment: implications for advance care planning. Ann Intern Med 127 (1997): 509-517. 27. Patton MQ. Qualitative research and evaluation methods. Third ed. Thousand Oaks, CA: Sage Publications;
2002. 28. Folstein MF, Folstein SE and McHugh PR. "Mini-mental state". A practical method for grading the
cognitive state of patients for the clinician. J Psychiatr Res 12 (1975): 189-198. 29. Pfeffer RI, Kurosaki TT, Harrah CH Jr, Chance JM and Filos S. Measurement of functional activities in
older adults in the community. J Gerontol 37 (1982): 323-329. 30. Degner LF, Sloan JA and Venkatesh P. The Control Preferences Scale. Can J Nurs Res 29 (1997): 21-43. 31. O'Connor AM. Validation of a decisional conflict scale. Med Decis Making 15 (1995): 25-30. 32. Brehaut JC, O'Connor AM, Wood TJ, Hack TF, Siminoff L, Gordon E and Feldman-Stewart D. Validation
of a decision regret scale. Med Decis Making 23 (2003): 281-292. 33. Connolly T and Zeelenber M. Regret in Decision Making. Current Directions in Psychological Science 11
(2002): 212-216. 34. Becerra-Perez MM, Menear M, Turcotte S, Labrecque M and Legare F. More primary care patients regret
health decisions if they experienced decisional conflict in the consultation: a secondary analysis of a
multicenter descriptive study. BMC Fam Pract 17 (2016): 156. 35. Fusch PI and Ness LR. The Qualitative Report 20 (2015): 1408-1416. 36. Strauss A and Corbin J. Grounded Theory Methodology. In: N. D. Y. Lincoln, ed. Handbook of Qualitative
Research Thousand Oaks: Sage Publications 1994: 217-285. 37. Holton JA. The coding process and its challenges. In: K. C. A. Bryant, ed. The Sage handbook of grounded
theory Thousand Oaks, CA: Sage Publications; 2007. 38. Gregoratos G, Abrams J, Epstein AE, Freedman RA, Hayes DL, et al. American College of
Cardiology/American Heart Association Task Force on Practice Guidelines American College of Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292]
24 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 Cardiology/American Heart Association/North American Society for P and Electrophysiology C. ACC/AHA/NASPE 2002 guideline update for implantation of cardiac pacemakers and antiarrhythmia
devices: summary article. Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292]
25 Citation: Nicole R. Fowler, C. Elizabeth Shaaban, Alexia M. Torke, Kathleen A. Lane, Samir Saba, and Am ber
E. Barnat. “I’m Not Sure We Had A Choice”: Decision Quality and The Use of Cardiac Im plantable Electronic
Devices In Older Adults With Cognitive Impairment. Cardiology and Cardiovascular Medicine 2 (2018): 010-026. References ACC/AHA/HRS 2008 Guidelines for Device-Based Therapy of Cardiac
Rhythm Abnormalities: a report of the American College of Cardiology/American Heart Association Task
Force on Practice Guidelines (Writing Committee to Revise the ACC/AHA/NASPE 2002 Guideline
Update for Implantation of Cardiac Pacemakers and Antiarrhythmia Devices) developed in collaboration
with the American Association for Thoracic Surgery and Society of Thoracic Surgeons. Heart Rhythm 5
(2008): e1-e62. 49. Hickman RL, Jr., Daly BJ and Lee E. Decisional conflict and regret: consequences of surrogate decision
making for the chronically critically ill. Appl Nurs Res 25 (2012): 271-275. 50. Matlock DD, Peterson PN, Heidenreich PA, Lucas FL, Malenka DJ, et al. Regional variation in the use of
implantable cardioverter-defibrillators for primary prevention: results from the National Cardiovascular
Data Registry. Circ Cardiovasc Qual Outcomes 4 (2011): 114-121. 25 DOI: 10.26502/fccm.92920032 DOI: 10.26502/fccm.92920032 Cardiol Cardiovascmed 2018; 2 (1): 010-026 Citation: Nicole R. Fowler, C. Elizabeth Shaaban, Alexia M. Torke, Kathleen A. Lane, Samir Saba, and Am ber
E. Barnat. “I’m Not Sure We Had A Choice”: Decision Quality and The Use of Cardiac Im plantable Electronic
Devices In Older Adults With Cognitive Impairment. Cardiology and Cardiovascular Medicine 2 (2018): 010-026. Citation: Nicole R. Fowler, C. Elizabeth Shaaban, Alexia M. Torke, Kathleen A. Lane, Samir Saba, and Am ber
E. Barnat. “I’m Not Sure We Had A Choice”: Decision Quality and The Use of Cardiac Im plantable Electronic
Devices In Older Adults With Cognitive Impairment. Cardiology and Cardiovascular Medicine 2 (2018): 010-026. This article is an open access article distributed under the terms and conditions of the This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license 4.0 Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292]
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Linkage, Evaluation and Analysis of National Electronic Healthcare Data: Application to Providing Enhanced Blood-Stream Infection Surveillance in Paediatric Intensive Care
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Linkage, Evaluation and Analysis of National Electronic
Healthcare Data: Application to Providing Enhanced
Blood-Stream Infection Surveillance in Paediatric
Intensive Care Katie Harron1*, Harvey Goldstein1,2, Angie Wade1, Berit Muller-Pebody3, Roger Parslow4, Ruth Gilbert1 Katie Harron1*, Harvey Goldstein1,2, Angie Wade1, Berit Muller-Pebody3, Roger P 1 Institute of Child Health, University College London, London, United Kingdom, 2 Graduate School of Education, University of Bristol, Bristol, United Kingdom,
3 Healthcare Associated Infection and Antimicrobial Resistance Department, Public Health England, London, United Kingdom, 4 Division of Epidemiology,
University of Leeds, Leeds, United Kingdom Abstract Background: Linkage of risk-factor data for blood-stream infection (BSI) in paediatric intensive care (PICU) with
bacteraemia surveillance data to monitor risk-adjusted infection rates in PICU is complicated by a lack of unique
identifiers and under-ascertainment in the national surveillance system. We linked, evaluated and performed
preliminary analyses on these data to provide a practical guide on the steps required to handle linkage of such
complex data sources. Methods: Data on PICU admissions in England and Wales for 2003-2010 were extracted from the Paediatric
Intensive Care Audit Network. Records of all positive isolates from blood cultures taken for children <16 years and
captured by the national voluntary laboratory surveillance system for 2003-2010 were extracted from the Public
Health England database, LabBase2. “Gold-standard” datasets with unique identifiers were obtained directly from
three laboratories, containing microbiology reports that were eligible for submission to LabBase2 (defined as
“clinically significant” by laboratory microbiologists). Reports in the gold-standard datasets were compared to those in
LabBase2 to estimate ascertainment in LabBase2. Linkage evaluated by comparing results from two classification
methods (highest-weight classification of match weights and prior-informed imputation using match probabilities) with
linked records in the gold-standard data. BSI rate was estimated as the proportion of admissions associated with at
least one BSI. Results: Reporting gaps were identified in 548/2596 lab-months of LabBase2. Ascertainment of clinically significant
BSI in the remaining months was approximately 80-95%. Prior-informed imputation provided the least biased
estimate of BSI rate (5.8% of admissions). Adjusting for ascertainment, the estimated BSI rate was 6.1-7.3%. Conclusion: Linkage of PICU admission data with national BSI surveillance provides the opportunity for enhanced
surveillance but analyses based on these data need to take account of biases due to ascertainment and linkage
error. This study provides a generalisable guide for linkage, evaluation and analysis of complex electronic healthcare
data. Citation: Harron K, Goldstein H, Wade A, Muller-Pebody B, Parslow R, et al. (2013) Linkage, Evaluation and Analysis of National Electronic Healthcare
Data: Application to Providing Enhanced Blood-Stream Infection Surveillance in Paediatric Intensive Care. PLoS ONE 8(12): e85278. doi:10.1371/
journal.pone.0085278 Editor: Caroline L Trotter, University of Cambridge, United Kingdom Copyright: © 2013 Harron 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. Citation: Harron K, Goldstein H, Wade A, Muller-Pebody B, Parslow R, et al. (2013) Linkage, Evaluation and Analysis of National Electronic Healthcare
Data: Application to Providing Enhanced Blood-Stream Infection Surveillance in Paediatric Intensive Care. PLoS ONE 8(12): e85278. doi:10.1371/
journal.pone.0085278 Copyright: © 2013 Harron 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. Received July 9, 2013; Accepted November 26, 2013; Published December 20, 2013
Copyright: © 2013 Harron et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by funding from the National Institute for Health Research Health Technology Assessment (NIHR HTA) programme
[project number 08/13/47]. The views and opinions expressed therein are those of the authors and do not necessarily reflect those of the HTA programme,
NIHR, NHS or the Department of Health. PICANet is funded by the National Clinical Audit and Patient Outcomes Programme via Healthcare Quality
Improvement Partnership (HQIP), Health Commission Wales Specialised Services, NHS Lothian / National Service Division NHS Scotland, the Royal
Belfast Hospital for Sick Children, Our Lady’s Children’s Hospital, Crumlin, Children’s University Hospital, Temple Street and The Harley Street Clinic,
London. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: Katie.harron.10@ucl.ac.uk Introduction processing, calculation of match weights or probabilities and
errors due to mis-classification in the linkage process can have
substantial effects on outcome measures[13–16]. We aim to
describe the steps involved in preparing and linking routine
data for enhanced BSI surveillance in PICU, which are
generalisable to other administrative data of this type. Blood-stream infection (BSI) is an important cause of
mortality, morbidity and substantial extra cost for paediatric
patients, and paediatric intensive care units (PICU) have one of
the highest rates of BSI of all specialties[1–4]. The national
laboratory surveillance system coordinated by Public Health
England (PHE, formerly the Health Protection Agency) collects
data on microorganisms submitted by hospital laboratories in
England and Wales[5]. Patient-level data on all children
admitted to paediatric intensive care units (PICU) in England
and Wales have been collected by the Paediatric Intensive
Care Audit Network (PICANet) since 2003[6]. To date, no
evaluation of the potential of linking these administrative data
sources for national monitoring of risk-adjusted BSI trends in
PICU has been performed[7,8]. Paediatric Intensive Care Infection Surveillance Paediatric Intensive Care Infection Surveillance Ethics Statement For PICANet, collection of personally identifiable data has
been approved by the National Information Governance Board
(Formerly the Patient Information Advisory Group) http://
www.nigb.nhs.uk/s251/registerapp
and
ethical
approval
granted by the Trent Medical Research Ethics Committee, ref. 05/MRE04/17. PICANet also has specific permission from the
National Research Ethics Service for linkage with the PHE
laboratory data on bloodstream infections using personal
identifiers and to share PICANet data with PHE. An exemption
under Section 251 of the NHS Act 2006 (previously Section 60
of the Health and Social Care Act 2001) allows PHE to receive
patient-identifiable data from other organisations without
patient consent in order to monitor infectious disease. Specific
permission for the PICANet-PHE linkage has been granted by
NIGB. Consent for the use of the data identifying individual
PICUs in this study was obtained by the relevant PICANet unit
leads. Data in PICANet and LabBase2 cannot be publicly
deposited as it is personally-identifiable. Access to an
anonymised form of the linked data may be requested from
http://www.picanet.org.uk/. There are two main obstacles to linkage for enhanced BSI
surveillance. Firstly, as a voluntary system, PHE’s surveillance
database (LabBase2) does not capture complete BSI data from
all laboratories[5]. Hospital laboratories are requested to report
any clinically significant bacterial infections and clinically
significant isolates from sterile sites such as blood, although
there are no specific guidelines for judgement of clinical
significance
and
non-clinically
significant
isolates
or
contaminants may also be present in the data. Data are not
always captured consistently, with staffing issues and IT
compatibility problems causing incomplete and variable
reporting over time. Ascertainment of MRSA and MSSA within
LabBase2 in 2008 was estimated at around 70% (based on
mandatory reports for methicillin-resistant and methicillin-
susceptible Staphylococcus aureus) although ascertainment for
all-cause bacteraemia in children is unknown[9]. Secondly, linkage between data sources is complicated due
to a lack of well-completed unique identifiers in LabBase2. For
data such as these, the method of choice for linkage is often to
calculate probabilistic match weights (or match probabilities)
that measure the similarity between records from different
sources, taking into account possible identifier errors or
missing values[10,11]. These weights or probabilities are then
used to classify record pairs as links or non-links. Linkage process Figure 1 displays the steps required for obtaining data for
enhanced BSI surveillance through linkage between PICANet
and LabBase2. Abstract Funding: This work was supported by funding from the National Institute for Health Research Health Technology Assessment (NIHR HTA) programme
[project number 08/13/47]. The views and opinions expressed therein are those of the authors and do not necessarily reflect those of the HTA programme,
NIHR, NHS or the Department of Health. PICANet is funded by the National Clinical Audit and Patient Outcomes Programme via Healthcare Quality
Improvement Partnership (HQIP), Health Commission Wales Specialised Services, NHS Lothian / National Service Division NHS Scotland, the Royal
Belfast Hospital for Sick Children, Our Lady’s Children’s Hospital, Crumlin, Children’s University Hospital, Temple Street and The Harley Street Clinic,
London. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist 1 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e85278 1 Data extraction and de-duplication Data on all PICU admissions for children <16 years for
2003-2010 were extracted from PICANet (n=109,654 records). Each PICANet record corresponded to an individual PICU
admission within one of twenty-two PICUs admitting more than
200 children per year in England and Wales. Records of all
positive isolates from blood culture captured by the national
surveillance system were extracted for children <16 years
between 2003-2010 extracted from LabBase2 (n=80,009). Classification is typically based on highest-weighted (HW)
pairs, where the candidate record with the highest weight is
accepted as a link, given it exceeds a pre-specified threshold. However, errors can be introduced if the highest-weighted
record is not the correct match (false-matches), or if no
candidate record exceeds the threshold (missed-matches). An
alternative classification method is prior-informed imputation,
which aims to avoid bias associated with these linkage errors. Prior-informed imputation works by accepting values for
variables of interest within a multiple imputation framework,
rather than by linking a complete record[12]. Values are
selected according to Information from a prior distribution
(based on match probabilities in candidate linking records)
combined with a likelihood derived from unequivocally-linked
records[12]. PICANet admission records could link to none, one or more
LabBase2 specimen records. If an admission record linked to
multiple specimens of the same organism (within 14 days), only
the first specimen was retained. LabBase2 specimen records
could link to more than one admission if a specimen fell within
the timeframe for two admissions at once (if the admissions
were consecutive). In this case, the specimen was linked to the
earlier of the two admissions, so that each LabBase2 record
linked to at most one admission record. There is a lack of practical guidance on the complex process
required to link and analyse national administrative data such
as PICANet and LabBase2. Methods used for data pre- December 2013 | Volume 8 | Issue 12 | e85278 PLOS ONE | www.plosone.org 2 Paediatric Intensive Care Infection Surveillance Figure 1. Steps involved in linkage of PICANet and LabBase2 for enhanced BSI surveillance in PICU. doi: 10.1371/journal.pone.0085278.g001 Figure 1. Steps involved in linkage of PICANet and LabBase2 for enhanced BSI surveillance in PICU. doi: 10 1371/journal pone 0085278 g001 Figure 1. Steps involved in linkage of PICANet and LabBase2 for enhanced BSI surveillance in PICU. Data extraction and de-duplication doi: 10.1371/journal.pone.0085278.g001 The representativeness of the gold-standard data was
assessed by comparing characteristics of laboratories and
PICUs providing gold-standard data with those that did not. Gold-standard data “Gold-standard” microbiology datasets were obtained directly
from three hospital laboratories: Birmingham Children’s
Hospital (BCH), Oxford University Hospital (OUH) and Royal
London Hospital (RLH). These laboratories were chosen as
they were able to provide microbiology data that were eligible
for reporting to LabBase2 (i.e. defined as clinically significant
my laboratory microbiologists) and included unique identifiers. The gold-standard datasets were used for two purposes: December 2013 | Volume 8 | Issue 12 | e85278 Ascertainment evaluation Incomplete reporting in LabBase2 was identified through
manual inspection of plots of the total number of reports (all
ages) of bacteraemia for individual laboratories by specimen
month. Data were inspected for all ages, as numbers for
children only were low, and reporting gaps were expected to
relate to the laboratory as a whole rather than to an individual
ward. Within individual laboratories, months during which no
reports were submitted were defined as a reporting gap. In
addition, months during which an unrealistically small number
of reports were present were defined as having incomplete
reporting. Unrealistic numbers of reports were identified
through careful manual inspection of reports over time: due to
the fluctuation of reports from month to month and variation in
size of laboratories, a consistent definition of incomplete
reporting could not be applied across all laboratories and so a
conservative judgement on incomplete reporting was made. 1. To estimate ascertainment in LabBase2 by comparing
records that were eligible for reporting to LabBase2 with
records that actually appeared in LabBase2 (BCH and
RLH) 2. To evaluate linkage error by comparing records linked
using incomplete identifiers within LabBase2 with records
linked using well-completed unique identifiers within the
gold-standard data (BCH and OUH). Any uncertain links in
the gold-standard data were verified with additional
information from the hospital. PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e85278 3 Paediatric Intensive Care Infection Surveillance Lab-months classified as incomplete reporting were excluded
from analysis. beginning with S, and the frequency-based weight represented
this difference. For the remaining months, ascertainment of clinically
significant BSI for children <16 years was estimated as the
proportion of eligible records in the gold-standard data from
BCH (March 2003-December 2010) and RLH (July 2006-
December 2009) captured within LabBase2. BCH and RLH
contained information on whether a specimen was deemed to
be clinically significant (and therefore eligible for submission to
LabBase2); this information was not available for OUH. The distribution of match weights for links and non-links was
plotted to assess the performance of the match weights at
separating links and non-links. Record pairs were then ordered
by match weight and manually inspected to identify obvious
non-links that had high weights, and probable links with low
weights. Subsequent training datasets were obtained by
retaining probable links identified through this review. Blocking The total number of pairwise comparisons between PICANet
and LabBase2 would produce of 80,009 x 109,654 =
8,773,306,886 comparison pairs. Comparison pairs were
therefore restricted to those where the specimen date fell within
3 days of a PICU admission, as errors in date variables were
assumed to be trivial (unpublished analysis showed <1% of
specimen dates were inconsistent with data upload dates). To
further reduce the number of comparison pairs, several
blocking variables were chosen, so that records were only
compared if they agreed on at least one of Soundex, initial,
postcode prefix, NHS number, hospital number or day of birth. This blocking scheme assumed that records not agreeing on
any of these blocking variables did not belong to the same
individual. For this project, manual review for uncertain links was not
possible. This was because no additional external data was
available, and uncertain links often contained only Soundex
and date of birth, which did not provide enough information to
positively determine link status by eye. Records were therefore
classified as links or non-links based on a single cut-off weight,
based on capturing probable links identified in the training
datasets. A sensitivity analysis was performed by repeating this
process with two different cut-offs. The first cut-off (relaxed
threshold) aimed to capture as many of the probable links as
possible. The second cut-off (conservative threshold) aimed to
exclude as many non-links as possible. Any records with a
match weight above the threshold was classified as a link, and
all others were classified as non-links. Match probability calculation Completeness of common identifiers for linking varied
between datasets and by time (identifiers were more complete
in recent years). For LabBase2, completeness of identifiers
varied by unit (Figure 2). For PICANet, date of birth and
hospital number were 100% complete and the majority of other
identifiers were >98% complete, with the exception of NHS
number (85% complete). For both datasets, cleaning and data
preparation were undertaken: NHS or hospital numbers such
as “Unknown” or “9999999999” were set to null; generic names
(e.g. “Baby”, “Twin 1”, “Infant Of”) were set to null; multiple
variables were created for multiple surname and first names;
postcodes beginning “ZZ” (indicating no UK postcode) were set
to null. Match probabilities P(M|agreement pattern) were calculated
to estimate the probability of a match given agreement on a
joint set of identifiers. This avoided the assumption of
independence between identifiers. Probabilities were derived
as the number of links divided by the total number of pairs for
each agreement pattern (based on probable links identified in
the training datasets). For example, if 378 comparison pairs
agreed on date of birth and Soundex but disagreed on sex, and
312 of these were probable links, the match probability for the
agreement pattern [ 1,1,0 ] was 312/378=0.825. Classification of links 1: Highest-weight (HW) classification. Traditionally,
candidate linking records are ordered by match weight, and
only the comparison pair with the highest probabilistic weight is
classified as a link. All remaining candidate records are
discarded (highest-weight classification). Comparison pairs are
classified into non-links, links and uncertain links, based on the
value of the match weight. Uncertain links are then classified
through manually inspecting the identifiers on each record, to
determine whether or not they belong to the same individual. Manual inspection makes use of the fact that the human eye
can recognise matches that a computer would discard (e.g. Liz
and Elizabeth) and can involve the use of additional identifiers
if available. Ascertainment evaluation This
process was iterated a number of times, until match weights
from consecutive training datasets were stable (Figure 3). Match weight calculation Match weight calculations were based on the Fellegi-Sunter
method[11,17]. ‘Training’ datasets of record pairs assumed to
be matches were used to estimate m- and u-probabilities
P(agreement|match)
and
P(agreement|non-match)
for
individual identifiers. The first training dataset took records
agreeing on NHS number or hospital number as assumed
matches. The same training dataset was used to create a list of
non-matches by cross-joining all record pairs and removing
those agreeing on NHS number or hospital number. Frequency-based weights were calculated for surname, first
name, sex and Soundex so that m- and u-probabilities were
allowed to vary according to how rare or common a value
was[18]. These were calculated by estimating m- and u-
probabilities within groups for each identifier. For example,
surnames beginning with Z were less common than surnames 2: Prior-informed imputation). Prior-informed imputation
was performed as proposed by Goldstein et al, using Stat-JR
software developed by the University of Bristol[12,19]. Linkage
between PICANet and LabBase2 was ‘incomplete’, as PICANet
records that did not have a BSI genuinely had no matching
record in LabBase2. This is a special case for prior-informed December 2013 | Volume 8 | Issue 12 | e85278 PLOS ONE | www.plosone.org 4 Paediatric Intensive Care Infection Surveillance Figure 2. Completeness of identifiers in LabBase2 by PICU. doi: 10 1371/journal pone 0085278 g002 Figure 2. Completeness of identifiers in LabBase2 by PICU. doi: 10.1371/journal.pone.0085278.g002 derived using the unequivocally linked records and a set of
PICANet predictor variables identified in previous analyses[7]. imputation, as all candidate records had the same outcome
(link = BSI). For the remaining (equivocal) PICANet records, a prior
distribution for BSI was created based on the maximum
probability of a BSI in the candidate records (Figure 4). If the
maximum candidate probability for BSI=1 if p, the probability
that BSI=0 is 1-p. Prior-informed imputation uses match probabilities, rather
than match weights, to avoid assuming independence between
identifiers. If a PICANet record had a match probability>0.9, it
was classed as “unequivocal” and the variable BSI was set
equal to 1 (Figure 4). If a PICANet record had no candidate
linking records (or the maximum candidate probability was
<0.1), it was also classed as “unequivocal”, but BSI was set
equal to 0. These cut-offs were based on previous simulation
work for prior-informed imputation. Match weight calculation A likelihood for BSI was A modified (posterior) probability distribution was created by
multiplying the above prior distribution by the likelihood and
scaling to 1. For each equivocal PICANet record, BSI was set
equal to 0 or 1 according to the highest modified probability for
that record. If no probability exceeded 0.1, BSI was treated as PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e85278 5 Paediatric Intensive Care Infection Surveillance Figure 3. Match weight calculation process. doi: 10.1371/journal.pone.0085278.g003 Figure 3. Match weight calculation process. doi: 10.1371/journal.pone.0085278.g003 doi: 10.1371/journal.pone.0085278.g003 subjective – for example, manual review or choice of
thresholds. In our study, we present results based on two
different probabilistic thresholds. missing and standard multiple imputation was used to impute a
value based on the likelihood only. Five imputed datasets were
produced and analysed separately, with results combined
using Rubin’s rules[20]. Thirdly, comparisons of linked and unlinked data can be
made, in order to identify potential sources of bias. This
process can help to identify groups of subjects who may be
missed from the linkage due to poor data quality. In our study,
differences in the characteristics of linked and unlinked records
were related to BSI: linked records corresponded to admissions
with BSI and therefore represented children who had risk-
factors for BSI. BSI rate: Evaluation of bias due to linkage error BSI rate was calculated as the percentage of admissions
linked to >=1 BSI (within three days either side of admission). This crude rate reflects the proportion of admissions associated
with an infection acquired either leading up to or during an
admission. Finally, statistical techniques can be used to handle
uncertainty in linkage within the analysis itself. In our study, we
used prior-informed imputation to account for linkage error
within analysis. There are a number of ways in which bias due to linkage
error can be evaluated[21]. Firstly, comparisons with gold-
standard data can be performed, based on the true match
status of any record pair (known in the gold-standard data). Such data could be in the form of an external dataset including
well-completed, unique identifiers, or a sample of records that
have been subjected to extensive manual review. In our study,
bias was estimated by comparing the BSI rate in gold-standard
data obtained directly from two laboratories, with that estimated
in the linked data, for each classification method. Match weight and probability calculation incomplete reporting for the entire study period (PICUs 9 and
10, Figure 5). Removing admissions during periods of
incomplete reporting reduced the total number of admissions
available for analysis from 109,654 to 78,525. Comparing the
number of BSI records within BCH and RLH gold-standard
datasets (defined as clinically significant by microbiologists)
with BSI records captured by LabBase2 gave an estimated
LabBase2 ascertainment (for clinically significant BSI in
children <16 years) of 81.5% (95% confidence interval (CI)
79.9-83.1%; 1872/2298) and 79.5% (95% CI 75.1-83.9%;
260/327) for BCH and RLH respectively. As non-clinically
significant specimens were also available in the gold-standard
data, we identified that 181/2054 (8.8%) of LabBase2 reports
for BCH were not clinically significant. incomplete reporting for the entire study period (PICUs 9 and
10, Figure 5). Removing admissions during periods of
incomplete reporting reduced the total number of admissions
available for analysis from 109,654 to 78,525. Comparing the
number of BSI records within BCH and RLH gold-standard
datasets (defined as clinically significant by microbiologists)
with BSI records captured by LabBase2 gave an estimated
LabBase2 ascertainment (for clinically significant BSI in
children <16 years) of 81.5% (95% confidence interval (CI)
79.9-83.1%; 1872/2298) and 79.5% (95% CI 75.1-83.9%;
260/327) for BCH and RLH respectively. As non-clinically
significant specimens were also available in the gold-standard
data, we identified that 181/2054 (8.8%) of LabBase2 reports
for BCH were not clinically significant. An initial three iterations of probabilistic weight calculation
were performed. However, match weights did not stabilise. This
was down to the failure of a number of assumptions
underpinning probabilistic weight calculation. Firstly, all records
should be equally likely to link. A small subset of LabBase2
records that did have well-completed data caused this
assumption to fail, and weight calculations were dominated by
agreement on NHS number, hospital number or name, making
it difficult to distinguish between records containing only
Soundex, date of birth and sex (Table 1). Secondly, agreement
between identifiers should be independent. However, records
with missing NHS number were also more likely to have
missing surname, meaning that records failing to agree on
NHS number were also likely to fail to agree on surname,
meaning the independence assumption failed. The same was
the case for Soundex and surname, and for separate elements
of date of birth (records that disagreed on date of birth were
disproportionately penalized). Finally, some weights produced
for missing values were counter-intuitive (e.g. Ascertainment Figure 5 shows the variation and fluctuation in the total
number of reports (all ages) submitted to LabBase2 per month
for laboratories serving individual PICUs between 2003-2010. Manual inspection of data identified a total of 548/2596 lab-
months with incomplete reporting. Two laboratories had Secondly, sensitivity analyses based on varying linkage
criteria can be used to provide a range of plausible results. This
is particularly useful when aspects of the linkage process are PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e85278 6 Paediatric Intensive Care Infection Surveillance Figure 4. Prior-informed imputation for ‘incomplete’ linkage between PICANet and LabBase2. Predictor variables: Length of
stay, age, admission type, admission source, renal status, quarter-year at admission. doi: 10.1371/journal.pone.0085278.g004 Figure 4. Prior-informed imputation for ‘incomplete’ linkage between PICANet and LabBase2. Predictor variables: Length of
stay, age, admission type, admission source, renal status, quarter-year at admission. doi: 10.1371/journal.pone.0085278.g004 Match weight and probability calculation a higher weight
for missing than agreement on day of birth). This was due to
small m- and u-probabilities (<0.01) for missing values
combined with the log-likelihood scaling convention. December 2013 | Volume 8 | Issue 12 | e85278 Blocking After removing record pairs that were not within the correct
timeframe (more than 3 days outside admission), there were
3,081,719 record pairs to be compared. Blocking on NHS
number, hospital number, day of birth, Soundex, initial and
postcode prefix, provided a total of 1,803,808 comparison
pairs. To deal with these problems, record pairs that included
completed NHS number, hospital number, first name, surname,
postcode and date of birth were extracted and a set of PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e85278 7 Paediatric Intensive Care Infection Surveillance Figure 5. Total number of reports (all ages) submitted to LabBase2 for laboratories serving PICUs between 2003-2010. doi: 10.1371/journal.pone.0085278.g005 Figure 5. Total number of reports (all ages) submitted to LabBase2 for laboratories serving PICUs between 2003-2010. doi: 10.1371/journal.pone.0085278.g005 Figure 5. Total number of reports (all ages) submitted to LabBase2 for laboratories serving PICUs between 2003-2010. doi: 10 1371/journal pone 0085278 g005 er of reports (all ages) submitted to LabBase2 for laboratories serving PICUs between 2003-2010. mber of reports (all ages) submitted to LabBase2 for laboratories serving PICUs between 2003-2010
85278.g005 deterministic rules applied (Table 2). Pairs agreeing on a
deterministic rule were manually reviewed to remove false-
matches based on disagreement between other identifiers,
which was possible due to the high-level of identifier
completeness. This deterministic process identified 6001 links. Table 1. Initial weight estimates based on first training
dataset (records agreeing on NHS number or hospital
number). Match weight
Agreement
between
identifiers
Disagreement
between identifiers
One or both
identifiers missing
NHS number
12.58
-7.94
-0.17
Hospital number
12.80
-2.23
0.10
Surname
6.20
-3.88
0.26
Soundex
5.26
-3.78
-0.46
First name
5.19
-3.22
0.25
Day of birth
1.28
-6.08
1.66
Month of birth
1.18
-6.81
1.66
Year of birth
0.91
-6.68
1.66
Sex
0.92
-5.63
-0.39
doi: 10.1371/journal.pone.0085278.t001 Final match weights were then calculated for the remaining
records pairs, based on agreement or disagreement on date of
birth (combined variable), Soundex, sex and location (Table 3). The relaxed threshold was set at 5, which was chosen to
include the majority of probable and possible links whilst not
including many non-links (Figure 6). The conservative
threshold was set at 10, which was chosen to exclude the
majority of non-links, whilst not excluding many probable or
possible links. Calculation of joint match probabilities confirmed that the
independence assumption did not hold (Table 4). BSI rate: Evaluation of bias due to linkage error A total of 6001 (deterministic), 6787 (highest-weighted
conservative) and 8490 (highest-weighted relaxed) links were
identified. Retaining only the first episode per admission and
removing admissions within reporting gaps resulted in 3626,
4651 and 4043 admissions with BSI using each method. Prior-
informed imputation identified 4549 admissions with BSI. Compared with gold-standard data, prior-informed imputation
provided the least biased estimate of BSI rate (Table 5). After adjusting for -0.5% linkage bias and 80-95% estimated
ascertainment, the crude BSI rate increased from 5.79% (initial
PII estimate) to 6.13-7.28% (adjusted estimate). Representativeness of gold-standard data BCH and OUH had lower than average BSI rate (2.69% and
4.64% for respective PICUs compared with 5.79% overall). The PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e85278 8 Paediatric Intensive Care Infection Surveillance Figure 6. Four iterations for match weight calculation. Lines=thresholds. doi: 10.1371/journal.pone.0085278.g006 Figure 6. Four iterations for match weight calculation. Lines=thresholds. doi: 10.1371/journal.pone.0085278.g006 Table 2. Identification of links through deterministic linkage. Deterministic rule
Number of
agreeing
pairs
Number of
links
Number of
non-links
NHS number or hospital number
4595
4586
9
First name, surname and date of birth
832
832
0
Postcode prefix and postcode suffix
538
416
122
Postcode prefix or postcode suffix
and date of birth
94
52
42
At least 2 elements of date of birth
and either first name or surname
1559
115
1444
Total reviewed
7618
6001
1617
doi: 10.1371/journal.pone.0085278.t002
Table 3. Final probabilistic match weights. Match weight
Agreement
Disagreement
Soundex
5.18
-4.05
Date of birth
4.66
-6.89
Sex
0.91
-4.70
Location (PICU / lab)
5.53
-1.06
doi: 10.1371/journal.pone.0085278.t003 Table 2. Identification of links through deterministic linkage. Table 2. Identification of links through deterministic linkage. Table 2. Identification of links through deterministic linkage. Table 4. Match probabilities under independence and
dependence assumptions. Table 2. Identification of links through deterministic linkage. Representativeness of gold-standard data Deterministic rule
Number of
agreeing
pairs
Number of
links
Number of
non-links
NHS number or hospital number
4595
4586
9
First name, surname and date of birth
832
832
0
Postcode prefix and postcode suffix
538
416
122
Postcode prefix or postcode suffix
and date of birth
94
52
42
At least 2 elements of date of birth
and either first name or surname
1559
115
1444
Total reviewed
7618
6001
1617
doi: 10.1371/journal.pone.0085278.t002 Agreement pattern
Assuming
dependence
Assuming
independence
Sex Soundex Dob Location P(NM|g)
P(M|g)
P(NM|g)
P(M|g)
0
0
0
0
1.000
0.000
1.000
0.000
0
0
0
1
1.000
0.000
1.000
0.000
0
0
1
0
1.000
0.000
1.000
0.000
0
0
1
1
1.000
0.000
0.998
0.002
0
1
0
0
1.000
0.000
1.000
0.000
0
1
0
1
0.993
0.007
0.999
0.001
0
1
1
0
0.883
0.117
0.957
0.043
0
1
1
1
0.600
0.400
0.530
0.470
1
0
0
0
1.000
0.000
0.999
0.001
1
0
0
1
1.000
0.000
1.000
0.000
1
0
1
0
0.998
0.002
0.999
0.001
1
0
1
1
0.949
0.051
0.883
0.117
1
1
0
0
1.000
0.000
1.000
0.000
1
1
0
1
0.985
0.015
0.980
0.020
1
1
1
0
0.175
0.825
<0
>1
1
1
1
1
0.009
0.991
<0
>1
doi: 10.1371/journal.pone.0085278.t004 Table 3. Final probabilistic match weights. Match weight
Agreement
Disagreement
Soundex
5.18
-4.05
Date of birth
4.66
-6.89
Sex
0.91
-4.70
Location (PICU / lab)
5.53
-1.06
doi: 10.1371/journal.pone.0085278.t003 Table 3. Final probabilistic match weights. distribution of identifiers within LabBase2 was also different for
these two PICUs. LabBase2 records from BCH were more distribution of identifiers within LabBase2 was also different for
these two PICUs. LabBase2 records from BCH were more December 2013 | Volume 8 | Issue 12 | e85278 December 2013 | Volume 8 | Issue 12 | e85278 December 2013 | Volume 8 | Issue 12 | e85278 9 9 Paediatric Intensive Care Infection Surveillance distributed non-randomly. Although non-random error can
introduce bias into results, prior-informed imputation has been
shown to be particularly effective at handling this type of
error[12]. Table 5. Estimated bias based on gold-standard data (BCH
and OUH). Table 5. Estimated bias based on gold-standard data (BCH
and OUH). Representativeness of gold-standard data Classification
Total links
BSI rate % Bias
Gold-standard
426
3.87%
Deterministic
125
1.14%
-70.5%
Highest-weighted: Relaxed threshold
492
4.47%
15.5
Highest weighted: Conservative threshold
418
3.80%
-1.9
Prior-informed imputation
424
3.85%
-0.5
doi: 10.1371/journal.pone.0085278.t005 Classification
Total links
BSI rate % Bias
Gold-standard
426
3.87%
Deterministic
125
1.14%
-70.5%
Highest-weighted: Relaxed threshold
492
4.47%
15.5
Highest weighted: Conservative threshold
418
3.80%
-1.9
Prior-informed imputation
424
3.85%
-0.5
doi: 10.1371/journal.pone.0085278.t005 This study demonstrates that linkage between routine
datasets is complex and requires a number of steps. Firstly,
calculation of appropriate match weights requires an iterative
process and time-consuming manual review. Calculation of
joint match probabilities avoids relying on independence
assumptions that often fail, but the most effective ways of
estimating such probabilities are still being debated. Current
work is investigating this issue. likely to have completed Soundex, date of birth and sex, but
less likely to have completed NHS number, name or postcode. LabBase2 records from OUH were more likely to have
completed NHS number and name, but less likely to have
completed postcode and date of birth. likely to have completed Soundex, date of birth and sex, but
less likely to have completed NHS number, name or postcode. LabBase2 records from OUH were more likely to have
completed NHS number and name, but less likely to have
completed postcode and date of birth. Secondly, evaluation of data and linkage quality is required
so that potential sources of bias can be identified. Bias due to
linkage error can have dramatic effects on analyses based on
linked data[13,14,16]. In particular, comparisons of units based
on linked data may be biased by differing data quality, and
such potential bias needs to be evaluated when using linked
data for this purpose. Acknowledgements The authors would like to thank Tom Fleming (PICANet), Jim
Gray, Ollie Bagshaw, Adrian Bigland (Birmingham Children’s
Hospital), John Finney, Sarah Walker, Tim Peto, David Wyllie,
Tariq Ali and Tim Whittington (Oxford University Hospital),
Michael Millar and David Ball (Royal London Hospital) for
helpful advice, contribution of data and facilitation of data
retrieval for this paper. We would like to thank all the staff in participating hospitals
who have collected data for PICANet. We are grateful to the
UK Paediatric Intensive Care Society for continued support and
to the members of the PICANet Steering Group and Clinical
Advisory Group who are listed on our website http://
www.picanet.org.uk/participants.html. The authors would also
like to thank the microbiology laboratories across England and
Wales for their efforts in reporting to LabBase2. Gold-standard data provide a convenient means for
evaluating both ascertainment and bias due to linkage error. This requires the assumption that the gold-standard datasets
are representative of the larger dataset of interest. Firstly, we
assumed that data capture from RLH and BCH reflected
ascertainment
in
LabBase2
more
generally,
and
that
ascertainment was relatively constant over time. However,
ascertainment based on these laboratories is likely to be
overestimated, as RLH and BCH consistently submit data. Therefore final estimates of BSI rate may be under-estimated. Secondly, we assumed that bias due to linkage error in BCH
and OUH was representative. Completeness of identifiers
differed between laboratories, and linkage error was therefore Discussion Gold-standard data is one way to measure linkage bias, and
this was practically possible in our study since linkage and
analysis could be performed within the same department (PHE
have permission to access patient-identifiable data for the
purposes of surveillance). However, this is a special case, as
clinical and identifiable data are often separated to protect
patient privacy[22]. Appropriate evaluation of linkage success
should be presented in reporting analysis based on linked data,
to
allow
meaningful
interpretation
of
results. Careful
coordination between linkage and analysis is required so that
research based on linked data can be reliable and transparent,
whilst data confidentiality is preserved. Our study demonstrates that linkage of PICU admission data
with national BSI surveillance is possible but that results based
on these data vary according to the degree of under-
ascertainment and bias introduced through linkage of
incomplete or imperfect identifiers. We show that reporting
gaps and under-ascertainment in national surveillance data
lead to under-estimated rates of BSI, but that this can be
measured through the use of gold-standard data. We also
show that errors occurring during linkage can cause under- or
over-estimated rates based on data linked using deterministic
linkage only or highest-weight classification, but that prior-
informed imputation can provide less biased results. Reasons for the under-ascertainment in LabBase2 cited by
laboratories are lack of staffing provision, IT system
compatibility
issues
and
upload
failures. We
took
a
conservative approach to removing data points within periods
of incomplete reporting, but this required a certain amount of
subjectivity due to the fluctuating nature of BSI reports. PHE is
currently developing a new surveillance system that aims to
improve data capture. However, this type of data quality issue
is relevant to many routinely collected datasets used for health
research, and analyses based on these data need to carefully
assess how poor data quality might affect results. References 11. Clark DE (2004) Practical introduction to record linkage for injury
research. Injury Prev 10: 186-191. doi:10.1136/ip.2003.004580. PubMed: 15178677. 1. Nosocomial Infection National Surveillance Service (2007) Surveillance
of Hospital-acquired Bacteraemia in English Hospitals. pp. 1997-2002. Public Health Laboratory Service. 1. Nosocomial Infection National Surveillance Service (2007) Surveillance
of Hospital-acquired Bacteraemia in English Hospitals. pp. 1997-2002. Public Health Laboratory Service. 12. Goldstein H, Harron K, Wade A (2012) The analysis of record-linked
data using multiple imputation with data value priors. Stat Med 31:
3481-3493. doi:10.1002/sim.5508. PubMed: 22807145. 2. Yogaraj JS, Elward AM, Fraser VJ (2002) Rate, risk factors, and
outcomes of nosocomial primary bloodstream infection in pediatric
intensive care unit patients. Pediatrics 110: 481-485. doi:10.1542/peds. 110.3.481. PubMed: 12205248. 13. Bohensky MA, Jolley D, Sundararajan V, Evans S, Pilcher DV et al. (2010) Data Linkage: A powerful research tool with potential problems. BMC Health Serv Res 10: 346-352. doi:10.1186/1472-6963-10-346. PubMed: 21176171. 3. Urrea M, Pons M, Serra M, Latorre C, Palomeque A (2003) Prospective
incidence study of nosocomial infections in a pediatric intensive care
unit. Pediatr
Infect
Dis
J
22:
490-494. doi:10.1097/01.inf. 0000069758.00079.d3. PubMed: 12799503. 14. Schmidlin K, Clough-Gorr KM, Spoerri A, Egger M, Zwahlen M (2013)
Impact of unlinked deaths and coding changes on mortality trends in
the Swiss National Cohort. BMC Med Inform Decis Mak 13: 1-11. doi:
10.1186/1472-6947-13-S2-S1. PubMed: 23289362. 4. Elward AM, Hollenbeak CS, Warren DK, Fraser VJ (2005) Attributable
cost of nosocomial primary bloodstream infection in pediatric intensive
care unit patients. Pediatrics 115: 868-872. doi:10.1542/peds. 2004-0256. PubMed: 15805357. 15. Ford JB, Roberts CL, Taylor LK (2006) Characteristics of unmatched
maternal and baby records in linked birth records and hospital
discharge data. Paediatr Perinat EP 20: 329-337. doi:10.1111/j. 1365-3016.2006.00715.x. PubMed: 16879505. 5. Wilson J, Elgohari S, Livermore DM, Cookson B, Johnson A et al. (2011) Trends among pathogens reported as causing bacteraemia in
England, 2004–2008. Clin Microbiol Infect 17: 451–458. doi:10.1111/j. 1469-0691.2010.03262.x. PubMed: 20491834. 16. Lariscy JT (2011) Differential Record Linkage by Hispanic Ethnicity and
Age in Linked Mortality Studies. J Aging Health 23: 1263-1284. doi:
10.1177/0898264311421369. PubMed: 21934120. 6. Universities of Leeds and Leicester (2012) Paediatric Intensive Care
Audit Network National Report 2009 - 2011 ISBN 9780853163121. Available:http://www.picanetorg.uk/Documents/General/
Annual_Report_2012/
Ninth_PICANet_Annual_Report_2009_2011_Summary_Report.pdf. Accessed 26 April 2013 17. Fellegi IP, Sunter AB (1969) A theory for record linkage. J Am Stat
Assoc 64: 1183-1210. doi:10.1080/01621459.1969.10501049. 18. Author Contributions Conceived and designed the experiments: KH HG AW BMP
RP RG. Performed the experiments: KH. Analyzed the data:
KH. Contributed reagents/materials/analysis tools: HG RP
BMP. Wrote the manuscript: KH HG AW BMP RP RG. December 2013 | Volume 8 | Issue 12 | e85278 PLOS ONE | www.plosone.org 10 Paediatric Intensive Care Infection Surveillance 10. Jaro MA (1995) Probabilistic linkage of large public health data files.
Stat
Med
14:
491-498.
doi:10.1002/sim.4780140510.
PubMed:
7792443. 22. Kelman CW, Bass AJ, Holman CDJ (2002) Research use of linked
health data—a best practice protocol. Aust Nz. J Publ Heal 26:
251-255. Paediatric Intensive Care Infection Surveillance References DuVall SL, Kerber RA, Thomas A (2010) Extending the Fellegi-Sunter
probabilistic record linkage method for approximate field comparators. J
Biomed Inform 43: 24-30. doi:10.1016/j.jbi.2009.08.004. PubMed:
19683070. 7. Harron K, Wade A, Muller-Pebody B, Goldstein H, Parslow R et al. (2013) Risk-adjusted monitoring of blood-stream infection in paediatric
intensive care: a data linkage study. Intensive Care Med 39:
1080-1087. doi:10.1007/s00134-013-2841-z. PubMed: 23404472. 19. Charlton CMJ, Michaelides DT, Cameron B, Szmaragd C, Parker RMA
et al. (2012) Stat-JR software. Center for Multilevel Modelling,
University of Bristol and Electronics and Computer Science. University
of Southampton. 8. García Álvarez L, Aylin P, Tian J, King C, Catchpole M et al. (2011)
Data linkage between existing healthcare databases to support hospital
epidemiology. J
Hosp
Infect
79:
231–235. doi:10.1016/j.jhin. 2011.06.016. PubMed: 21868128. 20. Rubin D (1987) Multiple imputation for nonresponse in surveys: Wiley. 21. Harron K, Wade A, Muller-Pebody B, Goldstein H, Gilbert R. (2012)
Opening the Black Box of Record Linkage. J Epidemiol Commun H
Online First 9. Pearson A, Chronias A, Murray M (2009) Voluntary and mandatory
surveillance for methicillin-resistant Staphylococcus aureus (MRSA)
and methicillin-susceptible S. aureus (MSSA) bacteraemia in England. J Antimicrob Chemother 64: i11-i17. doi:10.1093/jac/dkp260. PubMed:
19675013. 22. Kelman CW, Bass AJ, Holman CDJ (2002) Research use of linked
health data—a best practice protocol. Aust Nz. J Publ Heal 26:
251-255. 10. Jaro MA (1995) Probabilistic linkage of large public health data files. Stat
Med
14:
491-498. doi:10.1002/sim.4780140510. PubMed:
7792443. 10. Jaro MA (1995) Probabilistic linkage of large public health data files. Stat
Med
14:
491-498. doi:10.1002/sim.4780140510. PubMed:
7792443. PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e85278 11
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Native myeloperoxidase is required to make the experimental vasculitis model
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Arthritis research & therapy
| 2,019
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cc-by
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LETTER Open Access Funding This work was supported by a grant from Ono Pharmaceutical (Osaka,
Japan). Flow cytometry (FCM) using human neutrophils dem-
onstrated the presence of ANCA in sera of group 2 but
not group 1 (Fig. 1a). Correspondingly, sera of group 2
but not group 1 induced neutrophil extracellular traps
(NETs) from tumor necrosis factor (TNF)-primed neu-
trophils (Fig. 1b). Immunoblot of neutrophil lysates
demonstrated that antibody reactive with the MPO light
chain (14 kDa) was produced in group 1, whereas anti-
bodies reactive with the MPO heavy chain (59 kDa) and
light chain (14 kDa) were produced in group 2 (Fig. 1c). The collective findings indicated that the anti-MPO light
chain antibody produced in group 1 did not bind to
native
MPO. Renal
tissue
damage
represented
by Native myeloperoxidase is required to
make the experimental vasculitis model Mayu Nonokawa1, Ku Suzuki1, Hideyuki Hayashi1, Yuka Nishibata1, Sakiko Masuda1, Daigo Nakazawa2,
Satoshi Tanaka3, Utano Tomaru4 and Akihiro Ishizu1* Availability of data and materials The data sheets used and/or analyzed during the current study are available
from the corresponding author on reasonable request. Authors’ contributions
AI d
i
d h
d
M AI designed the study. MN, KS, HH, YN, SM, and AI acquired the data. All
authors were involved in the interpretation of data and approval of the
manuscript. UT and AI wrote the manuscript. Dear Editor, hematuria and erythrocyte casts in renal tubules was
evident in group 2 but not group 1 regardless of the
disease boost by LPS (Fig. 1d, e). The degree of
pulmonary hemorrhage that represents capillaritis in
the lungs tended to be severe in group 2 compared
to group 1 (Fig. 1f). Myeloperoxidase-antineutrophil cytoplasmic antibody
(MPO-ANCA) is a pathogenic autoantibody [1]. Wistar-
Kyoto (WKY) rats immunized with human native MPO
produce anti-human MPO antibody cross-reactive with
rat MPO, resulting in the development of MPO-ANCA-
associated vasculitis (MPO-AAV) [2]. MPO is a hetero-
tetramer composed of two light chains (14 kDa) and two
heavy chains (59 kDa) [3]. In this study, we examined if
immunization of WKY rats with the recombinant light
chain of human MPO could induce MPO-AAV. The majority of MPO-AAV patients produced MPO-
ANCA that recognizes an epitope in the heavy chain of
MPO, whereas a few number of patients produced
MPO-ANCA against an epitope in the light chain of
MPO [4, 5]. The collective findings suggested that the
recombinant light chain of human MPO has a low
potential to induce MPO-AAV in rats compared to na-
tive human MPO. WKY rats (4–5 weeks old) were immunized with the
recombinant light chain of human MPO (1600 μg/kg;
Cloud-Clone, Katy, TX, USA; group 1) or human native
MPO (1600 μg/kg; RayBiotech, Peachtree Corners, GA,
USA; group 2) on day 0. These rats were given an intra-
peritoneal injection of pertussis toxin (800 ng; Sigma-
Aldrich, St. Louis, MO, USA) on days 0 and 2. A sub-
group of group 1 was given an intraperitoneal injection
of
lipopolysaccharide
(LPS;
100
μM/week;
Sigma-
Aldrich) through days 7 to 35. Urine samples were col-
lected using a metabolic cage on day 40. All rats were
euthanized on day 42. Consent for publication
Not applicable Consent for publication
Not applicable. Competing interests The authors declare that they have no competing interests. * Correspondence: aishizu@med.hokudai.ac.jp
1Department of Medical Laboratory Science, Faculty of Health Sciences,
Hokkaido University, Kita-12, Nishi-5, Kita-ku, Sapporo 0600812, Japan
Full list of author information is available at the end of the article Nonokawa et al. Arthritis Research & Therapy (2019) 21:296
https://doi.org/10.1186/s13075-019-2084-7 Nonokawa et al. Arthritis Research & Therapy (2019) 21:296
https://doi.org/10.1186/s13075-019-2084-7 Author details
1 1Department of Medical Laboratory Science, Faculty of Health Sciences,
Hokkaido University, Kita-12, Nishi-5, Kita-ku, Sapporo 0600812, Japan. 2Department of Rheumatology, Endocrinology and Nephrology, Faculty of Ethics approval and consent to participate Experiments using rats were performed in accordance with the Guidelines
for the Care and Use of Laboratory Animals in Hokkaido University
(Permission No. 15-0034). Experiments using human materials were permit-
ted by the Ethics Committee of the Faculty of Health Sciences, Hokkaido Uni-
versity (Permission No. 18-34). © 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. Nonokawa et al. Arthritis Research & Therapy (2019) 21:296 Page 2 of 3 Fig. 1 (See legend on next page.) Fig. 1 (See legend on next page.) Page 3 of 3 Nonokawa et al. Arthritis Research & Therapy (2019) 21:296 (See figure on previous page.) (See figure on previous page.)
Fig. 1 Development of MPO-AAV. a ANCA detected by FCM. Human peripheral blood neutrophils were fixed with 4% paraformaldehyde, and
then the plasma membrane of neutrophils was penetrated using permeabilization wash buffer (BioLegend, San Diego, CA, USA). Cells (1 × 106/ml)
were allowed to react with 1:500 diluted rat sera for 30 min at room temperature (RT) followed by reaction with fluorescence-labeled secondary
antibody. Concerning day 42 sera of group 1, the reactivity of 1:100 and 1:20 dilutions was also examined. To show the reactivity of anti-MPO
heavy chain antibody to native MPO, a similar FCM was performed using the anti-MPO heavy chain monoclonal antibody (5 μg/ml; 4A4; Bio-Rad,
Tokyo, Japan) as primary antibody and mouse IgG2b (5 μg/ml; BioLegend) as isotype control. b NET-forming neutrophils detected by FCM. Human peripheral blood neutrophils (1 × 106/ml) were treated with 5 ng/ml TNF-α for 15 min at 37 °C and then exposed to 10% rat sera. After
incubation for 3 h at 37 °C, cells were next made to react with a plasma membrane-impermeable DNA-binding dye, SYTOX Green (Life
Technologies, Carlsbad, CA, USA). After filtering out the debris with a mesh, the percolated cells were subjected for FCM. Histograms highlighted
in green represent NET-forming neutrophils. The percentage of NET-forming neutrophils induced by group 2 sera was significantly higher than
that induced by group 1 sera. c ANCA detected by immunoblotting. Lysates of human neutrophils boiled under reducing condition were
electrophoresed (5 × 105 cells/lane) and then transferred to polyvinylidene difluoride membrane. References 1. Nakazawa D, Masuda S, Tomaru U, Ishizu A. Pathogenesis and therapeutic
interventions for ANCA-associated vasculitis. Nat Rev Rheumatol. 2019;15(2):
91–101. 1. Nakazawa D, Masuda S, Tomaru U, Ishizu A. Pathogenesis and therapeutic
interventions for ANCA-associated vasculitis. Nat Rev Rheumatol. 2019;15(2):
91–101. 2. Little MA, Smyth L, Salama AD, Mukherjee S, Smith J, Haskard D, et al. Experimental autoimmune vasculitis: an animal model of anti-neutrophil
cytoplasmic autoantibody-associated systemic vasculitis. Am J Pathol. 2009;
174(4):1212–20. 3. Fiedler TJ, Davey CA, Fenna RE. X-ray crystal structure and characterization
of halide-binding sites of human myeloperoxidase at 1.8 A resolution. J Biol
Chem. 2000;275(16):11964–71. 3. Fiedler TJ, Davey CA, Fenna RE. X-ray crystal structure and characterization
of halide-binding sites of human myeloperoxidase at 1.8 A resolution. J Biol
Chem. 2000;275(16):11964–71. 4. Suzuki K, Kobayashi S, Yamazaki K, Gondo M, Tomizawa K, Arimura Y, et al. Analysis of risk epitopes of anti-neutrophil antibody MPO-ANCA in vasculitis
in Japanese population. Microbiol Immunol. 2007;51(12):1215–20. 5. Gou SJ, Xu PC, Chen M, Zhao MH. Epitope analysis of anti-myeloperoxidase
antibodies in patients with ANCA-associated vasculitis. PLoS One. 2013;8(4):
e60530. After blocking the non-specific binding of
antibodies, the membrane was incubated in diluted rat sera (day 42; group 1, 1:200 dilution; group 2, 1:1000 dilution) overnight at 4 °C. After
rinsing with phosphate-buffered saline (PBS) with Tween 20 (PBS-T), the membrane was next incubated in the solution of horseradish peroxidase
(HRP)-conjugated secondary antibody for 1 h at RT. After rinsing with PBS-T, the HRP activity on the membrane was detected by
chemiluminescence using ImageQuant LAS 4000 (GE Healthcare, Little Chalfont, UK). Blue arrowhead, MPO heavy chain (59 kDa); red arrowheads,
MPO light chain (14 kDa). d Degree of hematuria assessed at urine sampling immediately by a dipstick (Siemens Healthineers, Erlangen, Germany). e Degree of renal tissue damage. Erythrocyte casts (yellow arrowheads) were counted in the maximum longitudinal section of the kidney. f
Degree of pulmonary hemorrhage. The foci of pulmonary hemorrhage were counted in the maximum longitudinal section of the lung. Mann-
Whitney U test was applied for statistical analyses between two non-parametric groups Medicine and Graduate School of Medicine, Hokkaido University, Sapporo,
Japan. 3Center for Cause of Death Investigation, Faculty of Medicine and
Graduate School of Medicine, Hokkaido University, Sapporo, Japan. 4Department of Pathology, Faculty of Medicine and Graduate School of
Medicine, Hokkaido University, Sapporo, Japan. Medicine and Graduate School of Medicine, Hokkaido University, Sapporo,
Japan. 3Center for Cause of Death Investigation, Faculty of Medicine and
Graduate School of Medicine, Hokkaido University, Sapporo, Japan. 4Department of Pathology, Faculty of Medicine and Graduate School of
Medicine, Hokkaido University, Sapporo, Japan. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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MOCHA: Real-Time Motion Characterization via Context Matching
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cc-by
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ABSTRACT characters performing a range of motions, which can serve as a
valuable resource for future research. characters performing a range of motions, which can serve as a
valuable resource for future research. Transforming neutral, characterless input motions to embody the
distinct style of a notable character in real time is highly compelling
for character animation. This paper introduces MOCHA, a novel
online motion characterization framework that transfers both mo-
tion styles and body proportions from a target character to an input
source motion. MOCHA begins by encoding the input motion into
a motion feature that structures the body part topology and cap-
tures motion dependencies for effective characterization. Central
to our framework is the Neural Context Matcher, which generates
a motion feature for the target character with the most similar
context to the input motion feature. The conditioned autoregres-
sive model of the Neural Context Matcher can produce temporally
coherent character features in each time frame. To generate the
final characterized pose, our Characterizer network incorporates
the characteristic aspects of the target motion feature into the in-
put motion feature while preserving its context. This is achieved
through a transformer model that introduces the adaptive instance
normalization and context mapping-based cross-attention, effec-
tively injecting the character feature into the source feature. We
validate the performance of our framework through comparisons
with prior work and an ablation study. Our framework can easily
accommodate various applications, including characterization with
only sparse input and real-time characterization. Additionally, we
contribute a high-quality motion dataset comprising six different • Computing methodologies →Animation; Motion process-
ing; Neural networks. • Computing methodologies →Animation; Motion process-
ing; Neural networks. ACM Reference Format: ACM Reference Format:
Deok-Kyeong Jang, Yuting Ye, Jungdam Won, and Sung-Hee Lee. 2023. MOCHA: Real-Time Motion Characterization via Context Matching. In
SIGGRAPH Asia 2023 Conference Papers (SA Conference Papers ’23), December
12–15, 2023, Sydney, NSW, Australia. ACM, New York, NY, USA, 11 pages. https://doi.org/10.1145/3610548.3618252 MOCHA: Real-Time Motion Characterization via Context
Matching Sung-Hee Lee∗
sunghee.lee@kaist.ac.kr
KAIST
South Korea Sung-Hee Lee∗
sunghee.lee@kaist.ac.kr
KAIST
South Korea Jungdam Won
jungdam@imo.snu.ac.kr
Seoul National University
South Korea Yuting Ye
yuting.ye@meta.com
Reality Labs, Meta
USA Jungdam Won
jungdam@imo.snu.ac.kr
Seoul National University
South Korea Deok-Kyeong Jang
shadofex@kaist.ac.kr
KAIST
South Korea
MOVIN Inc. South Korea Deok-Kyeong Jang
shadofex@kaist.ac.kr
KAIST
South Korea
MOVIN Inc. South Korea Figure 1: Our characterization framework transforms neutral motions to express distinct style of characters in real-time. ABSTRACT
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hi h Figure 1: Our characterization framework transforms neutral motions to express distinct style rization framework transforms neutral motions to express distinct style of characters in real-time. Figure 1: Our characterization framework transforms neutral motions to express distinct style of characters in real-time. ABSTRACT
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characters performing a range of motions, which can serve as a
valuable resource for future research s to express distinct style of characters in real-tim g
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ABSTRACT
Transforming neutral, characterless input motions to embody the
characters performing a range of motions, which can serve as a
valuable resource for future research. MOCHA: Real-Time Motion Characterization via Context
Matching
Deok-Kyeong Jang
shadofex@kaist.ac.kr
KAIST
South Korea
MOVIN Inc.
South Korea
Yuting Ye
yuting.ye@meta.com
Reality Labs, Meta
USA
Jungdam Won
jungdam@imo.snu.ac.kr
Seoul National University
South Korea
Sung-Hee Lee∗
sunghee.lee@kaist.ac.kr
KAIST
South Korea
Figure 1: Our characterization framework transforms neutral motions to express distinct style of characters in real-time.
ABSTRACT
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f
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t
l
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ti
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b d th
characters performing a range of motions, which can serve as a
valuable resource for future research MOCHA: Real-Time Motion Characterization via Context
Matching
Deok-Kyeong Jang
shadofex@kaist.ac.kr
KAIST
South Korea
MOVIN Inc. South Korea
Yuting Ye
yuting.ye@meta.com
Reality Labs, Meta
USA
Jungdam Won
jungdam@imo.snu.ac.kr
Seoul National University
South Korea
Sung-Hee Lee∗
sunghee.lee@kaist.ac.kr
KAIST
South Korea
Figure 1: Our characterization framework transforms neutral motions to express distinct style of characters in real-time. ABSTRACT
T
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characters performing a range of motions, which can serve as a
valuable resource for future research MOCHA: Real-Time Motion Characterization via Context
Matching KEYWORDS Motion style transfer, Motion synthesis, Character animation, Deep
learning 1
INTRODUCTION Transforming styleless motions to embody a particular character is
invaluable for animating characters in feature films or in VR. As the
popularity of interactive and immersive VR and AR applications
grows, so does the need for real-time user motion characterization. In this paper, we propose a novel technique for transforming a va-
riety of user motions into motions that express a specific character
(e.g., Princess, Clown, etc.) in real-time. ∗Corresponding author
This work is licensed under a Creative Commons Attribution International
4.0 License. SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia
© 2023 Copyright held by the owner/author(s). ACM ISBN 979-8-4007-0315-7/23/12. https://doi.org/10.1145/3610548.3618252 We adopt an example-based approach, presuming the availability
of a stylistic motion database for the target character. However, the
character’s body proportions will not be an exact match to the user’s,
and the character may not even possess a human-like physique. In
this work, we posit that a character’s distinctive movement style is
intricately linked with their unique body shape, so we need to solve
both motion stylization and motion retargeting problems jointly. ∗Corresponding author SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia
© 2023 Copyright held by the owner/author(s). ACM ISBN 979-8-4007-0315-7/23/12. https://doi.org/10.1145/3610548.3618252 Current data-driven motion stylization techniques often perceive
style as a consistent feature within data. One approach is to extract SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia Deok-Kyeong Jang, Yuting Ye, Jungdam Won, and Sung-Hee Lee • We release a high-quality character motion dataset that con-
tains a total of 6 characters performing various actions, with
each action conducted with 5 emotions. • We release a high-quality character motion dataset that con-
tains a total of 6 characters performing various actions, with
each action conducted with 5 emotions. • We release a high-quality character motion dataset that con-
tains a total of 6 characters performing various actions, with
each action conducted with 5 emotions. • We release a high-quality character motion dataset that con-
tains a total of 6 characters performing various actions, with
each action conducted with 5 emotions. style elements from the target examples and apply them to the
source motion [Aberman et al. 2020b; Jang et al. 2022]. Another
approach treats style as a conditioning variable for motion gen-
eration [Park et al. 2021; Tao et al. 2022a]. 2.1
Motion style transfer Research in motion style transfer centers around two fundamental
questions: the definition and representation of style. Convention-
ally, style is regarded as time-invariant variations within the same
motion content, structure, or context, which can vary based on
individual characteristics [Ma et al. 2010] or emotions [Amaya et al. 1996]. A lot of research investigate decomposition algorithms to
factorize and parameterize style from motion content [Brand and
Hertzmann 2000; Mason et al. 2018; Min et al. 2009; Rose et al. 1998;
Shapiro et al. 2006; Unuma et al. 1995; Yumer and Mitra 2016]. Style
can also be represented non-parametrically or as a label. For in-
stance, Gaussian Process has been used to learn a latent space of
pose styles [Grochow et al. 2004] or motion styles [Ikemoto et al. 2009; Wang et al. 2007]. More recently, explicit style labels are used
to condition generative models to stylize the output motion [Smith
et al. 2019; Tao et al. 2022b]. The style labels can also be latent
learned from data [Park et al. 2021]. g
To this end, we propose a novel framework that can characterize
a user’s diverse motions in real-time. The basic idea is that when
user performs a motion, we search for a target character’s motion
with the most similar context from a motion database and then
transfer the style elements of the found motion to the user’s motion. However, this approach has the disadvantages of storing character
motion database, searching a suitable motion in real-time, as well
as limiting target motions to the ones in the database. To improve
upon this, inspired by learned motion matching technique [Holden
et al. 2020], we train a neural network, dubbed Neural Context
Matcher (NCM), to generate target motion features suitable for
characterizing a source motion instead of searching a database. Modeled as a conditional VAE running autoregessively, the NCM
can generate temporally coherent motion features for the target
character suitable for the input motion, which is key to obtaining
high-quality motion characterization for diverse motions. Treating style as a static feature limits its representative power. These approaches can often capture stereotypical pose features, but
fall short at delivering complex and nuanced characteristics. Xia
et al. [2015] addressed this issue by adapting style parameters in
real-time based on local nearest-neighbors. They also contributed a
dataset that supported many follow up research. Style may also be
spatially varying. Motion Puzzle [Jang et al. 1
INTRODUCTION While these methods
perform well on stereotypical locomotion datasets, we challenge
their assumption by introducing a high quality and diverse dataset
of professional performances. Our data clearly illustrates style as
context-dependent. For example, the expression of "happiness" in a
jumping motion manifests differently than in a crawling motion
and their characteristics are not interchangeable. 2.1
Motion style transfer 2022] integrates style
elements based on body parts into a single character. In our work,
we consider style as non-parametric and context-dependent, and
therefore choose a different class of approach more akin to motion
matching [Clavet 2016].l In addition, the motions generated by our framework not only
reflect the motion style aspects but also the target character’s body
proportions, making additional motion retargeting to the target
character unnecessary. Hence we call our technique motion char-
acterization. This is possible because our framework encodes both
motion styles and body proportions into motion feature and ef-
fectively transfers them to the source motion. To the best of our
knowledge, our work is the first that performs motion stylization
and retagetting concurrently. Besides, we propose several crucial
ideas, such as introducing contrastive loss and incorporating adap-
tive instance normalization (AdaIN) [Huang and Belongie 2017]
into transformer decoder, that significantly enhance the motion
stylization quality. Recent research in motion style transfer is heavily influenced by
the vast body of work in image style transfer. In this context, motion
styles are analogous to image textures. The pioneering work of Neu-
ral Style Transfer [Gatys et al. 2015] introduced the use of the Gram
matrix of latent features for style representation, an approach later
adopted in the motion domain [Du et al. 2019; Holden et al. 2016]. Its successor, the Adaptive Instance Norm (AdaIN) layer [Huang
and Belongie 2017], has later become the predominant technique
with widespread adoption (e.g., [Aristidou et al. 2022]). Addition-
ally, Generative Adversarial Networks (GANs), in combination with
contrastive and cycle-consistency losses [Zhou et al. 2016] have
proven effective in self-supervised style learning [Aberman et al. 2020b; Dong et al. 2017], which we also apply in our work. Figure 1 shows snapshots of our characterization results. Various
motions, such as walking, sitting and jumping (white character), can
be characterized to match Zombie, Princess, and Clown. We demon-
strate the effectiveness of our framework through comparisons with
previous work and ablation study. Additionally, we showcase its
capability for real-time live characterization from streamed motion
data. We also show that our framework can accommodate sparse
inputs, enabling its application in VR tracker-based motion capture
systems. 4.1
Bodypart Encoder The bodypart encoder in Fig. 3 (a) consists of two components:
body patch embedding, which reduces the spatial and temporal
resolutions of the input motion while preserving bodypart structure,
and transformer encoder, which uses a transformer-based structure
to learn sequential motion dependencies. 4.1.1
Body patch embedding. Following the approach of flattening
2D patches input for standard vision transformer [Dosovitskiy
et al. 2020], we employ body patch embedding, which maintains
the graph structure of the human skeleton as much as possible. Specifically, we use spatial-temporal graph convolutional blocks
(STGCN) [Yan et al. 2018] to reduce the spatial (joint) and temporal
(frame) resolutions. STGCN blocks project an input motion into a
sequential feature embedding E. We define embedding process as
follows: 2.3
Puppeteering and Motion Retargeting Puppeteering and motion retargeting can be regarded as a special
case of motion style transfer, where the control or source motion
must be mapped and adapted to the target character’s design and
style. Automatically mapping between two arbitrary characters
remains a notable challenge with no unique answers. To address
this, heuristic-based solution have been developed to analyze either
the structure [Kry et al. 2009] or motion space between characters
[Dontcheva et al. 2003; Seol et al. 2013]. For the less complex task
of mapping between two bipedal skeletons - considered homeomor-
phic graphs - graph convolutional networks have been successful
[Aberman et al. 2020a; Park et al. 2021]. We therefore employ this
strategy to extra motion features, facilitating retargeting across a
diverse range of characters and styles. E = STGCN(X𝑖).𝑓𝑙𝑎𝑡𝑡𝑒𝑛(0, 1) + P ∈R( 𝑇
4 ∗𝑛𝑏𝑜𝑑𝑦)×𝐶,
(2) E = STGCN(X𝑖).𝑓𝑙𝑎𝑡𝑡𝑒𝑛(0, 1) + P ∈R( 𝑇
4 ∗𝑛𝑏𝑜𝑑𝑦)×𝐶,
(2) (2) where P denotes positional encoding with learnable parameters. E comprises a total of 𝑇
4 ∗𝑛𝑏𝑜𝑑𝑦patches with 𝐶channels, where
𝑛𝑏𝑜𝑑𝑦(= 6) is the number of bodyparts (head, spine, arms, and legs). X𝑖= [x]𝑖
𝑖−(𝑇−1), x = [𝑥𝑡
𝑗𝑥𝑟
𝑗¤𝑥𝑡
𝑗¤𝑥𝑟
𝑗],
Y𝑖= [y]𝑖
𝑖−(𝑇−1), y = [𝑦𝑡
𝑗𝑦𝑟
𝑗¤𝑦𝑡
𝑗¤𝑦𝑟
𝑗],
(1) (1) 4.1.2
Transformer encoder. We capture spatial-temporal dependen-
cies of body patches by using a transformer structure. We feed the
embedding sequence E into the transformer encoder 𝐸𝑏to gener-
ate a source feature 𝑧= 𝐸𝑏(E). Each layer of the encoder consists
of a multi-head self-attention module (MSA) and a feed-forward
network (FFN). where 𝑗denotes all joints (including the pelvis), 𝑥𝑡∈R3 and 𝑥𝑟∈
R6 (6 for two orthogonal axes) are joint translations and rotations,
¤𝑥𝑡
𝑗∈R3 and ¤𝑥𝑟
𝑗∈R3 are joint linear and angular velocities local to
root𝑖. Likewise, 𝑦𝑡, 𝑦𝑟, ¤𝑦𝑡
𝑗and ¤𝑦𝑟
𝑗denote joint translation, rotation,
and linear and angular velocities local to the parent, with respect
to root𝑖. As a result, the total dimensions of our human motion
feature with 𝑇frames are X ∈R𝑇×𝑛𝑗𝑜𝑖𝑛𝑡×15 and Y ∈R𝑇×𝑛𝑗𝑜𝑖𝑛𝑡×15,
where 𝑛𝑗𝑜𝑖𝑛𝑡is the number of joints. 𝑧′ = MSA(E) + E,
𝑧𝑖= FNN(𝑧′) + 𝑧′ ∈Z ⊂R( 𝑇
4 ∗𝑛𝑏𝑜𝑑𝑦)×𝐶. (3) 𝑧′ = MSA(E) + E, 𝑧= MSA(E) + E,
𝑧𝑖= FNN(𝑧′) + 𝑧′ ∈Z ⊂R( 𝑇
4 ∗𝑛𝑏𝑜𝑑𝑦)×𝐶. (3) (3) 𝑧𝑖= FNN(𝑧′) + 𝑧′ ∈Z ⊂R( 𝑇
4 ∗𝑛𝑏𝑜𝑑𝑦)×𝐶. 4.2
Neural Context Matcher After bodypart encoding, we search a character feature that shares
a similar context with the source feature. This character feature
will later be used to imbue its style aspects into the source feature. Inspired by learned motion matching (LMM) approach [Holden
et al. 2020], we train the neural context matcher (NCM) to generate
the best matching character feature. Unlike LMM, which performs
matching every few frames, NCM runs at every frame to maximize
responsiveness to the input motion, which is crucial for producing
temporally continuous character features. To this end, we model the
NCM using an autoregressive conditional variational autoencoder. 2.2
Motion Matching The major contributions of our work can be summarized as
follows: Motion matching [Clavet 2016] provides continuous and control-
lable animation in real-time for interactive gaming via nearest-
neighbor search of motion features. To improve its scalability in
memory and speed, learned motion matching [Holden et al. 2020]
approximates this process using a neural network. Our Neural Con-
tact Matcher (NCM) applies learned motion matching on learned
context features to find the closest matching target motion, ensur-
ing real-time and high quality output. • We present the first online motion characterization frame-
work that can transfer both the motion style aspects and
body proportions of characters to a variety of user motions. • Our C-VAE-based Neural Context Matcher model can effec-
tively generate the target character’s motion feature with
temporal coherency. SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia MOCHA: Real-Time Motion Characterization via Context Matching frame, the bodypart encoder transforms the input source motion
into a feature vector (dubbed source feature) that structures human
motion into six parts and captures sequential motion dependen-
cies. Subsequently, given a target character, the NCM generates
a corresponding feature of the target character (dubbed character
feature) that shares the most similar context with the source motion. Lastly, the characterizer uses the source feature and the context-
matched character feature to synthesize a characterized pose while
preserving the source motion’s context. Figure 2: Illustration of the motion
representation at frame 𝑖. We define
the character forward facing direc-
tion of current frame 𝑖as reference
frame, which is denoted as root𝑖. Figure 2: Illustration of the motion
representation at frame 𝑖. We define
the character forward facing direc-
tion of current frame 𝑖as reference
frame, which is denoted as root𝑖. The bodypart encoder and characterizer are character-agnostic,
allowing a single trained network to work for all characters. In
contrast, the NCM is trained separately for each target character,
so the number of NCMs increases linearly with the number of
characters. 3
MOTION DATA REPRESENTATION AND
PROCESSING Figure 2 illustrates the motion representation of our method. The
reference frame of a motion, denoted as root𝑖, is located at the
ground projection of the pelvis joint at current frame 𝑖and aligned
to the ground normal direction and the pelvis’ forward facing direc-
tion. A motion sequence at frame 𝑖is represented in two ways: X𝑖
and Y𝑖express each element with respect to root𝑖and with respect
to the parent, respectively. E = STGCN(X𝑖).𝑓𝑙𝑎𝑡𝑡𝑒𝑛(0, 1) + P ∈R( 𝑇
4 ∗𝑛𝑏𝑜𝑑𝑦)×𝐶,
(2) 4.3
Characterizer The NCM implicitly models a distribution of possible next character
features that match the current source context feature given pre-
vious character feature. Samples are drawn from this distribution
and passed through the NCM decoder to create a character feature
for next frame, one at a time in an autoregressive fashion (yellow
part in Fig. 3 (a)). The characterizer transfers the style aspects (e.g., skeleton pro-
portions, characteristic movements, etc.) of the character feature
to the source feature. For this, the character transformer decoder
with adaptive instance normalization (AdaIN) [Huang and Belongie
2017] and multi-head cross-attention generates characterized de-
coded feature 𝑧𝑑(dubbed translated feature), which is then up-
sampled with De-STGCN blocks to obtain the final characterized
motion. 4.2.1
Context mapping. The context feature 𝑓(𝑧𝑖𝑠𝑟𝑐) ∈R( 𝑇
4 ∗𝑛𝑏𝑜𝑑𝑦)×𝐶
is extracted from encoded feature 𝑧𝑖via context mapping net-
work. The context space made by the context mapping is character-
agnostic, capturing shared information on context across character
domains. It enables context matching between different characters,
which is a crucial step in characterizing. The context mapping is
learned from unlabeled motion data in an supervised manner with
a set of loss terms as will be discussed in Sec. 6.5. 4.3.1
Character transformer decoder. After context matching, we
feed the source feature and character feature into the transformer
decoder. The character transformer decoder generates translated
feature 𝑧𝑑= 𝐷𝑐(𝑧𝑖𝑠𝑟𝑐, 𝑧𝑖
𝑐ℎ𝑎) which merges the context of source
motion and the character (style) aspects of target character motion. 4.3.1
Character transformer decoder. After context matching, we
feed the source feature and character feature into the transformer
decoder. The character transformer decoder generates translated
feature 𝑧𝑑= 𝐷𝑐(𝑧𝑖𝑠𝑟𝑐, 𝑧𝑖
𝑐ℎ𝑎) which merges the context of source
motion and the character (style) aspects of target character motion. As shown in Fig. 3 (b), instead of employing traditional trans-
former decoder block, we model our decoder with the AdaIN, multi-
head cross-attention module (MCA) with context mapping function,
and FNN layer. The AdaIN module transfers the global statistics of
character feature, as in [Jang et al. 2022], by taking 𝑧𝑖𝑠𝑟𝑐as input
and injecting character feature 𝑧𝑖
𝑒,𝑐ℎ𝑎as: 4.2.2
Prior Net. The distribution over possible latent variable 𝑠𝑖∈
R𝐶for character feature 𝑧𝑖
𝑐ℎ𝑎is described by a learned prior [Rempe
et al. 4
MOCHA FRAMEWORK Figure 3 (a) illustrates our motion characterization framework at
runtime. Input to our framework is a motion sequence of length 𝑇
(1 second, 60 frames) from past to current frame 𝑖, represented with
X𝑖. For output, we generate a characterized motion sequence of the
same length, represented as Y𝑖, but only use the final characterized
pose y𝑖. Our framework comprises a bodypart encoder, a neural con-
text matcher (NCM), and a characterizer networks. At each time SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia Deok-Kyeong Jang, Yuting Ye, Jungdam Won, and Sung-Hee Lee Figure 3: Network configuration. (a) Overall architecture for motion characterization in run-time. Our framework consists of
bodypart encoder, neural context matcher, and characterizer networks. (b) Detail of the characterizer transformer decoder
block (𝐷𝑐). Figure 3: Network configuration. (a) Overall architecture for motion characterization in run-time. Our framework consists of
bodypart encoder, neural context matcher, and characterizer networks. (b) Detail of the characterizer transformer decoder
block (𝐷𝑐). 4.3
Characterizer 2021] conditioned on previous character feature 𝑧𝑖−1
𝑐ℎ𝑎and
current source context feature 𝑓(𝑧𝑖𝑠𝑟𝑐): 𝑝(𝑠𝑖|𝑧𝑖−1
𝑐ℎ𝑎, 𝑓(𝑧𝑖
𝑠𝑟𝑐))
= N (𝑠𝑖; 𝜇(𝑧𝑖−1
𝑐ℎ𝑎, 𝑓(𝑧𝑖
𝑠𝑟𝑐)), 𝜎(𝑧𝑖−1
𝑐ℎ𝑎, 𝑓(𝑧𝑖
𝑠𝑟𝑐))),
(4) 𝑧𝑑′′ = 𝛾(𝑧𝑖
𝑐ℎ𝑎)
𝑧𝑖𝑠𝑟𝑐−𝜇(𝑧𝑖𝑠𝑟𝑐)
𝜎(𝑧𝑖𝑠𝑟𝑐)
+ 𝛽(𝑧𝑖
𝑐ℎ𝑎),
(6) (4) (6) where 𝜎and 𝜇are the channel-wise mean and variance, respectively. AdaIN scales the normalized 𝑧𝑖𝑠𝑟𝑐with a learned affine transforma-
tion with scales 𝛾and biases 𝛽generated by 𝑧𝑖
𝑐ℎ𝑎. In the second
step, we feed the globally-stylized feature 𝑧𝑑′′ and character feature
𝑧𝑖
𝑐ℎ𝑎to MCA. We use the context feature 𝑓(𝑧𝑑′′) to generate the
query 𝑄, the character context feature 𝑓(𝑧𝑖
𝑐ℎ𝑎) to generate the key
𝐾, and the character feature 𝑧𝑖
𝑐ℎ𝑎to generate the value 𝑉: which parameterizes a Gaussian distribution with diagonal covari-
ance via a neural network. 4.2.3
NCM decoder. The character feature 𝑧𝑖
𝑐ℎ𝑎is predicted by the
NCM decoder, which takes as input the latent variables 𝑠𝑖while
being conditioned on previous character feature 𝑧𝑖−1
𝑐ℎ𝑎and current
source context feature 𝑓(𝑧𝑖𝑠𝑟𝑐): 𝑄= 𝑓(𝑧𝑑′′)𝑊𝑞,
𝐾= 𝑓(𝑧𝑖
𝑐ℎ𝑎)𝑊𝑘, 𝑉= 𝑧𝑖
𝑐ℎ𝑎𝑊𝑣,
(7) (7) 𝑧𝑖
𝑐ℎ𝑎= 𝐷𝑣(𝑠𝑖, 𝑧𝑖−1
𝑐ℎ𝑎, 𝑓(𝑧𝑖
𝑠𝑟𝑐)). (5) (5) where𝑊𝑞,𝑊𝑘,𝑊𝑣∈R𝐶×𝑑ℎ𝑒𝑎𝑑. Then, the output sequence 𝑧𝑑of the
transformer decoder is obtained by We use transformer based C-VAE model for the NCM. In training
phase, both NCM encoder and decoder are trained as detailed in
Section 5.2 while only the decoder is used for inference. 𝑧′
𝑑= MCA(𝑄, 𝐾,𝑉) + 𝑧𝑑′′,
𝑧𝑑= FNN(𝑧′
𝑑) + 𝑧′
𝑑. (8) (8) MOCHA: Real-Time Motion Characterization via Context Matching SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia Figure 4: Stage-1 training process. A source motion X𝑖𝑠𝑟𝑐and a
target motion X𝑖
𝑐ℎ𝑎are randomly selected from different char-
acters in the motion dataset, and the networks are trained to
make the characterized motion Y𝑖
𝑡𝑟𝑎preserve the context of
X𝑖𝑠𝑟𝑐while reflecting the characteristic aspects of X𝑖
𝑐ℎ𝑎. 5.1.1
Identity and cycle loss. To design identity and cycle consis-
tency losses, we first define a reconstruction loss that computes the
difference of two motions both in terms of X and Y [Holden et al. 2020] as well as their velocities. L𝑟𝑒𝑐(eX, X) = 𝜆𝑙𝑜𝑐EX[∥eX −X∥1] + 𝜆𝑟𝑡EY[∥eY −Y∥1]
+ 𝜆𝑙𝑣𝑒𝑙EX[∥𝑉(eX) −𝑉(X)∥1] + 𝜆𝑟𝑣𝑒𝑙EY[∥𝑉(eY) −𝑉(Y)∥1], L𝑟𝑒𝑐(eX, X) = 𝜆𝑙𝑜𝑐EX[∥eX −X∥1] + 𝜆𝑟𝑡EY[∥eY −Y∥1] + 𝜆𝑙𝑣𝑒𝑙EX[∥𝑉(eX) −𝑉(X)∥1] + 𝜆𝑟𝑣𝑒𝑙EY[∥𝑉(eY) where X = 𝐹𝐾(Y), 𝑉(X) = X0−X1
ℎ
, 𝑉(Y) = Y0−Y1
ℎ
, ℎis time step,
and 𝜆𝑙𝑜𝑐, 𝜆𝑟𝑡, 𝜆𝑙𝑣𝑒𝑙, 𝜆𝑟𝑣𝑒𝑙are the relative weights. 5
TRAINING Our training pipeline consists of two-stages; the bodypart encoder
and characterizer are trained first, followed by training the NCM. Thus, in the first stage, our framework learns to extract encoded
features from a motion and transfer style aspects of one motion
(e.g., motion style and skeleton proportions) to the other motion
to synthesize a characterized motion. At this stage, the context
mapping network is also trained as a part of transformer decoder. The first stage training is conducted in an unsupervised way. The
second stage trains the NCM in a supervised manner to generate
target character features corresponding to the input context features
from the source. To define the body patch-level context loss between 𝑓(𝑧𝑠𝑟𝑐) and
𝑓(𝑧𝑡𝑟𝑎), we use infoNCE loss [Oord et al. 2018]: 𝑙(ˆ𝑣, 𝑣+, 𝑣-) = −log
"
exp(ˆ𝑣· 𝑣+/𝜏)
exp(ˆ𝑣· 𝑣+/𝜏) + Í𝑁
𝑛=1 exp(ˆ𝑣· 𝑣-𝑛/𝜏)
#
,
(12) (12) where 𝜏is the temperature parameter, and 𝑣+ and 𝑣- denote positive
and negative for ˆ𝑣. We set pseudo positive samples between body patch-level con-
text of source 𝑓(𝑧𝑠𝑟𝑐) and characterized motion 𝑓(𝑧𝑡𝑟𝑎); for a body
patch 𝑓(𝑧𝑡𝑟𝑎)𝑏∈𝑓(𝑧𝑡𝑟𝑎), we set its positive patch 𝑓(𝑧𝑠𝑟𝑐)𝑏as
the patch in the same location in 𝑓(𝑧𝑠𝑟𝑐), and negative patches
𝑓(𝑧𝑠𝑟𝑐)𝐵\𝑏as all other patches.︁ 4.3
Characterizer To ensure that the characterized
output motion not only maintains the overall context 𝑓(𝑧𝑠𝑟𝑐) ∈
R( 𝑇
4 ∗𝑛𝑏𝑜𝑑𝑦)×𝐶of source motion X𝑠𝑟𝑐, but also preserves context
of body patches at a specific location between source and output,
we introduce a body patch contrastive loss. For example, in Figure
5, the context of a princess leg at frame 𝑖−𝑡should be closer to
that of input zombie leg at frame 𝑖−𝑡than the other patches of the
same input.i 4.3
Characterizer 𝑉(X) represents
the rate of change of X. Part of 𝑉(X) corresponds to the joint
accelerations. Figure 4: Stage-1 training process. A source motion X𝑖𝑠𝑟𝑐and a
target motion X𝑖
𝑐ℎ𝑎are randomly selected from different char-
acters in the motion dataset, and the networks are trained to
make the characterized motion Y𝑖
𝑡𝑟𝑎preserve the context of
X𝑖𝑠𝑟𝑐while reflecting the characteristic aspects of X𝑖
𝑐ℎ𝑎. The identity loss ensures that the input motion remains un-
changed when it is used for both the source and character motions: L𝑖𝑑= L𝑟𝑒𝑐(X𝑖𝑑
𝑠𝑟𝑐, X𝑠𝑟𝑐) + L𝑟𝑒𝑐(X𝑖𝑑
𝑐ℎ𝑎, X𝑐ℎ𝑎),
(10) (10) where X𝑖𝑑
𝑠𝑟𝑐= 𝐹𝐾(𝐶𝐻(𝑧𝑠𝑟𝑐, 𝑧𝑠𝑟𝑐)) and 𝑧𝑠𝑟𝑐= 𝐵𝐸(X𝑠𝑟𝑐). X𝑖𝑑
𝑠𝑟𝑐is
obtained by feeding X𝑠𝑟𝑐to both X𝑠𝑟𝑐and X𝑐ℎ𝑎in Fig. 4. Likewise,
X𝑖𝑑
𝑐ℎ𝑎= 𝐹𝐾(𝐶𝐻(𝑧𝑐ℎ𝑎, 𝑧𝑐ℎ𝑎)) with 𝑧𝑐ℎ𝑎= 𝐵𝐸(X𝑐ℎ𝑎). 4.3.2
Body patch expanding and output. The translated feature
𝑧𝑑∈R( 𝑇
4 ∗𝑛𝑏𝑜𝑑𝑦)×𝐶is upsampled with additional De-STGCN blocks,
which has symmetric architectures to that of STGCN, to yield out-
put translated motion Y𝑖
𝑡𝑟𝑎as follows: 𝑐ℎ𝑎
To guarantee that the resulting motion Y𝑡𝑟𝑎(hence X𝑡𝑟𝑎) pre-
serves the context of the source motion X𝑠𝑟𝑐and the characteristics
of the character motion X𝑐ℎ𝑎, we employ cycle consistency loss
[Choi et al. 2020]. Y𝑖
𝑡𝑟𝑎= De-STGCN(𝑅𝑒𝑠ℎ𝑎𝑝𝑒(𝑧𝑑)). (9) (9) L𝑐𝑦𝑐= L𝑟𝑒𝑐(X𝑐𝑦𝑐
𝑠𝑟𝑐, X𝑠𝑟𝑐) + L𝑟𝑒𝑐(X𝑐𝑦𝑐
𝑐ℎ𝑎, X𝑐ℎ𝑎),
(11)
where X𝑐𝑦𝑐
𝑠𝑟𝑐= 𝐹𝐾(𝐶𝐻(𝑧𝑡𝑟𝑎, 𝑧𝑠𝑟𝑐)), X𝑐𝑦𝑐
𝑐ℎ𝑎= 𝐹𝐾(𝐶𝐻(𝑧𝑐ℎ𝑎, 𝑧𝑡𝑟𝑎)),
𝑧𝑡𝑟𝑎= 𝐵𝐸(𝐹𝐾(Y𝑡𝑟𝑎)) and Y𝑡𝑟𝑎= 𝐶𝐻(𝑧𝑠𝑟𝑐, 𝑧𝑐ℎ𝑎). X𝑐𝑦𝑐
𝑠𝑟𝑐is obtained
by feeding X𝑠𝑟𝑐and X𝑐ℎ𝑎to top and bottom inputs in Fig 4 to get
Y𝑡𝑟𝑎, followed by feeding X𝑡𝑟𝑎(= 𝐹𝐾(Y𝑡𝑟𝑎)) and X𝑠𝑟𝑐to top and
bottom inputs in Fig. 4 to get Y𝑐𝑦𝑐
𝑠𝑟𝑐. L𝑐𝑦𝑐= L𝑟𝑒𝑐(X𝑐𝑦𝑐
𝑠𝑟𝑐, X𝑠𝑟𝑐) + L𝑟𝑒𝑐(X𝑐𝑦𝑐
𝑐ℎ𝑎, X𝑐ℎ𝑎),
(11)
where X𝑐𝑦𝑐
𝑠𝑟𝑐= 𝐹𝐾(𝐶𝐻(𝑧𝑡𝑟𝑎, 𝑧𝑠𝑟𝑐)), X𝑐𝑦𝑐
𝑐ℎ𝑎= 𝐹𝐾(𝐶𝐻(𝑧𝑐ℎ𝑎, 𝑧𝑡𝑟𝑎)),
𝑧𝑡𝑟𝑎= 𝐵𝐸(𝐹𝐾(Y𝑡𝑟𝑎)) and Y𝑡𝑟𝑎= 𝐶𝐻(𝑧𝑠𝑟𝑐, 𝑧𝑐ℎ𝑎). X𝑐𝑦𝑐
𝑠𝑟𝑐is obtained
by feeding X𝑠𝑟𝑐and X𝑐ℎ𝑎to top and bottom inputs in Fig 4 to get
Y𝑡𝑟𝑎, followed by feeding X𝑡𝑟𝑎(= 𝐹𝐾(Y𝑡𝑟𝑎)) and X𝑠𝑟𝑐to top and
bottom inputs in Fig. 4 to get Y𝑐𝑦𝑐
𝑠𝑟𝑐. (11) Finally, we pick the last frame pose y𝑖
𝑡𝑟𝑎from Y𝑡𝑟𝑎as final output. 4.3.3
Root motion. We maintain the root angular velocity of the
source motion while scaling the linear velocity according to the
ratio of the average hip velocities of the source motion X𝑖and
the output motion Y𝑖
𝑡𝑟𝑎. This strategy allows for maintaining the
overall shape of the root trajectory of the source motion, while
varying the global linear velocity to match the target character. 5.1.2
Body patch contrastive loss. 6.1
Datasets We constructed a high-quality character motion dataset with five
professional actors. The dataset comprises a total of 6 characters
(Clown, Ogre, Princess, Robot, Zombie, and AverageJoe) performing
various actions (Dance, Fight, Jump, Crawling, Run, Walk, and Sit)
with 5 emotion variations for each action (Angry, Happy, Neutral,
Sad, and Scared). Every character preserves the same body propor-
tions as the actors, except we scale the forearm of the Ogre character
to be twice as long to convey its style. The dataset contains a to-
tal of 573k frames, captured at 60 fps, resulting in approximately
159 minutes of data. Since different emotions are manifested with
unique styles, we consider a character-emotion pair as an individual
character, such as “Neutral Zombie” and “Happy Ogre”. Figure 6: Training architecture of Neural Context Matcher. Once trained, only the decoder is used for inference. We additionally tested our algorithm on the Adult2child dataset
[Dong et al. 2020] for quantitative evaluation.It consists of 17 sub-
jects including nine adults (older than 18 years) and eight children
(5-10 years old) performing actions such as Jump as high as you
can in place, Punch, Kick, Walk, and Hop Scotch. We treat each
subject as a character due to their personalized styles. Adult2child
dataset is suitable for our evaluation since the skeleton proportions
and motion styles vary depending on the subject. context loss work, with procedure to define positive and negative
samples. context loss work, with procedure to define positive and negative
samples. The total loss function of stage-1 is thus: (14) L𝑠𝑡𝑎𝑔𝑒1 = 𝜆𝑖𝑑L𝑖𝑑+ 𝜆𝑐𝑦𝑐L𝑐𝑦𝑐+ 𝜆𝑐𝑡𝑟L𝑐𝑡𝑟,
(14) where 𝜆𝑖𝑑, 𝜆𝑐𝑦𝑐, and 𝜆𝑐𝑡𝑟are weights. where 𝜆𝑖𝑑, 𝜆𝑐𝑦𝑐, and 𝜆𝑐𝑡𝑟are weights. 6
EVALUATION AND EXPERIMENTS We conduct comparisons with other methods, perform an ablation
study, and conduct additional experiments to demonstrate the utility
of our framework. For visual animation results, please refer to the
supplementary video. Figure 5: Illustration of body patch-level context contrastive
loss. Blue box indicates a positive sample, while yellow boxes
denote negative samples. Figure 6: Training architecture of Neural Context Matcher. Once trained, only the decoder is used for inference. 5.2
Stage-2 First, we qualitatively compare our method with two baselines:
Motion Puzzle [Jang et al. 2022] and replacing NCM in MOCHA
with nearest-neighbor search (Nearest Neighbor). Since Motion
Puzzle only works for a single body proportion, we have to re-
place its reconstruction loss with ours to accommodate skeleton
variations at training. Note that Motion Puzzle conducts an offline,
full sequence-to-sequence translation, while MOCHA is an online
translator. In stage-2, the NCM is trained to infer a target character feature
𝑧𝑐ℎ𝑎that shares the same context with a source feature 𝑧𝑠𝑟𝑐. Briefly,
this is achieved by providing the ground truth character feature
𝑧𝑐ℎ𝑎obtained from searching a target feature database D𝑡𝑎𝑟=
[Z𝑡𝑎𝑟, 𝑓(Z𝑡𝑎𝑟)] with a context metric ||𝑓(𝑧𝑡𝑎𝑟)−𝑓(𝑧𝑠𝑟𝑐)||. To begin
with, we roughly annotate the motion data with action labels (e.g.,
walk, jump, and crawl) to facilitate the nearest neighbor search;
limiting the search only on the relevant subset of the same action
label saves time and enhances accuracy of the results. Figure 7 compares the characterization result of Neutral Aver-
ageJoe as the source (1st row) among MOCHA, Nearest Neighbor,
and Motion Puzzle. Unlike MOCHA, both Motion Puzzle and the
Nearest Neighbor method require a specific reference motion as
input. Therefore, we manually selected a reference from each target
character performing the most similar action (2nd row) for them. Compared to Motion Puzzle, MOCHA better preserves the unique
style in each target character. Our results accurately capture the
limping legs of the Neutral Zombie, the energetic waves of the
Angry Clown, the elegantly crossed legs of the Princess, and the Figure 7 compares the characterization result of Neutral Aver-
ageJoe as the source (1st row) among MOCHA, Nearest Neighbor,
and Motion Puzzle. Unlike MOCHA, both Motion Puzzle and the
Nearest Neighbor method require a specific reference motion as
input. Therefore, we manually selected a reference from each target
character performing the most similar action (2nd row) for them. As the NCM operates autoregressively, training the NCM en-
coder and decoder is conducted with a sequence of source features
𝑧𝑖𝑠𝑟𝑐|𝑠
𝑖=0 and its corresponding sequence of target features 𝑧𝑖
𝑐ℎ𝑎|𝑠
𝑖=0
obtained with nearest neighbor search. These feature sequences
represent continuous motions in motion database. In the training,
the NCM transformer takes the previous character feature and cur-
rent source context feature as condition to reconstruct the context-
matched target character feature. 5.1
Stage-1 Figure 4 shows the training architecture in stage-1. Without the
NCM, we randomly choose a source motion X𝑖𝑠𝑟𝑐and a character
motion X𝑖
𝑐ℎ𝑎to train the bodypart encoder and the characterizer
jointly. In addition to using common loss terms for learning style
transfer, such as identity and cyclic consistency losses, we newly
introduce a contrastive loss to enhance local context preservation
both spatially and temporally. To simplify the notation, we use 𝐵𝐸(·)
and 𝐶𝐻(𝑧𝑠𝑟𝑐,𝑧𝑐ℎ𝑎) to denote body part encoder and characterizer,
and omit the superscript 𝑖. L𝑐𝑡𝑟= EX
𝐵
∑︁
𝑏
𝑙(𝑓(𝑧𝑡𝑟𝑎)𝑏, 𝑓(𝑧𝑠𝑟𝑐)𝑏, 𝑓(𝑧𝑠𝑟𝑐)𝐵\𝑏),
(13) (13) ︁
where 𝑏∈{1, 2, . . . 𝐵} and 𝐵(= 𝑇/4 ∗𝑛𝑏𝑜𝑑𝑦) is the number of
body patches. Figure 5 illustrates how body patch-level contrastive Deok-Kyeong Jang, Yuting Ye, Jungdam Won, and Sung-Hee Lee SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia Figure 5: Illustration of body patch-level context contrastive
loss. Blue box indicates a positive sample, while yellow boxes
denote negative samples. Figure 6: Training architecture of Neural Context Matcher. Once trained, only the decoder is used for inference. Figure 5: Illustration of body patch-level context contrastive
loss. Blue box indicates a positive sample, while yellow boxes
denote negative samples. shape the latent variable 𝑠as a standard normal distribution (𝜇𝑝𝑟,
𝜎𝑝𝑟) using 𝑃𝑟𝑖𝑜𝑟𝑁𝑒𝑡. This process is illustrated in Fig. 6. For details
on the training procedure, please see Algorithm 1 in supplementary
material. At run-time, the encoder is discarded and the decoder is used to
predict matched character features. At each time frame, we pass
𝑓(𝑧𝑖𝑠𝑟𝑐) through decoder to predict a matched character feature
𝑧𝑖
𝑐ℎ𝑎(Sec. 4.2). 5.2
Stage-2 At the same time, it attempts to Compared to Motion Puzzle, MOCHA better preserves the unique
style in each target character. Our results accurately capture the
limping legs of the Neutral Zombie, the energetic waves of the
Angry Clown, the elegantly crossed legs of the Princess, and the MOCHA: Real-Time Motion Characterization via Context Matching SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia Table 1: Frechet Motion Distance (FMD), context recognition
accuracy (CRA), and style recognition accuracy (SRA) on sam-
ples generated by each method as quantitative comparison. attention layer in the characterizing transformer decoder block (Fig-
ure 3 (b)). To assess its impact, we trained a Characterizer without
the AdaIN module. Results in Figure 8 shows that the ablated model
cannot capture the Ogre’s raising arms style without using AdaIN. Methods
FMD↓
SRA↑(%)
CRA↑(%)
Top1
Top5
Ground truth
43.61
84.76
-
Ours
34.99
36.51
73.68
-
Nearest Neighbor
32.80
23.62
69.06
-
Motion Puzzle
39.51
31.78
70.7
-
(a) MOCHA dataset. Methods
FMD↓
SRA↑(%)
CRA↑(%)
Top1
Top5
Top1
Top5
Ground truth
96.46
99.77
58.97
85.60
Ours
25.98
68.57
95.21
38.18
74.45
Nearest Neighbor
29.30
63.32
93.20
45.96
79.55
(b) Adult2child dataset. 6.4.2
Effect of contrastive loss. To examine the effect of patch-level
contrastive loss, we tested stage-1 training without the contrastive
loss. Since the advantage of our contrastive loss is to preserve
spatial-temporal context features across all characters, we randomly
picked a source and a character motion to generate output. Figure 9
shows that the left and right legs of the output Ogre character are
swapped (red circle) without patch-wise contrastive loss because
attention module alone may mismatch the left leg of the source with
the right leg of the character. In contrast, our model can distinguish
the left and right side of the body. Additionally, despite significant
differences of two motions, our method reasonably preserves the
arm swinging motion of the source (red arrow), which cannot be
achieved with the ablation model. 6.6
Applications 6.6.1
Input from unseen subjects. We test the effectiveness of our
framework for inputs from unseen subjects with varying heights. Figure 11 (a) shows that our framework works successfully even
when motions from unseen subjects with different body proportions
are given as input. 6.6.2
Sparse input. We supposed that our framework could work
with sparse inputs from the hip and end-effectors as they may
contain essential information about context and style. To verify this,
we reduced the input joints to only six (hip, head, hands, and feet)
and retrained the entire framework. As shown in Figure 11 (b), our
framework successfully characterizes motion as Neutral Princess
from sparse input. This experiment suggests that our framework can
accommodate 6-point tracker input data for real-time applications. 6.5
Context space analysisf wildly flailing arms of the Happy Zombie. In contrast, Motion Puzzle
dampens the target style in favor of the source motion. Successful context matching across different characters is possible
thanks to the shared character-agnostic context space constructed
by the context mapping module. To visualize its result, we randomly
sampled a character motion and computed its context feature as
a query, then searched another character’s context database for
the closest match. Figure 10 (a) shows semantically similar yet
diverse postures across four different characters that match the
query. In another experiment, given a source motion (X𝑠𝑟𝑐), we
searched for the nearest context feature (𝑧𝑐ℎ𝑎) of a target character
and generated a characterized motion (Y𝑡𝑟𝑎). Subsequently, both
source and characterized motions were mapped to the context space
as shown in Figure 10 (b). One can see that all three context feature
points (𝑓(𝐵𝐸(X𝑠𝑟𝑐)), 𝑓(𝑧𝑐ℎ𝑎), 𝑓(𝐵𝐸(Y𝑡𝑟𝑎))) are located very close
together, showing that the learned motion context indeed shares
the same space across characters. Nearest Neighbor exhibits noticeable discontinuity in the output,
due to the relatively sparse context space and insufficient temporal
features used in search. It cannot respect the source motion context
when similar context is not available for the target character. Thanks
to the autoregressive CVAE, our method using NCM can mitigate
these issues to produce smooth and consistent output. 6.3
Quantitative evaluation We quantitatively compare the degree of motion quality, context
preservation, and style reflection with Motion Puzzle and the Near-
est Neighbor method. Following prior work [Jang et al. 2022; Park
et al. 2021], we use Frechet Motion Distance (FMD), context recog-
nition accuracy (CRA), and style recognition accuracy (SRA) as
metrics. We train the CRA classifier and the SRA classifier using vi-
sion transformer [Dosovitskiy et al. 2020] for its superior accuracy. The two datasets, MOCHA and Adult2child, are each split into a
90% training set and a 10% test set. Results are detailed in Table 1. We didn’t apply CRA on the MOCHA dataset because it does not
contain action labels. We also have to remove Motion Puzzle from
the Adult2Child dataset comparison as it fails to train. When com-
pared to Motion Puzzle, our method yields superior FMD and SRA
scores, demonstrating higher motion quality and a more precise
style reflection. A comparison with the Nearest Neighbor method
underscores the significance of our NCM module, as we consistently
achieve higher SRA scores due to superior temporal coherence. The
better CRA score for Nearest Neighbor can be attributed to its direct
usage of features from the database. 7
DISCUSSION AND CONCLUSION Yuzhu Dong, Sachin Paryani, Neha Rana, Aishat Aloba, Lisa Anthony, and Eakta
Jain. 2017. Adult2Child: Dynamic Scaling Laws to Create Child-Like Motion. In
Proceedings of the 10th International Conference on Motion in Games. 13:1–13:10. https://doi.org/10.1145/3136457.3136460 We introduced MOCHA, a motion characterization framework that
can transform user motions to embody distinct style of characters
in real-time, and demonstrated its effectiveness through a number
of experiments and analysis. p
g
Mira Dontcheva, Gary Yngve, and Zoran Popović. 2003. Layered Acting for Character
Animation. ACM Trans. Graph. 22, 3 (jul 2003), 409–416. https://doi.org/10.1145/
882262.882285 Alexey Dosovitskiy, Lucas Beyer, Alexander Kolesnikov, Dirk Weissenborn, Xiaohua
Zhai, Thomas Unterthiner, Mostafa Dehghani, Matthias Minderer, Georg Heigold,
Sylvain Gelly, et al. 2020. An image is worth 16x16 words: Transformers for image
recognition at scale. arXiv preprint arXiv:2010.11929 (2020). Our method has several limitations that necessitates further
exploration. First, our method is most effective when a character
motion dataset contains a single characteristic style per context. If
multiple motions with different styles share the same context for a
target character, our NCM may encounter difficulties in generating
temporally consistent character features, leading to discontinuous
motions. We observed such phenomena when consolidating all
emotion sets of a character into one, thereby allowing emotional
style variations within the same motion. Future work is needed to
enable such style variations even within a single character. Han Du, Erik Herrmann, Janis Sprenger, Klaus Fischer, and Philipp Slusallek. 2019. Stylistic locomotion modeling and synthesis using variational generative models. In Motion, Interaction and Games. 1–10. https://doi.org/10.1145/3359566.3360083 p
g
Leon A. Gatys, Alexander S. Ecker, and Matthias Bethge. 2015. A Neural Algorithm of
Artistic Style. CoRR abs/1508.06576 (2015). http://dblp.uni-trier.de/db/journals/
corr/corr1508.html#GatysEB15a Keith Grochow, Steven L. Martin, Aaron Hertzmann, and Zoran Popović. 2004. Style-
Based Inverse Kinematics. ACM Trans. Graph. 23, 3 (aug 2004), 522–531. https:
//doi.org/10.1145/1015706.1015755 Daniel Holden, Oussama Kanoun, Maksym Perepichka, and Tiberiu Popa. 2020. Learned
motion matching. ACM Transactions on Graphics (TOG) 39, 4 (2020), 53–1. https:
//doi.org/10.1145/3386569.3392440 Second, while our unsupervised learning of context mapping is
effective without manual style labels, it is not flexible enough to
encompass all stylistic diversity of the character data. For instance,
Angry Ogre motions in our database has a unique style of running
on all fours and jumping sideways. Our algorihtm currently could
not link them to the running and jumping motions of other charac-
ters, as shown in the supplementary video. REFERENCESi Soomin Park, Deok-Kyeong Jang, and Sung-Hee Lee. 2021. Diverse motion stylization
for multiple style domains via spatial-temporal graph-based generative model. Proceedings of the ACM on Computer Graphics and Interactive Techniques 4, 3 (2021),
1–17. https://doi.org/10.1145/3480145 Kfir Aberman, Peizhuo Li, Dani Lischinski, Olga Sorkine-Hornung, Daniel Cohen-Or,
and Baoquan Chen. 2020a. Skeleton-aware networks for deep motion retargeting. ACM Transactions on Graphics (TOG) 39, 4 (2020), 62–1. https://doi.org/10.1145/
3386569.3392462i Davis Rempe, Tolga Birdal, Aaron Hertzmann, Jimei Yang, Srinath Sridhar, and
Leonidas J Guibas. 2021. Humor: 3d human motion model for robust pose es-
timation. In Proceedings of the IEEE/CVF international conference on computer vision. 11488–11499. Kfir Aberman, Yijia Weng, Dani Lischinski, Daniel Cohen-Or, and Baoquan Chen. 2020b. Unpaired motion style transfer from video to animation. ACM Transactions
on Graphics (TOG) 39, 4 (2020), 64–1. https://doi.org/10.1145/3386569.3392469 C. Rose, M.F. Cohen, and B. Bodenheimer. 1998. Verbs and adverbs: multidimensional
motion interpolation. IEEE Computer Graphics and Applications 18, 5 (1998), 32–40. https://doi.org/10.1109/38.708559 Kenji Amaya, Armin Bruderlin, and Tom Calvert. 1996. Emotion from Motion. In
Proceedings of the Graphics Interface 1996 Conference, May 22-24, 1996, Toronto,
Ontario, Canada. Canadian Human-Computer Communications Society, 222–229.i Yeongho Seol, Carol O’Sullivan, and Jehee Lee. 2013. Creature Features: Online
Motion Puppetry for Non-Human Characters. In Proceedings of the 12th ACM
SIGGRAPH/Eurographics Symposium on Computer Animation (SCA ’13). 213–221. https://doi.org/10.1145/2485895.2485903 Andreas Aristidou, Anastasios Yiannakidis, Kfir Aberman, Daniel Cohen-Or, Ariel
Shamir, and Yiorgos Chrysanthou. 2022. Rhythm is a dancer: Music-driven motion
synthesis with global structure. IEEE Transactions on Visualization and Computer
Graphics (2022). p
g
Ari Shapiro, Yong Cao, and Petros Faloutsos. 2006. Style components. In Proceedings
of Graphics Interface 2006 (Québec, Québec, Canada) (GI 2006). Canadian Human-
Computer Communications Society, Toronto, Ontario, Canada, 33–39.ffi Matthew Brand and Aaron Hertzmann. 2000. Style Machines. In Proceedings of the 27th
Annual Conference on Computer Graphics and Interactive Techniques (SIGGRAPH
’00). ACM Press/Addison-Wesley Publishing Co., USA, 183–192. https://doi.org/10. 1145/344779.344865 uter Communications Society, Toronto, Ontario, Canada, 33–39 Harrison Jesse Smith, Chen Cao, Michael Neff, and Yingying Wang. 2019. Efficient
neural networks for real-time motion style transfer. Proceedings of the ACM on
Computer Graphics and Interactive Techniques 2, 2 (2019), 1–17. https://doi.org/10. 1145/3340254 Yunjey Choi, Youngjung Uh, Jaejun Yoo, and Jung-Woo Ha. 2020. Stargan v2: Diverse
image synthesis for multiple domains. In Proceedings of the IEEE/CVF Conference on
Computer Vision and Pattern Recognition. 8188–8197. p
g
Simon
Clavet. 2016. The
road
to
Next
Gen
Animation. https://www.gdcvault.com/play/1023280/Motion-Matching-and-The-Road. T. Tao, X. ACKNOWLEDGMENTS Paul G Kry, Lionel Revéret, François Faure, and M-P Cani. 2009. Modal locomotion:
Animating virtual characters with natural vibrations. In Computer Graphics Forum,
Vol. 28. Wiley Online Library, 289–298. This work was conducted during Deok-Kyeong Jang’s internship at
Meta. Deok-Kyeong Jang would like to thank Michelle Hill for her
help in capturing the dataset and the Meta team for their mentor-
ship and support during his internship. Jungdam Won was partially
supported by the New Faculty Startup Fund from Seoul National
University, ICT (Institute of Computer Technology) at Seoul Na-
tional University. Sung-Hee Lee was partially supported by the
Technology Innovation Program (20011076) of KEIT. Wanli Ma, Shihong Xia, Jessica K Hodgins, Xiao Yang, Chunpeng Li, and Zhaoqi
Wang. 2010. Modeling style and variation in human motion. In Proceedings of
the 2010 ACM SIGGRAPH/Eurographics Symposium on Computer Animation. 21–30. https://doi.org/10.1109/IUCS.2010.5666642 Ian Mason, Sebastian Starke, He Zhang, Hakan Bilen, and Taku Komura. 2018. Few-
shot Learning of Homogeneous Human Locomotion Styles. In Computer Graphics
Forum, Vol. 37. Wiley Online Library, 143–153. https://doi.org/10.1111/cgf.13555 Jianyuan Min, Yen-Lin Chen, and Jinxiang Chai. 2009. Interactive Generation of Human
Animation with Deformable Motion Models. ACM Trans. Graph. 29, 1, Article 9
(dec 2009), 12 pages. https://doi.org/10.1145/1640443.1640452 y
g
Animation with Deformable Motion Models. ACM Trans. Graph. 29, 1, Article 9
(dec 2009), 12 pages. https://doi.org/10.1145/1640443.1640452 Aaron van den Oord, Yazhe Li, and Oriol Vinyals. 2018. Representation learning with
contrastive predictive coding. arXiv preprint arXiv:1807.03748 (2018). 7
DISCUSSION AND CONCLUSION More advanced methods
for context learning is desirable to improve the characterization
quality. Daniel Holden, Jun Saito, and Taku Komura. 2016. A deep learning framework for
character motion synthesis and editing. ACM Transactions on Graphics (TOG) 35, 4
(2016), 1–11. https://doi.org/10.1145/2897824.2925975 Xun Huang and Serge Belongie. 2017. Arbitrary style transfer in real-time with
adaptive instance normalization. In Proceedings of the IEEE International Conference
on Computer Vision. 1501–1510. Leslie Ikemoto, Okan Arikan, and David Forsyth. 2009. Generalizing Motion Edits
with Gaussian Processes. ACM Trans. Graph. 28, 1, Article 1 (feb 2009), 12 pages. https://doi.org/10.1145/1477926.1477927 Deok-Kyeong Jang, Soomin Park, and Sung-Hee Lee. 2022. Motion Puzzle: Arbitrary
Motion Style Transfer by Body Part. ACM Trans. Graph. 41, 3, Article 33 (jun 2022),
16 pages. https://doi.org/10.1145/3516429 6.4
Ablation study 6.6.3
Live characterization from streamed motion data. We demon-
strate the ability of our framework to characterize streamed motion
data in real-time. Figure 11 (c) shows a snapshot of live characteri-
zation of a streamed motion captured with Xsens Awinda sensor. The supplementary video shows that our method can produce suc-
cessfully characterized motion even with network delays and noisy We conducted ablation studies to demonstrate the effectiveness of
the AdaIN layer and the contrastive loss. For additional ablation
studies, please refer to the supplementary material. 6.4.1
Effect of AdaIN.. The AdaIN module facilitates successful
characterization by incorporating a global style before the cross Deok-Kyeong Jang, Yuting Ye, Jungdam Won, and Sung-Hee Lee SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia input. The overall inference time is under 16ms, achieving 60Hz or
higher frame rate with a 2080 Ti GPU. Yuzhu Dong, Andreas Aristidou, Ariel Shamir, Moshe Mahler, and Eakta Jain. 2020. Adult2child: Motion Style Transfer Using CycleGANs. In Proceedings of the 13th
ACM SIGGRAPH Conference on Motion, Interaction and Games. Article 13, 11 pages. https://doi.org/10.1145/3424636.3426909 T. Tao, X. Zhan, Z. Chen, and M. van de Panne. 2022a. Style-ERD: Responsive and
Coherent Online Motion Style Transfer. In 2022 IEEE/CVF Conference on Computer SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia REFERENCESi Zhan, Z. Chen, and M. van de Panne. 2022a. Style-ERD: Responsive and
Coherent Online Motion Style Transfer. In 2022 IEEE/CVF Conference on Computer MOCHA: Real-Time Motion Characterization via Context Matching Vision and Pattern Recognition (CVPR). IEEE Computer Society, Los Alamitos, CA,
USA, 6583–6593. https://doi.org/10.1109/CVPR52688.2022.00648 Vision and Pattern Recognition (CVPR). IEEE Computer Society, Los Alamitos, CA,
USA, 6583–6593. https://doi.org/10.1109/CVPR52688.2022.00648 p
g
Tianxin Tao, Xiaohang Zhan, Zhongquan Chen, and Michiel van de Panne. 2022b. Style-ERD: responsive and coherent online motion style transfer. In Proceedings of
the IEEE/CVF Conference on Computer Vision and Pattern Recognition. 6593–6603. Munetoshi Unuma, Ken Anjyo, and Ryozo Takeuchi. 1995. Fourier principles for
emotion-based human figure animation. In Proceedings of the 22nd annual conference
on Computer graphics and interactive techniques. 91–96. Jack M. Wang, David J. Fleet, and Aaron Hertzmann. 2007. Multifactor Gaussian
Process Models for Style-Content Separation. In Proceedings of the 24th International
Conference on Machine Learning (ICML ’07). 975–982. https://doi.org/10.1145/
1273496.1273619 Shihong Xia, Congyi Wang, Jinxiang Chai, and Jessica Hodgins. 2015. Realtime style
transfer for unlabeled heterogeneous human motion. ACM Transactions on Graphics
(TOG) 34, 4 (2015), 1–10. https://doi.org/10.1145/2766999 p
g
Sijie Yan, Yuanjun Xiong, and Dahua Lin. 2018. Spatial temporal graph convolu-
tional networks for skeleton-based action recognition. In Proceedings of the AAAI
Conference on Artificial Intelligence, Vol. 32. i
M Ersin Yumer and Niloy J Mitra. 2016. Spectral style transfer for human motion
between independent actions. ACM Transactions on Graphics (TOG) 35, 4 (2016),
1–8. https://doi.org/10.1145/2897824.2925955 p
g
T. Zhou, P. Krahenbuhl, M. Aubry, Q. Huang, and A. A. Efros. 2016. Learning Dense
Correspondence via 3D-Guided Cycle Consistency. In 2016 IEEE Conference on Com-
puter Vision and Pattern Recognition (CVPR). IEEE Computer Society, Los Alamitos,
CA, USA, 117–126. https://doi.org/10.1109/CVPR.2016.20 SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia Deok-Kyeong Jang, Yuting Ye, Jungdam Won, and Sung-Hee Lee Figure 7: Qualitative evaluation. Source motions of Neutral AverageJoe (top) are characterized with each method (3-5 rows). Reference motion is used for the Nearest Neighbor and Motion Puzzle. Figure 7: Qualitative evaluation. Source motions of Neutral AverageJoe (top) are characterized with each method (3-5 rows). Reference motion is used for the Nearest Neighbor and Motion Puzzle. MOCHA: Real-Time Motion Characterization via Context Matching SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia g
p
y
y
Figure 8: Ablation study on AdaIN. Result obtained without AdaIN fails to capture the jumping style of Ogre. REFERENCESi ation study on AdaIN. Result obtained without AdaIN fails to capture the jumping style of Ogre. Figure 8: Ablation study on AdaIN. Result obtained without AdaIN fails to capture the jumpi Figure 9: Contrastive loss test. Ogre’s left and right leg are mistakenly swapped (red circle) without the contrastive loss. Furthermore, the contrastive loss helps retain the left arm’s swing phase of the source motion to the characterized motion (red
arrows). Figure 9: Contrastive loss test. Ogre’s left and right leg are mistakenly swapped (red circle) without the contrastive loss. Furthermore, the contrastive loss helps retain the left arm’s swing phase of the source motion to the characterized motion (red
arrows). Figure 10: (a) Four best matching motions in the entire datasets given the white character’s motion (top: running, bottom:
walking). (b) A t-SNE visualization of the entire context features in the test dataset with colors differentiating characters. Three
context feature points corresponding to a source, matched target, and output motion are located very closely to each other. Figure 10: (a) Four best matching motions in the entire datasets given the white character’s motion (top: running, bottom:
walking). (b) A t-SNE visualization of the entire context features in the test dataset with colors differentiating characters. Three
context feature points corresponding to a source, matched target, and output motion are located very closely to each other. Figure 11: Applications of our framework. Characterization results from an unseen subject (left), with sparse input from 6
joints (middle), and from streamed motion data (right). Figure 11: Applications of our framework. Characterization results from an unseen subject (left), with sparse input from 6
joints (middle), and from streamed motion data (right).
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Àíàë³òèêà á³çíåñ–ð³øåíü ÿê ñêëàäîâà ä³ÿëüíîñò³
ï³äïðèºìñòâà: òåîðåòè÷íèé àñïåêò Ïðåäìåò äîñë³äæåííÿ.  ñòàòò³ äîñë³äæåíî òåîðåòè÷íèé àñïåêò àíàë³òèêè á³çíåñ–ð³øåíü ÿê
ñêëàäîâî¿ ä³ÿëüíîñò³ ï³äïðèºìñòâà â ñó÷àñíèõ óìîâàõ ãîñïîäàðþâàííÿ ñóá’ºêò³â ï³äïðèºìíèöòâà. Ìåòîþ íàïèñàííÿ ñòàòò³. Äîñë³äæåíî îñíîâíó ìåòó á³çíåñ–àíàë³òèêè òà âñòàíîâëåíî, ùî àíà-
ë³òèêà á³çíåñ–ð³øåíü â êîðîòêîìó ñåíñ³ ñëîâà – öå àíàë³òè÷íà ñêëàäîâà, ùî ñóïðîâîäæóºòüñÿ âè-
êîðèñòàííÿì ñó÷àñíèõ ï³äõîä³â ï³ä ÷àñ àíàë³çó ³íôîðìàö³¿ â îáëàñò³ ï³äãîòîâêè á³çíåñ–ð³øåíü. Ìåòîäîëîã³ÿ ïðîâåäåííÿ ðîáîòè.  øèðøîìó àñïåêò³, àíàë³òèêà á³çíåñ–ð³øåíü – öå çàãàëüíà
ñóêóïí³ñòü ïðèíöèï³â ìåòîäîëîã³÷íî–îðãàí³çàö³éíîãî, à òàêîæ òåõíîëîã³÷íîãî ð³çíîãî ðîäó ðîçóìîâî¿
ä³ÿëüíîñò³, ùî äîçâîëÿº åôåêòèâíî ïðîâîäèòè îáðîáêó ³íôîðìàö³¿ ç ö³ëëþ ï³äâèùåííÿ ÿêîñò³ íàÿâ-
íèõ ³ îòðèìàííÿ íîâèõ çíàíü, à òàêîæ ï³äãîòîâêè ³íôîðìàö³éíî–³íòåëåêòóàëüíî¿ áàçè äëÿ ïðèéíÿòòÿ
ðàö³îíàëüíèõ óïðàâë³íñüêèõ á³çíåñ–ð³øåíü â ï³äïðèºìíèöüê³é ä³ÿëüíîñò³ ñóá’ºêò³â ãîñïîäàðþâàííÿ. Ðåçóëüòàòè ðîáîòè. Ðîçãëÿíóòî îñíîâí³ ïðîöåñè, ÿê³ âèçíà÷àþòü àíàë³òèêó á³çíåñ–ð³øåíü ÿê
ñïåöèô³÷íèé âèä ä³ÿëüíîñò³ íà ï³äïðèºìñòâ³. Âñòàíîâëåíî, ùî âèá³ð àëüòåðíàòèâè â õîä³ îáðàííÿ á³çíåñ–ð³øåííÿ çàëåæèòü â³ä: ìîæëèâîñò³
îö³íèòè åôåêòèâí³ñòü àëüòåðíàòèâíîãî ð³øåííÿ ³ íåîáõ³äí³ âèòðàòè äëÿ éîãî ðåàë³çàö³¿; ìåòîä³â, ÿê³
âèêîðèñòîâóâàëèñÿ â ï³äïðèºìíèöüê³é ä³ÿëüíîñò³, à òàêîæ ðîçðîáëåí³ äî öüîãî ÷àñó ìîäåë³ äîñë³-
äæåííÿ ï³äïðèºìíèöüêèõ îïåðàö³é; ìåòîä³â, ÿê³ äîçâîëèëè á àíàë³çóâàòè ñõîæ³ ïðîáëåìè.  ñòàòò³ ïðåäñòàâëåí³ ï³äõîäè äî âèíà÷åííÿ ïîíÿòòÿ «àíàë³òèêà á³çíåñ–ð³øåíü» òà òðè íàéâàæ-
ëèâ³ø³ ñêëàäîâ³, ÿê³ îá’ºäíóº àíàë³òèêà á³çíåñ ð³øåíü. Äîñë³äæåíî îñíîâí³ ïðèíöèïè òà ñêëàäîâ³ àíàë³çó äàíèõ â á³çíåñ–àíàë³òèö³. Âèñíîâîê. Îðãàí³çàö³ÿ ïðîöåñó ïðèéíÿòòÿ á³çíåñ–ð³øåííÿ çàëåæèòü â³ä ïðîåêòóâàííÿ ³íôîðìà-
ö³éíèõ òåõíîëîã³é äëÿ á³çíåñ–àíàë³òèêè â ï³äïðèºìíèöüê³é ä³ÿëüíîñò³ òà ìຠáàçóâàòèñÿ íà ³íòåãðàö³¿
¿õ ó ñèñòåìó óïðàâë³ííÿ á³çíåñ–ïðîöåñàìè ï³äïðèºìñòâà òà â³äïîâ³äàòè á³çíåñ–ìîäåë³ êîæíîãî ï³ä-
ïðèºìñòâà ç óðàõóâàííÿì ìîæëèâîñò³ ¿õ ³íòåãðóâàííÿ ó íàÿâí³ ³íôîðìàö³éí³ ñèñòåìè ï³äïðèºìñòâà. Êëþ÷îâ³ ñëîâà: ï³äïðèºìñòâî, àíàë³ç, àíàë³òèêà, á³çíåñ–ð³øåííÿ, ñóá’ºêò ãîñïîäàðþâàííÿ, àíàë³òèê. ÅÊÎÍÎ̲×Ͳ ÏÐÎÁËÅÌÈ ÐÎÇÂÈÒÊÓ ÃÀËÓÇÅÉ ÒÀ ÂÈIJ ÅÊÎÍÎ̲×Íί IJßË Î̲×Ͳ ÏÐÎÁËÅÌÈ ÐÎÇÂÈÒÊÓ ÃÀËÓÇÅÉ ÒÀ ÂÈIJ ÅÊÎÍÎ̲×Íί IJßËÜÍÎÑÒ Óñòàíîâëåíî, ÷òî âûáîð àëüòåðíàòèâû â õîäå èçáðàíèÿ áèçíåñ–ðåøåíèÿ çàâèñèò îò: âîçìîæ-
íîñòè îöåíèòü ýôôåêòèâíîñòü àëüòåðíàòèâíîãî ðåøåíèÿ è íåîáõîäèìûå ðàñõîäû äëÿ åãî ðåàëè-
çàöèè; ìåòîäîâ, êîòîðûå èñïîëüçîâàëèñü â ïðåäïðèíèìàòåëüñêîé äåÿòåëüíîñòè, à òàêæå ðàçðà-
áîòàííûå äî ñèõ ïîð ìîäåëè èññëåäîâàíèÿ ïðåäïðèíèìàòåëüñêèõ îïåðàöèé; ìåòîäîâ, êîòîðûå
ïîçâîëèëè áû àíàëèçèðîâàòü ñõîæèå ïðîáëåìû. Â ñòàòüå ïðåäñòàâëåíû ïîäõîäû ê îïðåäåëåíèþ ïîíÿòèÿ «àíàëèòèêà áèçíåñ–ðåøåíèé» è òðè
âàæíåéøèõ ñêëàäîâûõ, êîòîðûå îáúåäèíÿåò àíàëèòèêà áèçíåñ ðåøåíèé. Èññëåäîâàíî îñíîâíûå ïðèíöèïû è ñîñòàâëÿþùèå àíàëèçà äàííûõ â áèçíåñ–àíàëèòèêå. Âûâîä. Îðãàíèçàöèÿ ïðîöåññà ïðèíÿòèÿ áèçíåñ–ðåøåíèÿ çàâèñèò îò ïðîåêòèðîâàíèÿ èí-
ôîðìàöèîííûõ òåõíîëîãèé äëÿ áèçíåñ–àíàëèòèêè â ïðåäïðèíèìàòåëüñêîé äåÿòåëüíîñòè è äî-
ëæíà áàçèðîâàòüñÿ íà èíòåãðàöèè èõ â ñèñòåìó óïðàâëåíèÿ áèçíåñ–ïðîöåññàìè ïðåäïðèÿòèÿ è
ñîîòâåòñòâîâàòü áèçíåñ–ìîäåëè êàæäîãî ïðåäïðèÿòèÿ ñ ó÷åòîì âîçìîæíîñòè èõ èíòåãðàöèè â
èìåþùèåñÿ èíôîðìàöèîííûå ñèñòåìû ïðåäïðèÿòèÿ. Êëþ÷åâûå ñëîâà: ïðåäïðèÿòèå, àíàëèç, àíàëèòèêà, áèçíåñ–ðåøåíèÿ, ñóáúåêò õîçÿéñòâîâàíèÿ,
àíàëèòèê. Êëþ÷åâûå ñëîâà: ïðåäïðèÿòèå, àíàëèç, àíàëèòèêà, áèçíåñ–ðåøåíèÿ, ñóáúåêò õîçÿéñòâîâàíèÿ,
àíàëèòèê ñëîâà: ïðåäïðèÿòèå, àíàëèç, àíàëèòèêà, áèçíåñ–ðåøåíèÿ, ñóáúåêò õîçÿéñòâîâàíèÿ, IVANYUTA T.M. 62
´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕÂÄÍÎÑÈͳÊÐÀÍ Àíàëèòèêà áèçíåñ–ðåøåíèé êàê ñîñòàâëÿþùàÿ
ïðåäïðèÿòèÿ: òåîðåòè÷åñêèé àñïåêò Ïðåäìåò èññëåäîâàíèÿ.  ñòàòüå èññëåäîâàí òåîðåòè÷åñêèé àñïåêò àíàëèòèêè áèçíåñ–ðå-
øåíèé êàê äåÿòåëüíîñòè ïðåäïðèÿòèÿ â ñîâðåìåííûõ óñëîâèÿõ õîçÿéñòâîâàíèÿ ñóáúåêòîâ ïðåä-
ïðèíèìàòåëüñòâà. Öåëüþ íàïèñàíèÿ ñòàòüè. Èññëåäîâàíî îñíîâíóþ öåëü áèçíåñ–àíàëèòèêè è óñòàíîâëåíî, ÷òî
àíàëèòèêà áèçíåñ–ðåøåíèé â êðàòêîì ñìûñëå ñëîâà – ýòî àíàëèòè÷åñêàÿ ñîñòàâëÿþùàÿ, êîòî-
ðàÿ ñîïðîâîæäàåòñÿ èñïîëüçîâàíèåì ñîâðåìåííûõ ïîäõîäîâ ïðè àíàëèçå èíôîðìàöèè â îáëàñòè
ïîäãîòîâêè áèçíåñ–ðåøåíèé. Ìåòîäîëîãèÿ ïðîâåäåíèÿ ðàáîòû. Â áîëåå øèðîêîì àñïåêòå, àíàëèòèêà áèçíåñ–ðåøåíèé
– ýòî îáùàÿ ñîâîêóïíîñòü ïðèíöèïîâ ìåòîäîëîãè÷åñêè–îðãàíèçàöèîííîãî, à òàêæå òåõíîëîãè-
÷åñêîãî ðàçíîãî ðîäà óìñòâåííîé äåÿòåëüíîñòè, ÷òî ïîçâîëÿåò ýôôåêòèâíî ïðîâîäèòü îáðàáîòêó
èíôîðìàöèè ñ öåëüþ ïîâûøåíèÿ êà÷åñòâà ñóùåñòâóþùèõ è ïîëó÷åíèÿ íîâûõ çíàíèé, à òàêæå ïî-
äãîòîâêè èíôîðìàöèîííî–èíòåëëåêòóàëüíîé áàçû äëÿ ïðèíÿòèÿ ðàöèîíàëüíûõ óïðàâëåí÷åñêèõ
áèçíåñ–ðåøåíèé â ïðåäïðèíèìàòåëüñêîé äåÿòåëüíîñòè ñóáúåêòîâ õîçÿéñòâîâàíèÿ. Ðåçóëüòàòû ðàáîòû. Ðàññìîòðåíû îñíîâíûå ïðîöåññû, êîòîðûå îïðåäåëÿþò àíàëèòèêó áèç-
íåñ–ðåøåíèé êàê ñïåöèôè÷åñêèé âèä äåÿòåëüíîñòè íà ïðåäïðèÿòèè. © ²ÂÀÍÞÒÀ Ò.Ì., 2021 61 ´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕÂÄÍÎÑÈͳÊÐÀÍ Business decision analysis as a component
of enterprise activity: theoretical aspect The subject of research. The article examines the theoretical aspect of business decision analysis
as a component of the enterprise in modern business conditions of business entities. The purpose of writing. The main purpose of business analytics is studied and it is established
that business decision analytics in the short sense of the word is an analytical component, which is
accompanied by the use of modern approaches in the analysis of information in the field of business
decision preparation. Methodology of work In a broader sense, business decision analytics is a general set of principles
of methodological and organizational, as well as technological various kinds of mental activity, which
allows you to effectively process information to improve the quality of existing and new knowledge, as
well as preparing information and intellectual base for adoption rational managerial business decisions
in business activities of business entities. The results of the work. The main processes that define the analysis of business decisions as a
specific type of activity in the enterprise are considered. It is established that the choice of alternatives during the selection of a business solution depends
on: the ability to assess the effectiveness of the alternative solution and the necessary costs for its
implementation; methods used in business activities, as well as models developed so far to study
business operations; methods that would analyze similar problems. The article presents approaches to the introduction of the concept of «business decision analyst»
and the three most important components that unite business decision analytics. Researched the basic principles and components of data analysis in business intelligence. Conclusions. We concluded that the organization of the business decision–making process depends
on the design of information technology for business intelligence in business and should be based on their
integration into the business process management system of the enterprise and meet the business model
of each enterprise taking into account their integration into existing information enterprise systems. Keywords: enterprise, analysis, analytics, business decisions, business entity, analyst. ³íôîðìàö³éíèõ ïîòîê³â, ÿê³ äîçâîëÿþòü, â çà-
ëåæíîñò³ â³ä ¿õ ïîâíîòè, ïðèéìàòè åêîíîì³÷í³ òà
óïðàâë³íñüê³ á³çíåñ–ð³øåííÿ. Ïîñòàíîâêà ïðîáëåìè. ijÿëüí³ñòü ï³äïðè-
ºìñòâ â ðèíêîâèõ êîíêóðåíòíèõ óìîâàõ ãîñïî-
äàðþâàííÿ çàëåæèòü â³ä âíóòð³øí³õ òà çîâí³øí³õ 62
´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕÂÄÍÎÑÈͳÊÐÀÍ Î̲×Ͳ ÏÐÎÁËÅÌÈ ÐÎÇÂÈÒÊÓ ÃÀËÓÇÅÉ ÒÀ ÂÈIJ ÅÊÎÍÎ̲×Íί IJßËÜÍÎÑÒ Ïðèéíÿòòÿ á³çíåñ–ð³øåííÿ áóäå çàëåæàòè â³ä
ÿêîñò³, ñâîº÷àñíîñò³ íàäõîäæåííÿ ïîòîêó ³íôîð-
ìàö³¿ é ñïåö³àë³ñòà, ÿêèé ïðîâîäèòü á³çíåñ–àíà-
ë³ç, ç ìåòîþ çàäîâîëåííÿ ïîòðåá ï³äïðèºìñòâà
(åêîíîì³÷íîãî ñóá’ºêòà). Business decision analysis as a component
of enterprise activity: theoretical aspect øåíü ñïðè÷èíþþòü íåìîæëèâ³ñòü åôåêòèâíîãî
óïðàâë³ííÿ ï³äïðèºìñòâîì áåç âèêîðèñòàííÿ ñó-
÷àñíèõ ³íôîðìàö³éíèõ òåõíîëîã³é, çîêðåìà, ìî-
äåëåé, ìåòîä³â ³ çàñîá³â á³çíåñ–àíàë³òèêè. Íà ñüîãîäí³, åêîíîì³÷í³é ä³ÿëüíîñò³ ï³äïðè-
ºìñòâ ïðèòàìàíí³ òàê³ îçíàêè, ÿê: êðèòè÷íèé ð³-
âåíü êîíêóðåíö³¿ íà ðèíêó ³ ¿õ ÷óòëèâ³ñòü íà âïëèâ
åêîíîì³÷íèõ ôàêòîð³â ³ ÷èííèê³â. Â÷åíèìè âñòàíîâëåíî, ùî á³çíåñ–àíàë³ç º íà-
áîðîì çàâäàíü òà ìåòîäèê, ùî âèêîðèñòîâóºòü-
ñÿ ç ö³ëëþ çàáåçïå÷èòè ðîçóì³ííÿ çàö³êàâëåíèìè
ñòîðîíàìè ñòðóêòóðè, ïðàâèë òà ðîáîòè îðãàí³-
çàö³¿ é çàïðîïîíóâàòè ð³øåííÿ, ÿê³ äîçâîëÿòü îð-
ãàí³çàö³¿ äîñÿãòè ïîñòàâëåíèõ ö³ëåé.  ê³íöåâîìó ðåçóëüòàò³ – íåñòàá³ëüí³ñòü ñòàíó
íà ðèíêó, ÿêà âèìàãຠîïåðàòèâíèõ ³ âèñîêîÿê³ñ-
íèõ ð³øåíü ìåíåäæåð³â íà âñ³õ ð³âíÿõ óïðàâë³ííÿ
ï³äïðèºìñòâîì. Òîìó ïèòàííÿ àíàë³òèêè á³çíåñ–ð³øåíü ÿê
ñêëàäîâî¿ ä³ÿëüíîñò³ ï³äïðèºìñòâà º àêòóàëüíè-
ìè ³ ïîòðåáóþòü óâàãè ç áîêó â÷åíèõ. Îñíîâîþ äëÿ ïðèéíÿòòÿ óïðàâë³íñüêèõ á³ç-
íåñ– ð³øåíü º ïîâíà ³íôîðìàö³ÿ ïðî ñòàí á³çíå-
ñó ³ òåíäåíö³é ðîçâèòêó ðèíêó, à êåðóâàííÿ òàêîþ
³íôîðìàö³ºþ – ñêëàäíèé ïðîöåñ, ùî âèìàãàº
êîìïëåêñíîãî ï³äõîäó. Äîñë³äæóâàíà òåìà íàóêîâî¿ ñòàòò³ âàðòà óâà-
ãè á³çíåñ–àíàë³òèê³â, íàóêîâö³â òà ôàõîâèõ ñïå-
ö³àë³ñò³â. Îñíîâíà ìåòà àíàë³òèêè á³çíåñ–ð³øåíü – öå
îð³ºíòàö³ÿ óïðàâë³íñüêîãî ïðîöåñó íà äîñÿãíåí-
íÿ ö³ëåé, ùî ñòîÿòü ïåðåä ñóá’ºêòîì ãîñïîäà-
ðþâàííÿ (ï³äïðèºìñòâîì, îðãàí³çàö³ºþ, ô³ðìîþ
òà ³í.), çà ðàõóíîê çì³ùåííÿ àêöåíòó ç ä³àãíîñ-
òèêè ìèíóëèõ ðåòðîñïåêòèâ íà ïðîãíîç ìàéáóò-
í³õ ïåðñïåêòèâ, â³äñòåæåííÿ 䳿 âïëèâó âíóòð³øí³õ
³ çîâí³øí³õ ôàêòîð³â íà ä³ÿëüí³ñòü ï³äïðèºìñòâà
ç ìåòîþ ïðèéíÿòòÿ ðàö³îíàëüíèõ á³çíåñ–ð³øåíü,
³íôîðìàö³éíî¿ òà êîíñóëüòàö³éíî¿ ï³äòðèìêè ïðè-
éíÿòòÿ óïðàâë³íñüêèõ á³çíåñ–ð³øåíü [2]. Àíàë³ç îñòàíí³õ äîñë³äæåíü òà ïóáë³êàö³é. Îñíîâíîþ ì³ñ³ºþ äëÿ åêîíîì³êè êîæíî¿ êðà¿íè º çà-
áåçïå÷åííÿ âèïåðåäæàþ÷èõ òåìï³â åêîíîì³÷íîãî
çðîñòàííÿ ï³äïðèºìñòâ, ùî äîñÿãàºòüñÿ â õîä³ ïðè-
éíÿòòÿ åôåêòèâíèõ á³çíåñ–ð³øåíü, ÿê³ ìîæóòü áóòè
ïðèéíÿò³ â õîä³ ïðàâèëüíîãî ï³äáîðó ³íôîðìàö³¿ òà
îáðàííÿ àëüòåðíàòèâíîãî ð³øåííÿ, ùî çàëåæèòü â
ï³äïðèºìíèöòâ³ é â³ä òåõíîëîã³÷íèõ ³ííîâàö³é. Ïî÷èíàþ÷è ç 1980 ðîêó â çàõ³äí³é á³çíåñ–
ïðàêòèö³ ç’ÿâèëîñÿ ïîíÿòòÿ á³çíåñ àíàë³òèêè
(Business Analytics), ÿêå é íèí³ ïîâí³ñòþ îòîòîæ-
íþºòüñÿ äåÿêèìè íàóêîâöÿìè ç á³çíåñ àíàë³çîì. Ïî÷èíàþ÷è ç 1980 ðîêó â çàõ³äí³é á³çíåñ–
ïðàêòèö³ ç’ÿâèëîñÿ ïîíÿòòÿ á³çíåñ àíàë³òèêè
(Business Analytics), ÿêå é íèí³ ïîâí³ñòþ îòîòîæ-
íþºòüñÿ äåÿêèìè íàóêîâöÿìè ç á³çíåñ àíàë³çîì. Äî òåïåð³øíüîãî ÷àñó öå ïîíÿòòÿ îòðèìàëî äå-
ÿêå òåîðåòè÷íå îá´ðóíòóâàííÿ, ùî çàñâ³ä÷óº âå-
ëèêà ê³ëüê³ñòü íàóêîâèõ ïðàöü ³íîçåìíèõ â÷åíèõ ³
ïðàêòèê³â ç ö³º¿ ïðîáëåìàòèêè [1–5]. Àíàë³ç (ãðåö. ´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕÂÄÍÎÑÈͳÊÐÀÍ Business decision analysis as a component
of enterprise activity: theoretical aspect – ðîçïàä, ðîç÷ëåíóâàííÿ, ðîçá³ð)
– öå ëîã³÷íèé ïðèéîì, ìåòîä äîñë³äæåííÿ, ÿêèé
ïîëÿãຠâ òîìó, ùî äîñë³äæóâàíèé ïðåäìåò ïî-
äóìêè ðîçä³ëÿºòüñÿ íà ñêëàäîâ³ åëåìåíòè, ÿê³ ïî-
ò³ì äîñë³äæóþòüñÿ îêðåìî ÿê ÷àñòèíà ðîçïîä³ëå-
íîãî ö³ëîãî, äëÿ òîãî, ùîá âèä³ëåí³ â õîä³ àíàë³çó
åëåìåíòè ç’ºäíàëèñÿ ç äîïîìîãîþ ³íøîãî ëîã³÷-
íîãî ïðèéîìó – ñèíòåçó – â ö³ëå, çáàãà÷åíå íî-
âèìè çíàííÿìè [3]. Äî òåïåð³øíüîãî ÷àñó öå ïîíÿòòÿ îòðèìàëî äå-
ÿêå òåîðåòè÷íå îá´ðóíòóâàííÿ, ùî çàñâ³ä÷óº âå-
ëèêà ê³ëüê³ñòü íàóêîâèõ ïðàöü ³íîçåìíèõ â÷åíèõ ³
ïðàêòèê³â ç ö³º¿ ïðîáëåìàòèêè [1–5]. Äî â³äîìèõ ïðåäñòàâíèê³â åêîíîì³÷íî¿ äóìêè,
ÿê³ â ö³ëîìó äîñë³äæóâàëè òåìó ïðèñâÿ÷åíó àíà-
ë³òèö³ á³çíåñ–ð³øåíü â åêîíîì³ö³ ìîæíà â³äíåñòè
Áàðèëåíêî Â.²., Áîíäàðåíêî Â.Ã., Êîíðàä Êàðë-
áåðãà, Ëÿøåíêî Î., Áàá³ÿ Ñ. Â., Øòåôàíà Á [1–5]. Ìåòà ñòàòò³. Äîñë³äèòè òåîðåòè÷íèé àñïåêò
àíàë³òèêè á³çíåñ–ð³øåíü ÿê ñêëàäîâî¿ ä³ÿëüíîñò³
ï³äïðèºìñòâà â ñó÷àñíèõ óìîâàõ ãîñïîäàðþâàí-
íÿ ñóá’ºêò³â ï³äïðèºìíèöòâà. Äî â³äîìèõ ïðåäñòàâíèê³â åêîíîì³÷íî¿ äóìêè,
ÿê³ â ö³ëîìó äîñë³äæóâàëè òåìó ïðèñâÿ÷åíó àíà-
ë³òèö³ á³çíåñ–ð³øåíü â åêîíîì³ö³ ìîæíà â³äíåñòè
Áàðèëåíêî Â.²., Áîíäàðåíêî Â.Ã., Êîíðàä Êàðë-
áåðãà, Ëÿøåíêî Î., Áàá³ÿ Ñ. Â., Øòåôàíà Á [1–5]. Òîìó àíàë³òèêà – öå êîìïëåêñíà íàóêîâà ñêëà-
äîâà, â ÿê³é ìîæíà âèêîðèñòàòè ïðèíöèïîâî íîâ³,
óð³çíîìàí³òíåíí³ ï³äõîäè ïðè àíàë³ç³ ³íôîðìàö³¿ â
êîíòåêñò³ ï³äãîòîâêè óïðàâë³íñüêèõ ð³øåíü. Ìåòà ñòàòò³. Äîñë³äèòè òåîðåòè÷íèé àñïåêò
àíàë³òèêè á³çíåñ–ð³øåíü ÿê ñêëàäîâî¿ ä³ÿëüíîñò³
ï³äïðèºìñòâà â ñó÷àñíèõ óìîâàõ ãîñïîäàðþâàí-
íÿ ñóá’ºêò³â ï³äïðèºìíèöòâà. Àíàë³òèêà á³çíåñ–ð³øåíü – öå àíàë³òè÷íà ñêëà-
äîâà, ùî ñóïðîâîäæóºòüñÿ âèêîðèñòàííÿì ñó-
÷àñíèõ ï³äõîä³â ï³ä ÷àñ àíàë³çó ³íôîðìàö³¿ â îá-
ëàñò³ ï³äãîòîâêè á³çíåñ–ð³øåíü. Âèêëàä îñíîâíîãî ìàòåð³àëó. Ôóíêö³îíóâàí-
íÿ ñó÷àñíèõ ï³äïðèºìñòâ ð³çíèõ ãàëóçåé åêîíîì³êè
ó ðåàëüíèõ óìîâàõ õàðàêòåðíà ïîñò³éíîþ áîðîòü-
áîþ ç íàðîñòàþ÷îþ êîíêóðåíö³ºþ, ÿêà ïîñò³éíî
âèìàãຠâ³ä óïðàâë³íö³â ï³äâèùåííÿ îïåðàòèâíî-
ñò³ ³ ÿêîñò³ ïðèéíÿòòÿ óïðàâë³íñüêèõ ð³øåíü. Ìîæíà çàçíà÷èòè, ùî ³ñíóþòü îñíîâí³ ïðîöåñè,
ÿê³ âèçíà÷àþòü àíàë³òèêó á³çíåñ–ð³øåíü ÿê ñïåöè-
ô³÷íèé âèä ä³ÿëüíîñò³ íà ï³äïðèºìñòâ³, ñåðåä ÿêèõ: Âåëèê³ îáñÿãè ðèíêîâî¿ ³íôîðìàö³¿, ÿêó íåîáõ³ä-
íî ìîí³òîðèòè â õîä³ ïðèéíÿòòÿ óïðàâë³íñüêèõ ð³- 1. Àíàë³ç ö³ëåé óïðàâë³ííÿ ³ îá´ðóíòóâàííÿ çà-
âäàíü ³íôîðìàö³éí–àíàë³òè÷íî¿ ðîáîòè. 63 ´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕÂÄÍÎÑÈͳÊÐÀÍ ²×Ͳ ÏÐÎÁËÅÌÈ ÐÎÇÂÈÒÊÓ ÃÀËÓÇÅÉ ÒÀ ÂÈIJ ÅÊÎÍÎ̲×Íί IJßËÜÍÎÑÒ² 2. Óïðàâë³ííÿ çáîðîì ³íôîðìàö³¿ ³ âèçíà÷åííÿ
çíàíü äëÿ âèð³øåííÿ óïðàâë³íñüêèõ á³çíåñ–çà-
äà÷ â óìîâàõ ì³íëèâî¿ åêîíîì³÷íî¿ ñèòóàö³¿. åôåêòèâíî ïðîâîäèòè îáðîáêó ³íôîðìàö³¿ ç ö³ë-
ëþ ï³äâèùåííÿ ÿêîñò³ íàÿâíèõ ³ îòðèìàííÿ íî-
âèõ çíàíü, à òàêîæ ï³äãîòîâêè ³íôîðìàö³éíî–³í-
òåëåêòóàëüíî¿ áàçè äëÿ ïðèéíÿòòÿ ðàö³îíàëüíèõ
óïðàâë³íñüêèõ á³çíåñ–ð³øåíü â ï³äïðèºìíèöüê³é
ä³ÿëüíîñò³ ñóá’ºêò³â ãîñïîäàðþâàííÿ. 3. Àíàë³ç ³ îö³íêà îòðèìàíî¿ ³íôîðìàö³¿ ³ çíàíü
â êîíòåêñò³ çàâäàíü óïðàâë³ííÿ, âèÿâëåííÿ ñóò³
ñïîñòåðåæóâàíèõ ïðîöåñ³â ³ ÿâèù. 4. Business decision analysis as a component
of enterprise activity: theoretical aspect Ïîáóäîâà ìîäåëåé ïðåäìåòíî¿ ³ ïðîáëåìíî¿
îáëàñòåé, îá’ºêòà ñåðåäîâèùà éîãî ôóíêö³îíóâàí-
íÿ, ïåðåâ³ðêà àäåêâàòíîñò³ ìîäåë³ ³ ¿¿ êîîðäèíàö³ÿ. 4. Ïîáóäîâà ìîäåëåé ïðåäìåòíî¿ ³ ïðîáëåìíî¿
îáëàñòåé, îá’ºêòà ñåðåäîâèùà éîãî ôóíêö³îíóâàí-
íÿ, ïåðåâ³ðêà àäåêâàòíîñò³ ìîäåë³ ³ ¿¿ êîîðäèíàö³ÿ. Íàéãîëîâí³øèì ïðè âèð³øåíí³ çàâäàíü àíàë³-
òèêè á³çíåñ–ð³øåíü, íåçàëåæíî â³ä ñôåðè ¿¿ çà-
ñòîñóâàííÿ, çì³ñòó ³ õàðàêòåðó, º âèðîáëåííÿ
íàéêðàùîãî óïðàâë³íñüêîãî á³çíåñ–ð³øåííÿ. ßê
íàñë³äîê, âèá³ð àëüòåðíàòèâè â õîä³ îáðàííÿ á³ç-
íåñ–ð³øåííÿ çàëåæèòü â³ä: 5. Ïëàíóâàííÿ ³ ïðîâåäåííÿ ìîäåëüíèõ åêñïå-
ðèìåíò³â. 6. Ñèíòåç íîâèõ çíàíü, íåîáõ³äíèõ äëÿ âèð³øåíü
óïðàâë³íñüêèõ á³çíåñ–çàäà÷. • ìîæëèâîñò³ îö³íèòè åôåêòèâí³ñòü àëüòåðíàòèâ-
íîãî ð³øåííÿ ³ íåîáõ³äí³ âèòðàòè äëÿ éîãî ðåàë³çàö³¿;
• ìåòîä³â, ÿê³ âèêîðèñòîâóâàëèñÿ â ï³äïðèºì-
íèöüê³é ä³ÿëüíîñò³, à òàêîæ ðîçðîáëåí³ äî öüîãî ÷à-
ñó ìîäåë³ äîñë³äæåííÿ ï³äïðèºìíèöüêèõ îïåðàö³é;
• ìåòîä³â, ÿê³ äîçâîëèëè á àíàë³çóâàòè ñõî-
æ³ ïðîáëåìè, ÿê îäíå ö³ëå, ùî çàáåçïå÷óâàëè á
ïåðåãëÿä âåëèêî¿ ê³ëüêîñò³ àëüòåðíàòèâ, êîæíà ç
ÿêèé áóëà á îïèñàíà âåëèêîþ ê³ëüê³ñòþ çì³ííèõ, Àíàë³òèêà á³çíåñ–ð³øåíü ïîâ’ÿçàíà ç ³íòåëåê-
òóàëüíî–òåõíîëîã³÷íîþ ä³ÿëüí³ñòþ, ÿêà ñïðÿìî-
âàíà íà âèð³øåííÿ çàâäàíü óïðàâë³ííÿ ³ ñèíòåçó
íîâèõ çíàíü. Business decision analysis as a component
of enterprise activity: theoretical aspect  øèðøîìó àñïåêò³, àíàë³òèêà á³çíåñ–ð³øåíü
– öå çàãàëüíà ñóêóïí³ñòü ïðèíöèï³â ìåòîäîëî-
ã³÷íî–îðãàí³çàö³éíîãî, à òàêîæ òåõíîëîã³÷íîãî
ð³çíîãî ðîäó ðîçóìîâî¿ ä³ÿëüíîñò³, ùî äîçâîëÿº 64
´ÎÐÌÓÂÀÍÍß ÐÈÍÊÎÂÈÕ ÂÄÍÎÑÈÍ Â ³ÊÐÀÍ
¹
Ñóòí³ñòü ï³äõîäó
1
Ìåòîäîëîã³÷íà îñíîâà ïðîöåñó îáðîáêè ³íôîðìàö³¿
2
Ìåòîäîëîã³ÿ ï³çíàííÿ, ùî âèêîðèñòîâóº äëÿ îòðèìàííÿ íîâîãî çíàííÿ ÿê ñòðîãî íàóêîâ³, òàê ³
³íòó¿òèâí³ ìåòîäè
3
Ôîðìà ìèñëåííÿ ³ ñâ³òîâ³ä÷óòòÿ, ùî ñïèðàºòüñÿ íà íàóêîâèé ï³äõ³ä
4
Ñóòí³ñíå çíàííÿ ïðî ïðîöåñè ðåàëüíîãî ñâ³òó
5
Çàñ³á ïåðåòâîðåííÿ ³íòó¿òèâíèõ óÿâëåíü ó ëîã³÷íèé, ðàö³îíàëüíèé ïëàí ìèñëåííÿ
6
Ôîðìà íàóêîâîãî çíàííÿ, ùî çàñòîñîâóºòüñÿ â ïðîöåñàõ óïðàâë³ííÿ, íàñàìïåðåä – äëÿ âèðîáëåííÿ
óïðàâë³íñüêèõ ð³øåíü
7
Ñóêóïí³ñòü ìåòîä³â, çà äîïîìîãîþ ÿêèõ ìîæíà âèÿâëÿòè ïðèõîâàí³ ñìèñëè ó òåêñòàõ ³ ðåàëüíèõ
ñîö³àëüíî–ïîë³òè÷íèõ òà åêîíîì³÷íèõ ïðîöåñàõ
8
ßäðî íàóêîâî–äîñë³äíî¿ ðîáîòè
9
Ïîòóæíèé ïëàñò ³íòåëåêòóàëüíî¿ êóëüòóðè, ùî âèêîðèñòîâóºòüñÿ åë³òîþ áóäü–ÿêîãî ñóñï³ëüñòâà äëÿ
óïðàâë³ííÿ ñîö³óìîì
10 Êîðîòêà íàçâà ³íôîðìàö³éíî–àíàë³òè÷íî¿ ðîáîòè
11 Ñèíîí³ì ä³àëåêòè÷íî¿ ëîã³êè
12 Ñèíîí³ì ñèñòåìíîãî àíàë³çó
13 Ïðîöåñ óçàãàëüíåííÿ òà àíàë³çó ðîçð³çíåíèõ, íåïîâíèõ ³ ÷àñòî ñóïåðå÷ëèâèõ äàíèõ ïðî îá’ºêò
äîñë³äæåííÿ
14 Ïðîöåñ âèÿâëåííÿ ïðè÷èííî–íàñë³äêîâèõ çàëåæíîñòåé ³ ïðîñòîðîâî÷àñîâèõ çâ’ÿçê³â ó áóäü–ÿêèõ
îá’ºêòàõ
15 Ïðîöåñ ñèñòåìàòèçàö³¿ çì³ñòó çà äîïîìîãîþ ñõåìàòèçàö³¿, êîíñòðóþâàííÿ òà ìîäåëþâàííÿ
ñóòí³ñíèõ åëåìåíò³â ³ çâ’ÿçê³â
16 Ïðîöåñ ïîä³ëó îá’ºêòà íà ñêëàäîâ³ ÷àñòèíè ³ ïîäàëüøîãî ¿õíüîãî îá’ºäíàííÿ ó ïåâíó ñèñòåìó
17 Ïðîöåñ âèÿâëåííÿ ïðîòèð³÷ â îá’ºêò³ ï³çíàííÿ, çâåäåííÿ ñêëàäíîãî äî ïðîñòîãî
18 Ïðèíöèï êîíñòðóêòèâíîãî ñïðîùåííÿ äëÿ âèÿâëåííÿ ôîðì âçàºìî䳿 åëåìåíò³â ö³ëîãî ³ ðîçêðèòòÿ
âíóòð³øíüî¿ ñòðóêòóðè áóäü–ÿêîãî îá’ºêòà äîñë³äæåííÿ
19 Ñâîºð³äí³ çàñîáè äëÿ âèâ÷åííÿ ñóò³ ÿâèù, ïðåäìåò³â ³ ïðîöåñ³â ðåàëüíîñò³, ÿäðî áóäü–ÿêî¿ äîñë³äíî¿
ïðîãðàìè
Äæåðåëî: ñêëàäåíî àâòîðîì íà îñíîâ³ äæåðåë 1–5]. Business decision analysis as a component
of enterprise activity: theoretical aspect ϳäõîäè äî âèçíà÷åííÿ ïîíÿòòÿ «àíàë³òèêà á³çíåñ–ð³øåíü» 64
´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕÂÄÍÎÑÈͳÊÐÀÍ
¹
Ñóòí³ñòü ï³äõîäó
1
Ìåòîäîëîã³÷íà îñíîâà ïðîöåñó îáðîáêè ³íôîðìàö³¿
2
Ìåòîäîëîã³ÿ ï³çíàííÿ, ùî âèêîðèñòîâóº äëÿ îòðèìàííÿ íîâîãî çíàííÿ ÿê ñòðîãî íàóêîâ³, òàê ³
³íòó¿òèâí³ ìåòîäè
3
Ôîðìà ìèñëåííÿ ³ ñâ³òîâ³ä÷óòòÿ, ùî ñïèðàºòüñÿ íà íàóêîâèé ï³äõ³ä
4
Ñóòí³ñíå çíàííÿ ïðî ïðîöåñè ðåàëüíîãî ñâ³òó
5
Çàñ³á ïåðåòâîðåííÿ ³íòó¿òèâíèõ óÿâëåíü ó ëîã³÷íèé, ðàö³îíàëüíèé ïëàí ìèñëåííÿ
6
Ôîðìà íàóêîâîãî çíàííÿ, ùî çàñòîñîâóºòüñÿ â ïðîöåñàõ óïðàâë³ííÿ, íàñàìïåðåä – äëÿ âèðîáëåííÿ
óïðàâë³íñüêèõ ð³øåíü
7
Ñóêóïí³ñòü ìåòîä³â, çà äîïîìîãîþ ÿêèõ ìîæíà âèÿâëÿòè ïðèõîâàí³ ñìèñëè ó òåêñòàõ ³ ðåàëüíèõ
ñîö³àëüíî–ïîë³òè÷íèõ òà åêîíîì³÷íèõ ïðîöåñàõ
8
ßäðî íàóêîâî–äîñë³äíî¿ ðîáîòè
9
Ïîòóæíèé ïëàñò ³íòåëåêòóàëüíî¿ êóëüòóðè, ùî âèêîðèñòîâóºòüñÿ åë³òîþ áóäü–ÿêîãî ñóñï³ëüñòâà äëÿ
óïðàâë³ííÿ ñîö³óìîì
10 Êîðîòêà íàçâà ³íôîðìàö³éíî–àíàë³òè÷íî¿ ðîáîòè
11 Ñèíîí³ì ä³àëåêòè÷íî¿ ëîã³êè
12 Ñèíîí³ì ñèñòåìíîãî àíàë³çó
13 Ïðîöåñ óçàãàëüíåííÿ òà àíàë³çó ðîçð³çíåíèõ, íåïîâíèõ ³ ÷àñòî ñóïåðå÷ëèâèõ äàíèõ ïðî îá’ºêò
äîñë³äæåííÿ
14 Ïðîöåñ âèÿâëåííÿ ïðè÷èííî–íàñë³äêîâèõ çàëåæíîñòåé ³ ïðîñòîðîâî÷àñîâèõ çâ’ÿçê³â ó áóäü–ÿêèõ
îá’ºêòàõ
15 Ïðîöåñ ñèñòåìàòèçàö³¿ çì³ñòó çà äîïîìîãîþ ñõåìàòèçàö³¿, êîíñòðóþâàííÿ òà ìîäåëþâàííÿ
ñóòí³ñíèõ åëåìåíò³â ³ çâ’ÿçê³â
16 Ïðîöåñ ïîä³ëó îá’ºêòà íà ñêëàäîâ³ ÷àñòèíè ³ ïîäàëüøîãî ¿õíüîãî îá’ºäíàííÿ ó ïåâíó ñèñòåìó
17 Ïðîöåñ âèÿâëåííÿ ïðîòèð³÷ â îá’ºêò³ ï³çíàííÿ, çâåäåííÿ ñêëàäíîãî äî ïðîñòîãî
18 Ïðèíöèï êîíñòðóêòèâíîãî ñïðîùåííÿ äëÿ âèÿâëåííÿ ôîðì âçàºìî䳿 åëåìåíò³â ö³ëîãî ³ ðîçêðèòòÿ
âíóòð³øíüî¿ ñòðóêòóðè áóäü–ÿêîãî îá’ºêòà äîñë³äæåííÿ
19 Ñâîºð³äí³ çàñîáè äëÿ âèâ÷åííÿ ñóò³ ÿâèù, ïðåäìåò³â ³ ïðîöåñ³â ðåàëüíîñò³, ÿäðî áóäü–ÿêî¿ äîñë³äíî¿
ïðîãðàìè
Äæåðåëî: ñêëàäåíî àâòîðîì íà îñíîâ³ äæåðåë 1–5]. ϳäõîäè äî âèçíà÷åííÿ ïîíÿòòÿ «àíàë³òèêà á³çíåñ–ð³øåíü» 64 Î̲×Ͳ ÏÐÎÁËÅÌÈ ÐÎÇÂÈÒÊÓ ÃÀËÓÇÅÉ ÒÀ ÂÈIJ ÅÊÎÍÎ̲×Íί IJßËÜÍÎÑÒ Äîñë³äèìî îñíîâí³ ïðèíöèïè òà ñêëàäîâ³ àíàë³-
çó äàíèõ â á³çíåñ–àíàë³òèö³: ÿê³ äàâàëè á ïîâíîòó êîæíî¿ àëüòåðíàòèâè òà áóëè
âèì³ðþâàíèìè, à òàêîæ äàâàëè ìîæëèâ³ñòü ÿê³ñ-
íî â³äîáðàæàòè íåâèçíà÷åí³ñòü ³ ðèçèêîâàí³ñòü
îáðàíî¿ àëüòåðíàòèâè. Ïðîöåñ àíàë³çó (â ³íôîðìàö³éíîìó ï³äõîä³ äî
àíàë³çó äàíèõ íåîáõ³äíèì º íàÿâí³ñòü òðüîõ êëþ-
÷îâèõ ñêëàäîâèõ: åêñïåðò, ã³ïîòåçà ³ àíàë³òèê). Äîñë³äæåííÿ òà çãðóïóâàííÿ ð³çíèõ òî÷îê çîðó,
ùîäî àíàë³çó ³íôîðìàö³¿, ÿêà ïðèçâîäèòü äî áåç-
ë³÷³ ï³äõîä³â äî âèçíà÷åííÿ ïîíÿòòÿ «àíàë³òèêè
á³çíåñ–ð³øåíü», ïðåäñòàâëåíî â òàáëèö³. Âèá³ð ³ â³çóàë³çàö³ÿ äàíèõ (÷àñòî, ùîá îòðèìà-
òè íîâ³ çíàííÿ ïðî ÿêèéñü îá’ºêò ÷è ÿâèùå, çîâ-
ñ³ì íå îáîâ’ÿçêîâà ïîáóäîâà ñêëàäíèõ ìîäåëåé). Ìîäåëþâàííÿ (ïðîöåñ ïîáóäîâè ìîäåëåé â á³ç-
íåñ–àíàë³òèö³ ñêëàäàºòüñÿ ç ïåâíèõ ïîñë³äîâíèõ
åòàï³â). Çàëåæíî â³ä ñïåöèô³êè ñâî¿õ äîñë³äæåíü, ð³çí³
çàðóá³æí³ òà â³÷òèçíÿí³ â÷åí³ ³ ïðàêòèêè âêëàäàëè â
ñâîº ðîçóì³ííÿ àíàë³òè÷íî¿ ä³ÿëüíîñò³ ð³çíèé çì³ñò. Òàê, ÷àñòèíà öèõ âèçíà÷åíü â³äíîñèòüñÿ íå äî
ñóò³, à äî ôóíêö³é àíàë³òèêè á³çíåñ–ð³øåíü òà
ôîðì ¿¿ ïðîÿâó. Âèñíîâîê Âèõîäÿ÷è ç âèùå âèêëàäåíîãî, ìîæíà ñïî-
ñòåð³ãàòè, ùî ³íòåãðàö³ÿ ³íôîðìàö³éíèõ çàñîá³â
â çàãàëüíó ³íôîðìàö³éíó ñèñòåìó ï³äïðèºìñòâà
òà ³íôîðìàö³éíèõ òåõíîëîã³é, äàñòü ìîæëèâ³ñòü
ïðîâîäèòè àíàë³òèêó åêîíîì³÷î¿ ³íôîðìàö³¿ òà
äîçâîëèòü ïðèéìàòè åôåêòèâí³ á³çíåñ–ð³øåííÿ
é øâèäêî âèçíà÷èòèñÿ ³ç àëüòåðíàòèâîþ. Á³çíåñ–àíàë³òèêà «íàãîëîøóº» íà òîìó, ùî ïðîöåñ
îðãàí³çàö³¿ ³ àíàë³çó âæå ³ñíóþ÷èõ äàíèõ äëÿ ïðèéíÿò-
òÿ åôåêòèâíèõ á³çíåñ–ð³øåíü º îäíèì ç íàéâàæ÷èõ ³
îñíîâíèõ çàâäàíü ìåíåäæìåíòó (óïðàâë³ííÿ). Á³çíåñ–àíàë³òèêà «íàãîëîøóº» íà òîìó, ùî ïðîöåñ
îðãàí³çàö³¿ ³ àíàë³çó âæå ³ñíóþ÷èõ äàíèõ äëÿ ïðèéíÿò-
òÿ åôåêòèâíèõ á³çíåñ–ð³øåíü º îäíèì ç íàéâàæ÷èõ ³
îñíîâíèõ çàâäàíü ìåíåäæìåíòó (óïðàâë³ííÿ). Ïîñò³éíå óäîñêîíàëåííÿ ³íòåëåêòóàëüíîãî àïà-
ðàòó ñó÷àñíîãî óïðàâë³ííÿ ñòຠïåðøî÷åðãîâîþ
ïðîáëåìîþ òåî𳿠³ ïðàêòèêè óïðàâë³ííÿ ï³äïðè-
ºìñòâîì. Îðãàí³çàö³ÿ ïðîöåñó ïðèéíÿòòÿ á³çíåñ–ð³øåííÿ
çíà÷íî çàëåæèòü â³ä çàñòîñóâàííÿ ³íôîðìàö³éíèõ
òåõíîëîã³é äëÿ á³çíåñ–àíàë³òèêè â ï³äïðèºìíèöü-
ê³é ä³ÿëüíîñò³ òà ìຠáàçóâàòèñÿ íà ³íòåãðàö³¿ ¿õ ó
ñèñòåìó óïðàâë³ííÿ á³çíåñ–ïðîöåñàìè ï³äïðèºì-
ñòâà òà â³äïîâ³äàòè á³çíåñ–ìîäåë³ êîæíîãî ï³äïðè-
ºìñòâà ç óðàõóâàííÿì ìîæëèâîñò³ ¿õ ³íòåãðóâàííÿ ó
íàÿâí³ ³íôîðìàö³éí³ ñèñòåìè ï³äïðèºìñòâà. Êåð³âíèêè, ÿê³ óñâ³äîìëþþòü, ùî ñòðàòåã³÷í³
àñïåêòè óïðàâë³ííÿ ñó÷àñíèì àâòîìàòèçîâàíèì
ï³äïðèºìñòâîì, ³íòåãðîâàíèì ç çîâí³øí³ì ñå-
ðåäîâèùåì, ïîòðåáóþòü ³íøî¿ òåîðåòè÷íî¿ áàçè
â ïîð³âíÿíí³ ç, íàïðèêëàä, ïðèíöèïàìè óïðàâë³í-
ñüêîãî ðàö³îíàë³çìó, òà çìîæóòü çíàéòè äîäàòêî-
â³ ìîæëèâîñò³ âäîñêîíàëåííÿ âëàñíî¿ ä³ÿëüíîñò³
â êîíöåïö³¿ âñåçàãàëüíî¿ åêîíîì³÷íî¿ íàóêè [4]. Âàãîìîþ º ³íòåãðàö³ÿ á³çíåñ–àíàë³òèêè ç ñòðà-
òåã³÷íèì àíàë³çîì, îñíîâíå ïðèçíà÷åííÿ ÿêîãî –
öå ï³äãîòîâêà îñíîâè äëÿ ïðèéíÿòòÿ åêîíîì³÷íèõ
òà óïðàâë³íñüêèõ á³çíåñ–ð³øåíü ç âèðîáëåííÿ ì³-
ñ³¿, ö³ëåé, ñòðàòå㳿 ðîçâèòêó ï³äïðèºìñòâà ÷è ô³ðìè.  ïðîöåñ³ ñòðàòåã³÷íîãî àíàë³çó íåîáõ³äíî âè-
ÿâèòè êîíêóðåíòí³ ïåðåâàãè îá’ºêòà äîñë³äæåííÿ,
ùî çàáåçïå÷óþòü éîãî ê³íöåâ³ äîñÿãíåííÿ. Ó çàðóá³æí³é ïðàêòèö³ º äåÿêèé àðñåíàë ìåòî-
ä³â ðèíêîâîãî àíàë³çó ÿêèé ìîæíà çàïîçè÷èòè ³ Êåð³âíèêè, ÿê³ óñâ³äîìëþþòü, ùî ñòðàòåã³÷í³
àñïåêòè óïðàâë³ííÿ ñó÷àñíèì àâòîìàòèçîâàíèì
ï³äïðèºìñòâîì, ³íòåãðîâàíèì ç çîâí³øí³ì ñå-
ðåäîâèùåì, ïîòðåáóþòü ³íøî¿ òåîðåòè÷íî¿ áàçè
â ïîð³âíÿíí³ ç, íàïðèêëàä, ïðèíöèïàìè óïðàâë³í-
ñüêîãî ðàö³îíàë³çìó, òà çìîæóòü çíàéòè äîäàòêî-
â³ ìîæëèâîñò³ âäîñêîíàëåííÿ âëàñíî¿ ä³ÿëüíîñò³
â êîíöåïö³¿ âñåçàãàëüíî¿ åêîíîì³÷íî¿ íàóêè [4]. Business decision analysis as a component
of enterprise activity: theoretical aspect Íà îñíîâ³ âèùåñêàçàíîãî ïîòð³áíî çàçíà÷è-
òè ³ óçàãàëüíèòè, ùî îáîâ’ÿçêîâîþ ³íôîðìàö³é-
íîþ ñêëàäîâîþ â õîä³ àíàë³òèêè á³çíåñ–ð³øåíü
íà ï³äïðèºìñòâ³ º çàñòîñóâàííÿ ³íôîðìàö³éíèõ
òåõíîëîã³é, ÿê³ âèð³øóâàòèìóòü ð³çíîìàí³òí³ çà-
äà÷³ á³çíåñ–àíàë³òèêè. Õîò³ëîñÿ á ñêàçàòè, ùî àíàë³òèêà ïîñòຠÿê ³
åêîíîì³÷íà äèñöèïë³íà, ÿêà âêëþ÷ຠòðè íàé-
âàæëèâ³øèõ ñêëàäîâ³: 1. Ìåòîäîëîã³þ ³íôîðìàö³éíî–àíàë³òè÷íî¿ ðîáîòè. Ñó÷àñí³ ³íôîðìàö³éí³ òåõíîëî㳿 äàþòü ìîæëè-
â³ñòü çíà÷íî ñïðîùóâàòè ïðîöåñ àíàë³çó, à òàêîæ
ï³äâèùóâàòè éîãî ïðîäóêòèâí³ñòü ³ ÿê³ñü. 2. Îðãàí³çàö³éíå çàáåçïå÷åííÿ ïðîöåñó àíàë³-
òè÷íî¿ ðîáîòè. 3. Òåõíîëîãî–ìåòîäîëîã³÷íå
çàáåçïå÷åííÿ
ðîçðîáêè ³ ñòâîðåííÿ ³íñòðóìåíòàëüíèõ çàñîá³â
äëÿ âåäåííÿ àíàë³òè÷íî¿ ðîáîòè íà ï³äïðèºìñòâ³. ´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕÂÄÍÎÑÈͳÊÐÀÍ Data about author
Tetyana Ivanyuta, 4. Lyashenko O., Babij S. V. Novi informacijno–
anality’chni texnologiyi doslidzhennya sy’stem uprav- 4. Lyashenko O., Babij S. V. Novi informacijno–
anality’chni texnologiyi doslidzhennya sy’stem uprav- Äàííûå îá àâòîðå 3. Konrad Karlberg. By’znes–analy’z s y’spol’zovany’em
Excel. Resheny’e by’znes–zadach, 4–e y’zdany’e = Busi-
ness Analysis: Microsoft Excel. Moskva : «Vy’l’yams»,
2013. – 576 s. 3. Konrad Karlberg. By’znes–analy’z s y’spol’zovany’em
Excel. Resheny’e by’znes–zadach, 4–e y’zdany’e = Busi-
ness Analysis: Microsoft Excel. Moskva : «Vy’l’yams»,
2013. – 576 s. Ñïèñîê âèêîðèñòàíèõ äæåðåë Âàãîìîþ º ³íòåãðàö³ÿ á³çíåñ–àíàë³òèêè ç ñòðà-
òåã³÷íèì àíàë³çîì, îñíîâíå ïðèçíà÷åííÿ ÿêîãî –
öå ï³äãîòîâêà îñíîâè äëÿ ïðèéíÿòòÿ åêîíîì³÷íèõ
òà óïðàâë³íñüêèõ á³çíåñ–ð³øåíü ç âèðîáëåííÿ ì³-
ñ³¿, ö³ëåé, ñòðàòå㳿 ðîçâèòêó ï³äïðèºìñòâà ÷è ô³ðìè. 1. Áàðèëåíêî Â.È. Áèçíåñ–àíàëèç êàê âàæíûé âèä
êîíñàëòèíãîâûõ óñëóã. ÐÈÑÊ: Ðåñóðñû, Èíôîðìàöèÿ,
Ñíàáæåíèå, Êîíêóðåíöèÿ. ¹ 4. 2012 – Ñ. 202–207. 2. Áîíäàðåíêî Â.Ã. Ñó÷àñí³ ³ ìàéáóòí³ ³íôîêîìóí³êàö³éí³
òåõíîëî㳿 Óêðà¿íè: íàâ÷. ïîñ³á. [äëÿ ñòóäåíò³â âèùèõ íàâ÷. çàêë. çà íàïðÿìîì Òåëåêîìóí³êàö³¿ll] / Â.Ã. Áîíäàðåíêî,
Â.Î. Ãðåáåíí³êîâ. – Ê.: Ðàä³îàìàòîð, 2013. – 160 ñ.  ïðîöåñ³ ñòðàòåã³÷íîãî àíàë³çó íåîáõ³äíî âè-
ÿâèòè êîíêóðåíòí³ ïåðåâàãè îá’ºêòà äîñë³äæåííÿ,
ùî çàáåçïå÷óþòü éîãî ê³íöåâ³ äîñÿãíåííÿ. 3. Êîíðàä Êàðëáåðã. Áèçíåñ–àíàëèç ñ èñïîëüçî-
âàíèåì Excel. Ðåøåíèå áèçíåñ–çàäà÷, 4–å èçäàíèå
= Business Analysis: Microsoft Excel. Ìîñêâà : «Âè-
ëüÿìñ», 2013. – 576 ñ. Ó çàðóá³æí³é ïðàêòèö³ º äåÿêèé àðñåíàë ìåòî-
ä³â ðèíêîâîãî àíàë³çó, ÿêèé ìîæíà çàïîçè÷èòè ³
ââåñòè â ïðàêòèêó ñòðàòåã³÷íîãî àíàë³çó. 65 ´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕÂÄÍÎÑÈͳÊÐÀÍ ÂÈÒÊÓ ÃÀËÓÇÅÉ ÒÀ ÂÈIJ ÅÊÎÍÎ̲×Íί IJßËÜÍÎÑÒ² ²×Ͳ ÏÐÎÁËÅÌÈ ÐÎÇÂÈÒÊÓ ÃÀËÓÇÅÉ ÒÀ ÂÈIJ ÅÊÎÍÎ̲×Íί IJßËÜÍÎÑÒ² linnya pidpry’yemstvamy’ //Innovacijna ekonomika. –
2012. – #. 3. – S. 20–25. linnya pidpry’yemstvamy’ //Innovacijna ekonomika. –
2012. – #. 3. – S. 20–25. 5. Shtefan B. Model’ informacijnoyi texnologiyi
anality’chnogo zabezpechennya procesu vy’robny’cztva
novoyi produkciyi / B.M. Shtefan // Ekonomichny’j
analiz: zb. nauk. pr. 2015. – # 2. – T. 21. – S. 229–236. linnya pidpry’yemstvamy’ //Innovacijna ekonomika. –
2012. – #. 3. – S. 20–25. linnya pidpry’yemstvamy’ //Innovacijna ekonomika. –
2012. – #. 3. – S. 20–25. 4. Ëÿøåíêî Î., Áàá³é Ñ. Â. Íîâ³ ³íôîðìàö³éíî–àíà-
ë³òè÷í³ òåõíîëî㳿 äîñë³äæåííÿ ñèñòåì óïðàâë³ííÿ ï³ä-
ïðèºìñòâàìè //²ííîâàö³éíà åêîíîì³êà. – 2012. – ¹. 3. – Ñ. 20–25. 4. Ëÿøåíêî Î., Áàá³é Ñ. Â. Íîâ³ ³íôîðìàö³éíî–àíà-
ë³òè÷í³ òåõíîëî㳿 äîñë³äæåííÿ ñèñòåì óïðàâë³ííÿ ï³ä-
ïðèºìñòâàìè //²ííîâàö³éíà åêîíîì³êà. – 2012. – ¹. 3. – Ñ. 20–25. 5. Shtefan B. Model’ informacijnoyi texnologiyi
anality’chnogo zabezpechennya procesu vy’robny’cztva
novoyi produkciyi / B.M. Shtefan // Ekonomichny’j
analiz: zb. nauk. pr. 2015. – # 2. – T. 21. – S. 229–236. 5. Shtefan B. Model’ informacijnoyi texnologiyi
anality’chnogo zabezpechennya procesu vy’robny’cztva
novoyi produkciyi / B.M. Shtefan // Ekonomichny’j
analiz: zb. nauk. pr. 2015. – # 2. – T. 21. – S. 229–236. 5. Øòåôàí Á. Ìîäåëü ³íôîðìàö³éíî¿ òåõíîëî㳿 àíà-
ë³òè÷íîãî çàáåçïå÷åííÿ ïðîöåñó âèðîáíèöòâà íîâî¿
ïðîäóêö³¿ / Á.Ì. Øòåôàí // Åêîíîì³÷íèé àíàë³ç: çá. íàóê. ïð. 2015. – ¹ 2. – Ò. 21. – Ñ. 229–236. ´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕÂÄÍÎÑÈͳÊÐÀÍ References ê.å.í., äîöåíò, Íàö³îíàëüíîãî óí³âåðñèòåòó õàð÷îâèõ
òåõíîëîã³é ê.å.í., äîöåíò, Íàö³îíàëüíîãî óí³âåðñèòåòó õàð÷îâèõ
òåõíîëîã³é 1. Bary’lenko V.Y’. By’znes–analy’z kak vazhnûj vy’d
konsalty’ngovûx uslug. RY’SK: Resursû, Y’nformacy’ya,
Snabzheny’e, Konkurency’ya. # 4. 2012 – S. 202–207. 1. Bary’lenko V.Y’. By’znes–analy’z kak vazhnûj vy’d
konsalty’ngovûx uslug. RY’SK: Resursû, Y’nformacy’ya,
Snabzheny’e, Konkurency’ya. # 4. 2012 – S. 202–207. e–mail: te_ta_na@ukr.net e–mail: te_ta_na@ukr.net 2. Bondarenko V.G. Suchasni i majbutni infokomuni-
kacijni texnologiyi Ukrayiny’: navch. posib. [dlya studentiv
vy’shhy’x navch. zakl. za napryamom Telekomunikaciyill]
/ V.G. Bondarenko, V.O. Grebennikov. – K.: Radioama-
tor, 2013. – 160 s. Äàííûå îá àâòîðå
Èâàíþòà Òàòüÿíà Íèêîëàåâíà,
ê.ý.í., äîöåíò, Íàöèîíàëüíîãî óíèâåðñèòåòà ïè-
ùåâûõ òåõíîëîãèé
e–mail: te_ta_na@ukr.net
Data about author
Tetyana Ivanyuta,
Ph.D., Associate Professor, National University of Ïðåäìåò äîñë³äæåííÿ – ìåõàí³çìè ñòðàòåã³÷íîãî âòðó÷àííÿ áàãàòîð³âíåâîãî ñóá’ºêòèâíîãî
ôàêòîðà â ñîö³àëüíî–åêîíîì³÷í³ ïðîöåñè. Ïðåäìåò äîñë³äæåííÿ – ìåõàí³çìè ñòðàòåã³÷íîãî âòðó÷àííÿ áàãàòîð³âíåâîãî ñóá’ºêòèâíîãî
ôàêòîðà â ñîö³àëüíî–åêîíîì³÷í³ ïðîöåñè. Ïðåäìåò äîñë³äæåííÿ – çì³íà ïåðåäóìîâ îñâ³òíüîãî ïðîöåñó â ïîë³òèêî–åêîíîì³÷íîìó â³ä-
òâîðåíí³. Ìåòîþ íàïèñàííÿ ñòàòò³ º âèÿâëåííÿ ìîæëèâîñòåé ³ âèìîã äî íàâ÷àííÿ äëÿ çðîñòàííÿ 䳺âîñò³
íàóêîâî–îñâ³òíüî–âèðîáíè÷èõ êëàñòåð³â ³ ñåðåäîâèùà çì³í. Ìåòîäîëîã³ÿ ïðîâåäåííÿ ðîáîòè áàçóºòüñÿ íà ïîºäíàíí³ ëîã³÷íîãî òà ³ñòîðè÷íîãî ï³äõîä³â â àíà-
ë³ç³ ñîö³àëüíî–åêîíîì³÷íèõ ïðîöåñ³â ÿê îñíîâè ðåãóëÿòèâíèõ âïëèâ³â. Êîíöåíòðàö³ÿ çì³í, ùî äîç-
âîëÿº õàðàêòåðèçóâàòè ñóñï³ëüñòâî ³ éîãî åêîíîì³êó, ÿê «çíàííÿ», «ï³çíàííÿ», «³íôîðìàö³¿» ³ òàê
äàë³, çì³ùóº íå ò³ëüêè òðèâàë³ñòü, àëå ³ ôóíêö³¿, à òàêîæ çàâäàííÿ íàâ÷àííÿ. ßêùî ðàí³øå ³ñòîð³ÿ
ïðèâ÷èëà äî ñèòóàö³¿, äå àêòèâíó ó÷àñòü â ïðîöåñàõ íàâ÷àííÿ áóëî äîñèòü êîðîòêèì ïåð³îäîì ³í-
òåíñèâíî¿ ï³äãîòîâêè äî æèòòÿ â ñóñï³ëüñòâ³, à ðîëü ïåäàãîãà ÷àñîì êîíöåíòðóâàëàñÿ íàâêîëî ïå-
ðåäà÷³ êèìîñü óæå âèðîáëåíèõ çíàíü, òî íîâà ðåàëüí³ñòü âèìàãຠâ³ä ëþäèíè ïîñò³éíîãî íàâ÷àííÿ
(â òîìó ÷èñë³ ñàìîíàâ÷àííÿ), à ÿê³ñòü ä³ÿëüíîñò³ ïåäàãîãà íåâ³ä’ºìíî â³ä éîãî îñîáèñòî¿ (çîêðåìà,
äóõîâíî–³íòåëåêòóàëüíî¿) òâîð÷îñò³. Äîñòàòîê ³íôîðìàö³¿ ï³äâèùóº ö³íí³ñòü ìåòîäîëîã³÷íîãî, êîí-
öåïòóàëüíîãî çíàííÿ, ðîçóì³ííÿ ñèòóàö³éíèõ ³ ïðîöåñóàëüíèõ õàðàêòåðèñòèê, à òàêîæ âì³ííÿ êîí-
âåðòóâàòè ¿õ â ñâ³äîìó àêòèâí³ñòü, ïåðø çà âñå, – ó âèðîáíè÷îìó æèòò³ òà åêîíîì³ö³ îñâ³òè. Ðåçóëüòàòè ðîáîòè – çä³éñíåíî àíàë³ç ìîæëèâîñòåé ï³äâèùåííÿ ðîë³ íàâ÷àííÿ ó ôîðìóâàíí³
íàóêîâî–îñâ³òíüî–âèðîáíè÷èõ êëàñòåð³â ³ ðîçâèòêó ñåðåäîâèùà çì³í. © ØÅÄßÊΠÂ.ª., 2021 66 ´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕÂÄÍÎÑÈͳÊÐÀÍ
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https://openalex.org/W2969819020
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https://www.e3s-conferences.org/10.1051/e3sconf/201911301008/pdf
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English
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Integration of heat pump and gas turbine combined cycle: market and climatic conditions for power plant flexibility enhancement
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E3S web of conferences
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| ERROR: type should be string, got "https://doi.org/10.1051/e3sconf/201911301008 https://doi.org/10.1051/e3sconf/201911301008 E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons \nAttribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Integration of heat pump and gas turbine \ncombined cycle: market and climatic conditions \nfor power plant flexibility enhancement \nAndrea Giugno1, Alessandro Sorce1, Alessandra Cuneo2* and Stefano Barberis2 \n1 Thermochemical Power Group, University of Genoa, Via Montallegro 1, 16145 Genoa, Italy \n2 RINA Consulting, Via San Nazaro 19, 16145, Genoa, Italy Abstract. The increasing share of electricity produced from renewable \nenergy sources (RES), with the consequent strong penetration in the current \nenergy network, is causing a growing need of balancing power to \ncompensate power supply from such fluctuating sources. For these reasons, \nnowadays the power plants are requested to improve their operational \nflexibility, together with their global efficiency in part-load operation, for \nancillary services and to sustain the grid operability. A possible solution for \nflexibility enhancement is characterized by a highly efficient heat pump \nintegrated in a conventional natural gas combined cycle (CC). Such concept \ncan be applied both to power oriented combined cycle (POCC), to modify \nthe compressor intake temperature with a consequent increase or decrease \nof the power production, and both to cogeneration CC in association with \nDistrict Heating Network (DHN). In this work, a statistical analysis of \nclimatic data and their correlations with energy market condition will be \nperformed considering Italian context, to understand which the more \nsuitable conditions for such integrated system are. The analysis will be \nperformed on seasonal and daily basis. The final aim of this work is to \nidentify how such integrated system can be operated at its best in the \ndifferent Italian markets and climatic frames. E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 https://doi.org/10.1051/e3sconf/201911301008 E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 An innovative concept based on the coupling of a fast-cycling highly efficient Heat Pump \n(HP) with the CCGTs is here proposed [4], within the framework of an H2020 EU project \ncalled PUMP-HEAT [5]. This possible solution for flexibility enhancement is characterized \nby a highly efficient heat pump integrated in a CCGT, featuring cold/warm thermal storage \nand advanced control. Such concept can be applied both to power oriented combined cycle \n(POCC), to modify the compressor intake temperature with a consequent increase or decrease \nof the power production, and both to cogeneration CCGT in association with District Heating \nNetwork (DHN). In this work, a statistical analysis of climatic data and their correlations \nwith energy market condition will be performed considering the Italian market scenario, to \nunderstand which is the more suitable conditions for such integrated systems. The final aim is to identify how PUMP HEAT concept can be operated and the influences \nthat different Italian price zones and climatic conditions can have on its profitability. The \nPower Oriented (PO) combined cycle configuration is taken into consideration during this \nanalysis, with a particular attention on the continuous cooling mode (Figure 1). Such \noperational mode allows to modify the compressor intake temperature thanks to the cold \nenergy produced by the heat pump. The cooling of the inlet temperature increases the \nmaximum power output of the CC (about 10%) during peak hours, with also an increase of \nthe yearly efficiency and production. Figure 1. Power oriented HP-CC concept scheme working in continuous cooling Figure 1. Power oriented HP-CC concept scheme working in continuous cooling The heat pump produces cold energy using ambient air through a dedicate heat exchanger \n(ambient HX in Figure 1) as energy source. The performances of the heat pump in terms of \nCOP and the temperature that can be reached at the compressor inlet are dependent on the \nambient temperature. However, to consistently evaluate the HP performance, both ambient \ntemperature and humidity were considered. In addition, in this work, it was always \nconsidered to work at the maximum load of the heat pump, not considering off-design \nconditions. In fact, from the optimization analysis performed during the PUMP-HEAT \nproject, it resulted that this would be the condition that maximizes the economic benefit. However, a minimum threshold value of 5°C was set at the compressor inlet to avoid the risk \nof ice formation. E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 The CCGT model was validated in previous work with respect to the effect \nof ambient temperature. the effect of GT intake humidity on the CCGT overall performance \nwas neglected as conservative assumption as explained in [4]. 1 Introduction In the last years, a consistent evolution of combined cycle performance was shown, mainly \ncommanded by the market. The intermittent renewable capacity has increased dramatically \nin the last 10 years, due also to the EU target [1], having a major impact on markets. The \ngrowth of renewable energy sources is, of course, highly beneficial to the environment but \nintroduces intermittency into grid management. This rapid growth in variable generation is \ndriving the need for a more flexible power system and for a research and development \nstrategy to help achieve that. In such new energy scenario, the combined cycle gas turbines (CCGT) have to change \nthe way they operate: from a baseload to backup [2]. CCGT must now respond rapidly to \nvariations due to renewable supply and demand, requiring a more flexible cyclic pattern of \nuse [3]. Making conventional power plants more flexible can be a key strategy to integrate \nlarge shares of renewables more effectively in power systems [1,2]. 2 Italian electricity scenario The volatility of minimum and maximum prices \nof this region is much higher than in the rest of Italy and the other macro regions [8] compared to 2016), for 12.8% by hydroelectric production and for the remaining 16.3% by \ngeothermal, wind and photovoltaic sources (with an increase of 10.3% respect to the previous \nyear) [7]. The Italian electricity market operates in three different periods: (i) daily market \n(MGP), which trades most of the electricity purchase and sale transactions (ii) intra-day \nmarket (MI) consisting of seven sessions which allow market participants to modify the \nprograms defined in the daily market by sending additional sales or purchase offers and (iii) \nthe market for continuous trading of daily products (MPEG). The electricity system is divided \ninto zones classified into geographical zones, national virtual zones and foreign virtual zones. The geographical zones represent a part of the national network; there are currently six active \nzones: Northern Italy (NORD), Central-Northern Italy (CNOR), Central-Southern Italy \n(CSUD), Southern Italy (SUD), Sicily (SICI) and Sardinia (SARD). The MGP market is a \nmarginal market in which the price and volume of each hour are established from the point \nof equilibrium between supply and demand. Matched purchase offers referring to units of \nconsumption belonging to Italian geographical areas are valued at the single national price \n(PUN). The PUN price is equal to the average of the prices of the zones, by zonal \nconsumption and represents the purchase price for end customers. In the last years, due to \nthe increase of RES and to a reduction of the natural gas price, the PUN in all the zones has \ndecreased consistently (Figure 2). The Sicily zone, however, seems to be the most interesting \none where to study continuous cooling concept, with an average zonal price always higher \nrespect to the national PUN (+13% in 2017). The volatility of minimum and maximum prices \nof this region is much higher than in the rest of Italy and the other macro regions [8] Figure 2. Annual average zonal price on MGP (left) and monthly average zonal price in 2017 (right) \n[9] Figure 2. Annual average zonal price on MGP (left) and monthly average zonal price in 2017 (right) \n[9] Figure 2. 2 Italian electricity scenario Annual average zonal price on MGP (left) and monthly average zonal price in 2017 (rig\n[9] In this market scenario, the use of a heat pump (HP) to decrease the temperature at the \ncompressor inlet do not depend only on the electricity price but it is also related to the ambient \ntemperature. In the following sections, a statistical analysis to correlate the price and the \nambient temperature is performed, as well as a first order economic analysis of the proposed \nintegrated system (CCGT+HP). 2 Italian electricity scenario As part of the new National Energy Strategy, Italy set itself the goal of the phase out of coal \nby 2025, aiming to expand the share of renewable energy in final energy consumption to 28% \nby 2030 [6]. The Italian wholesale electricity market started to operate as an Exchange in \n2005 with the liberalization of the demand side bidding. In 2017 gross national production \namounting to 295.8 TWh was satisfied for 70.8% by thermoelectric production (+5.0% 2 https://doi.org/10.1051/e3sconf/201911301008 E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 compared to 2016), for 12.8% by hydroelectric production and for the remaining 16.3% by \ngeothermal, wind and photovoltaic sources (with an increase of 10.3% respect to the previous \nyear) [7]. The Italian electricity market operates in three different periods: (i) daily market \n(MGP), which trades most of the electricity purchase and sale transactions (ii) intra-day \nmarket (MI) consisting of seven sessions which allow market participants to modify the \nprograms defined in the daily market by sending additional sales or purchase offers and (iii) \nthe market for continuous trading of daily products (MPEG). The electricity system is divided \ninto zones classified into geographical zones, national virtual zones and foreign virtual zones. The geographical zones represent a part of the national network; there are currently six active \nzones: Northern Italy (NORD), Central-Northern Italy (CNOR), Central-Southern Italy \n(CSUD), Southern Italy (SUD), Sicily (SICI) and Sardinia (SARD). The MGP market is a \nmarginal market in which the price and volume of each hour are established from the point \nof equilibrium between supply and demand. Matched purchase offers referring to units of \nconsumption belonging to Italian geographical areas are valued at the single national price \n(PUN). The PUN price is equal to the average of the prices of the zones, by zonal \nconsumption and represents the purchase price for end customers. In the last years, due to \nthe increase of RES and to a reduction of the natural gas price, the PUN in all the zones has \ndecreased consistently (Figure 2). The Sicily zone, however, seems to be the most interesting \none where to study continuous cooling concept, with an average zonal price always higher \nrespect to the national PUN (+13% in 2017). 3 Statistical Analysis The distribution of the electricity price plays a major role over the profitability of CCGT \npower plants, presenting an average production cost, i.e. the Cost of Electricity, COE, aligned \nwith the average value of the PUN. The overall profitability of the proposed power \naugmentation application, continuous cooling by a HP, can be related to the electricity peak \nabsolute price and to the temperature and price peak coupling. In a broader approach, \nconsidering the use of a Thermal Energy Storage (TES) to accumulate the cold energy during \nthe off-peak periods, also the respective temperature and price minima must be considered. The daily temperature and price variation can increase the benefit related to TES installation, \nin particular when electrical price and temperatures maxima or minima occurs at the same 3 E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 https://doi.org/10.1051/e3sconf/201911301008 time. Also the width of the positive and negative peaks, i.e how many hours can be accounted \nas “high range” and “low range” can be related to the system profitability. Such extreme \nconditions were defined with respect to the daily variation ∆dailyx = max(𝑥) −min(𝑥), as: time. Also the width of the positive and negative peaks, i.e how many hours can be accounted \nas “high range” and “low range” can be related to the system profitability. Such extreme \nconditions were defined with respect to the daily variation ∆dailyx = max(𝑥) −min(𝑥), as: 𝐻𝑖𝑔ℎ𝑟𝑎𝑛𝑔𝑒𝑥= [max(𝑥) −∆dailyx\n20\n, max(𝑥)] \n𝐿𝑜𝑤𝑟𝑎𝑛𝑔𝑒𝑥= [min(𝑥) , min(𝑥) + ∆dailyx\n20\n] \n(1) (1) This definition allows preserving at least 90% of the ∆dailyx between high and low range \nof the variable, as depicted in Figure 3 for the temperature for a representative day. The points \nwith the x markers and ▼ are respectively for high and low range and are counted to \nevaluated the peak extension, in this case respectively 4 and 5 hours. Figure 3. Example of the peak analysis: red line, ambient temperature [°C; x high range value; ▼ low \nrange value \n∆dailyT \nHighrangeT \nLowrangeT \n90%∆dailyT LowrangeT Figure 3. Example of the peak analysis: red line, ambient temperature [°C; x high range value; ▼ low \nrange value The statistical analysis considers six price zones and six geographical location, as \nreported in Table 1, while price data were acquired by the Italian Gestore del Mercato \nElettrico [9]. Table 1. Meteorological data measurement point [10] Table 1. 3 Statistical Analysis Meteorological data measurement point [10] \nZones \nLocation \nPosition [N lat., E long.] \nAltitude [m] \nNorth \nTurin / Bric della Croce (airport) \n45° 02', 07° 44' \n709 \nCNorth \nAncona / Falconara (airport) \n43° 37', 13° 22' \n12 \nCSouth \nRome / Fiumicino (airport) \n41° 48', 12° 14' \n2 \nSouth \nBari \n41° 8.17', 16° 45.78' \n42 \nSard \nCapo Caccia \n40° 34', 08° 10' \n200 \nSici \nTrapani-Birgi (airport) \n37° 54.7’, 12° 29.6' \n7 Figure 4 represents the average maximum and the minimum value of the electrical price \nwithin the six Italian zones. The size of the marker is proportional to the number of hours in \nthe high and low range (i.e. the peak extension). The distance between the two marker \ncentres, in the same month, is equal to the average ∆dailyx. The maximum price values are \nregistered in the North and in Center North during the first two months of the year, while the \nSouth presents always lower prices. This could be related to the high number of CCGT 4 E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 https://doi.org/10.1051/e3sconf/201911301008 production sites and wind farm installed in a region with a relative low power demand. It can \nbe noticed that the prices in all the zones experience a reduction until April (loosing up to 54 \n€/MWh) and then a recovery. The maximum price of Sicily shows a less pronounced \nreduction in price until April (Pmax=85.6 €/MWh) and then remains above 90 €/MWh. In \nthis zone, also the width of the peak price is different respect to the other zones in Italy. In \nSicily, the average of the high range is up to 6h/day during the hot season and the yearly \naverage is around 4 hours versus the average of 1.66 h/day of the other zones. Looking at the \ndaily price variation, for the first five zones the changes appear to be related to a seasonal \ntrend with a strong reduction during the hot months. production sites and wind farm installed in a region with a relative low power demand. It can \nbe noticed that the prices in all the zones experience a reduction until April (loosing up to 54 \n€/MWh) and then a recovery. The maximum price of Sicily shows a less pronounced \nreduction in price until April (Pmax=85.6 €/MWh) and then remains above 90 €/MWh. 3.1 Peak coupling of the ambient temperature and the electrical price 3.1 Peak coupling of the ambient temperature and the electrical price Under the proposed application perspective, another important effect that increases the \nbenefit of the inlet cooling system, is the location of the price and temperature peak within \nthe daytime. Figure 5. Monthly averaged daily peak positions: blue circle Zonal Electricity Price peak (max) value \n(size proportional to price), blue lines price peak extension; red circle ambient temperature peak (max) \nvalue (size proportional to temperature), red lines temperature peak extension Figure 5. Monthly averaged daily peak positions: blue circle Zonal Electricity Price peak (max) value \n(size proportional to price), blue lines price peak extension; red circle ambient temperature peak (max) \nvalue (size proportional to temperature), red lines temperature peak extension It can be noticed that the maximum daily temperature is registered around the first two \nhours after noon, apart from the northern locations in which the peak is delayed up to 5 pm. The duration of the peak is intense in Sicily (a year average of 3.1 h/day) and in Center-North \nlocations (2.89 h/day), with a slightly higher value in the summer months. The price peaks occur later in the day moving from North to South, from 5pm in the North \nzone and 5.30pm in the Center-North, to 6pm in the South. The Sicily zone differs also for \nthis parameter, with an average price peak that occurs at 7.25pm. However, the price enters \nthe high range at about 6pm, with a longer persistence in the summer months up to 11pm. The temperature and price peaks appear to be separated in time of 1-2 hours, except for the \nnorthern regions in which there is a partial overlap. 3 Statistical Analysis In \nthis zone, also the width of the peak price is different respect to the other zones in Italy. In \nSicily, the average of the high range is up to 6h/day during the hot season and the yearly \naverage is around 4 hours versus the average of 1.66 h/day of the other zones. Looking at the \ndaily price variation, for the first five zones the changes appear to be related to a seasonal \ntrend with a strong reduction during the hot months. Figure 4. Monthly averaged max daily peak and min off-peak zonal price, the size of the marker is \nproportional to the peak extension hours. Figure 4. Monthly averaged max daily peak and min off-peak zonal price, the size of the marker is \nproportional to the peak extension hours. The daily ambient temperature variation, which is evaluated as the difference between \nthe maximum and the minimum temperature value in the same month, presented in Table 2, \nshows a trend that increases during summer due to the increase of the solar radiance, except \nfor the South location. This is beneficial to the TES application, as they are usually employed \nduring the hot months. The monthly average maximum and minimum temperatures, not \nreported here, as expected, increase during the summer period. Table 2. Monthly average daily variation of ambient Temperature [K] Table 2. Monthly average daily variation of ambient Temperature [K] \nZones \nJan \nFeb \nMar \nApr \nMay \nJun \nJul \nAug \nSep \nOct \nNov \nDec \nNorth \n4.0 \n3.5 \n5.5 \n6.8 \n7.0 \n7.0 \n6.7 \n6.3 \n5.8 \n4.8 \n3.3 \n3.5 \nCNorth \n6.7 \n7.7 \n10.2 \n10.1 \n9.6 \n10.3 \n10.3 \n10.3 \n8.5 \n9.8 \n8.0 \n8.5 \nCSouth \n9.0 \n8.4 \n10.2 \n10.7 \n10.8 \n9.7 \n10.5 \n11.1 \n8.9 \n10.7 \n8.9 \n9.4 \nSouth \n5.0 \n6.1 \n6.0 \n6.3 \n5.6 \n5.3 \n5.3 \n6.0 \n6.9 \n6.4 \n6.5 \n5.7 \nSard \n4.5 \n4.5 \n4.6 \n4.9 \n5.5 \n5.4 \n6.0 \n6.5 \n4.4 \n4.3 \n3.5 \n3.1 \nSici \n7.1 \n7.7 \n9.0 \n9.2 \n9.0 \n10.5 \n9.2 \n10.0 \n7.4 \n7.6 \n6.8 \n6.7 5 5 E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 https://doi.org/10.1051/e3sconf/201911301008 SUPEHR19 Volume 1 4 Economic analysis The continuous inlet cooling system performance were calculated taking into account five \ndifferent heat-pump (HP) sizes [1, 3, 4, 5 and 7.5 MWe] and the six geographical zones \ndescribed in the previous section. Such locations were used as possible sites for the 6 6 6 E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 https://doi.org/10.1051/e3sconf/201911301008 installation of the CC+HP layout to perform the continuous cooling of the turbine intake to \nenhance the performance of the system and so evaluate the earnings that such system would \nguarantee. The HP are considered to operate to reach a target inlet temperature of the GT \nintake of 5°C, which has been identified as the minimum reachable temperature without the \nice formation risk. installation of the CC+HP layout to perform the continuous cooling of the turbine intake to \nenhance the performance of the system and so evaluate the earnings that such system would \nguarantee. The HP are considered to operate to reach a target inlet temperature of the GT \nintake of 5°C, which has been identified as the minimum reachable temperature without the \nice formation risk. The CC net power and efficiency dependence on the inlet GT temperature was obtained \nfrom the Gate Cycle calculation presented in [4] and so the earnings that such system would \nproduce over the year, taking 2017 as reference, were computed as: 𝐸𝑎𝑟𝑛𝑖𝑛𝑔𝑠 = ∑𝐸𝑝𝑟𝑜𝑑,𝑖\n8760\n𝑖=1\n∙(𝑃𝑍,𝑖−(𝐶𝑓𝑖𝑥+𝐶𝑔𝑎𝑠\n𝜂𝐶𝐶\n)); \n𝑖𝑓𝑃𝑧> 𝐶𝑓𝑖𝑥+𝐶𝑔𝑎𝑠\n𝜂𝐶𝐶\n[€] \n(2) (2) Where 𝐸𝑝𝑟𝑜𝑑,𝑖 is the energy produced by the CC at the i-th hour as function of the \ntemperature, Pz is the zonal price, Cfix and Cgas represent the fixed costs of the plant and \nthe gas cost based on [9,11], and 𝜂𝐶𝐶 is the CC efficiency as function of the temperature. To \nestimate the cooling potential of a given climatic profile, the concept of Continuous \nEquivalent Cooling Degree Hours (ECDH) is introduced, based on [12], and evaluated as: 𝐸𝐶𝐷𝐻 = ∑(𝑇𝑎𝑚𝑏,𝑖\n8760\n𝑖=1\n−𝑇𝑟𝑒𝑓)𝑖𝑓𝑇𝑎𝑚𝑏,𝑖>𝑇𝑟𝑒𝑓 \n(3) (3) (3) Where 𝑇𝑟𝑒𝑓 for this study is set to 5 °C for the reasons explained before. From Figure 6b \nit can be observed that the ΔEarnings, calculated as percentage variation from the earnings \nthat the same CC would guarantee in ambient conditions, tend to increase while moving from \nNorth to South of Italy, due to the temperature increase over the year which would allow a \nhigher exploitation of the continuous cooling system proposed. 5 Conclusions The statistical analysis of climatic data and their correlations with energy market condition \nwas performed considering Italian context for six different electricity market zones. The \nanalysis was also carried out on monthly and daily basis introducing a technique to identify \nthe range of extreme values of the daily distribution on the base of the daily price variation. It was highlighted how the daily price variation reduces during the hot months, while the \ntemperature daily variation increases, as expected, due to the increase in solar radiant energy \nduring the summer time. Finally, Sicily market zone, shows a strongly different behaviour, \ncharacterized by high and persistent maximum prices, with a yearly average of 20 €/MWh \nover the other zonal prices. Focusing on the Italian market, the profitability of a continuous inlet cooling system \nbecomes more attractive moving from north to south due to the increase of the average \ntemperatures. On the other side, the South electricity price, the lowest in the peninsula, has a \nlow profitability compared to the meteorological potential. The gain in power of the proposed \nsystem is proportional to the climatic condition summarized by the Continuous Equivalent \nCooling Degree Hours, plus the effect of the zonal price. Under the actual condition the Sicily \nmarket has the largest potential, maximizing both the temperature and electrical price effect. This project has received funding from the European Union’s Horizon 2020 research \nand innovation programme under Grant Agreement No 764706, PUMP-HEAT. (http://www.pumpheat.eu). 4 Economic analysis However, it can also be \nhighlighted that the South area (SUD) presents high ECDH, but low percentage earnings \ncompared to the other areas considered, due to the lower Pz. (a) \n(b) \nFigure 6 : (a) percentage power production and (b) percentage earnings over ambient conditions \nfor five different HP sizes and six different areas of Italy (a) (b) (b) (a) Figure 6 : (a) percentage power production and (b) percentage earnings over ambient conditions \nfor five different HP sizes and six different areas of Italy This trend becomes more evident increasing the HP size over 1 MW, as ΔEarnings \nbecomes lower than all the other areas apart from the North (NORD) one. This effect is 7 7 E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 https://doi.org/10.1051/e3sconf/201911301008 mostly related to the PUN of the South being lower than the national mean and other areas \nconsidered, which does not allow a good exploitation of the power produced (Figure 6a). mostly related to the PUN of the South being lower than the national mean and other areas \nconsidered, which does not allow a good exploitation of the power produced (Figure 6a). The high PUN of Sicily allows to benefit more from this layout not only thanks to the \nhigher average temperatures, resulting in a maximum ΔEarnings of about 4% using a 7.5MW \nHP. On the other hand, from Figure 6a it can be observed that the percentage power variation \nconsidering Sicily and a 1MW HP is lower than SUD and SARD. This is due to the fact that \ntemperatures in Sicily are particularly high and then a 1MW HP would not be able to reach \nthe target temperature of 5°C, resulting in a power augmentation lower than the actual \npossibility. Indeed, also the worst result, obtained from using a 1MW HP in Northern Italy, \nwould guarantee an increase of earnings of about 0.8% for the year considered. Moreover, it can be highlighted that the lines corresponding to the HPs of 5 and 7.5MW \nbecome very close in terms of earnings, suggesting that when the investment cost comes in \nplace within the calculations, it would probably be more profitable to buy a 5MW HP which \nwould result in lower capital and installation costs. [ ]\ngo\ne g ewe de,\n7,\ny\n.\n[3] \nPrina, M. G., Garegnani, G., Moser, D., Oberegger, U. F., Vaccaro, R., Sparber, \nW., Gazzani, M., and Manzolini, G., 2015, “Economic and Environmental Impact of \nPhotovoltaic and Wind Energy High Penetration towards the Achievement of the Italian 20-\n20-20 Targets,” 2015 10th International Conference on Ecological Vehicles and Renewable \nEnergies, EVER 2015. This project has received funding from the European Union’s Horizon 2020 research \nand innovation programme under Grant Agreement No 764706, PUMP-HEAT. \n(http://www.pumpheat.eu). [4] \nSorce, A., Giugno, A., Marino, D., Piola, S., and Guedez, R., 2019, “Analysis of a [1] \nEuropean Parliament, 2018, Directive (EU) 2018/2001 of the European \nParliament and of the Council of 11 December 2018 on the Promotion of the Use of Energy \nfrom Renewable Sources. [1] \nEuropean Parliament, 2018, Directive (EU) 2018/2001 of the European \nParliament and of the Council of 11 December 2018 on the Promotion of the Use of Energy \nfrom Renewable Sources. \n[2] \nAgora Energiewende, 2017, Flexibility in Thermal Power Plants. \n[3] \nPrina, M. G., Garegnani, G., Moser, D., Oberegger, U. F., Vaccaro, R., Sparber, \nW., Gazzani, M., and Manzolini, G., 2015, “Economic and Environmental Impact of \nPhotovoltaic and Wind Energy High Penetration towards the Achievement of the Italian 20-\n20-20 Targets,” 2015 10th International Conference on Ecological Vehicles and Renewable \nEnergies, EVER 2015. \n[4] \nSorce, A., Giugno, A., Marino, D., Piola, S., and Guedez, R., 2019, “Analysis of a References [1] \nEuropean Parliament, 2018, Directive (EU) 2018/2001 of the European \nParliament and of the Council of 11 December 2018 on the Promotion of the Use of Energy \nfrom Renewable Sources. [1] \nEuropean Parliament, 2018, Directive (EU) 2018/2001 of the European \nParliament and of the Council of 11 December 2018 on the Promotion of the Use of Energy \nfrom Renewable Sources. [2] \nAgora Energiewende, 2017, Flexibility in Thermal Power Plants. [3] \nPrina, M. G., Garegnani, G., Moser, D., Oberegger, U. F., Vaccaro, R., Sparber, \nW., Gazzani, M., and Manzolini, G., 2015, “Economic and Environmental Impact of \nPhotovoltaic and Wind Energy High Penetration towards the Achievement of the Italian 20-\n20-20 Targets,” 2015 10th International Conference on Ecological Vehicles and Renewable \nEnergies, EVER 2015. [4] \nSorce, A., Giugno, A., Marino, D., Piola, S., and Guedez, R., 2019, “Analysis of a 8 https://doi.org/10.1051/e3sconf/201911301008 E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 Combined Cycle Exploiting Inlet Conditioning Technologies for Power Modulation,” Combined Cycle Exploiting Inlet Conditioning Technologies for Power Modulation,” \nProceedings of ASME Turbo Expo 2019: Turbomachinery Technical Conference and Combined Cycle Exploiting Inlet Conditioning Technologies for Power Modulation,” \nProceedings of ASME Turbo Expo 2019: Turbomachinery Technical Conference and \nExposition GT2019. Combined Cycle Exploiting Inlet Conditioning Technologies for Power Modulation,” \nProceedings of ASME Turbo Expo 2019: Turbomachinery Technical Conference and \nExposition GT2019. [5] \n“Pump-Heat” [Online]. Available: www.pumpheat.eu. [Accessed: 30-Apr-2019] [5] \n“Pump-Heat” [Online]. Available: www.pumpheat.eu. [Accessed: 30-Apr-2019]\n[6]\nMinistero dello Sviluppo Economico, Proposta Di Piano Nazionale Integrato pe [5] \n“Pump-Heat” [Online]. Available: www.pumpheat.eu. [Accessed: 30-Apr-2019]. [6] \nMinistero dello Sviluppo Economico, Proposta Di Piano Nazionale Integrato per \nl’Energia Ed Il Clima. [7]\nTERNA 2018 D ti St ti ti i S ll’\ni El tt i\ni It li [ ]\np\n[\n]\np\np\n[\np\n]\n[6] \nMinistero dello Sviluppo Economico, Proposta Di Piano Nazionale Integrato per\nl’Energia Ed Il Clima. [7] \nTERNA, 2018, Dati Statistici Sull’energia Elettrica in Italia. [8] \nAntonelli, M., Desideri, U., and Franco, A., 2018, “Effects of Large Scale \nPenetration of Renewables: The Italian Case in the Years 2008–2015,” Renew. Sustain. Energy Rev. [9] \nGestore Mercati Energetici, “Dati Storici Mercati Gas” [Online]. Available: \nhttp://www.mercatoelettrico.org/It/download/DatiStoriciGas.aspx. [Accessed: 07-Apr-\n2019]. [10] \n“Weather in the World” [Online]. Available: https://rp5.ru/Weather_in_the_world. [Accessed: 30-Apr-2019]. [11] \nRicerca sul Sistema Energetico - RSE Spa, 2017, Energia Elettrica, Anatomia Dei \nCosti. [12] \nAncona, M. References A., Bianchi, M., Melino, F., and Peretto, A., 2015, “Power \nAugmentation Technologies: Part II — Thermo-Economic Analysis.” 9 9"
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Algumas Considerações Sobre Ensino e Aprendizagem na Disciplina Laboratório de Eletromagnetismo
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Revista Brasileira de Ensino de F
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471
Algumas Consideraco~es Sobre Ensino e Aprendizagem
na Disciplina Laborat
orio de Eletromagnetismo
Considerations About Learning and Teaching in the Electromagnetism Laboratory
J. Humberto Dias da Silva
Faculdade de Ci^
encias, Departamento de F
sica
Universidade Estadual Paulista, CEP 17033-360, Bauru, SP
Recebido em 8 de maio, 2002. Aceito em 26 de setembro, 2002.
Baseados por um lado em avancos conceituais recentes das pesquisas em ensino de ci^encias, e
por outro em nossa experi^encia docente, propomos algumas re ex~oes sobre as praticas de ensino
e aprendizagem na disciplina Laboratorio de Eletromagnetismo. Apresentamos e interpretamos
resultados e comentarios dos estudantes, os quais forneceram elementos para a analise da efetividade
de propostas de novo enfoque para esta disciplina.
Based on recent research in science education, and on teaching experience, we analyze the learning
and teaching process in the Electromagnetism laboratory for undergraduate physics students. Data
and reasoning obtained from the answers of some groups of students are used to help the analysis
of the e ectiveness of the changes proposed in this physics laboratory.
I Introduc~ao
Professores e pesquisadores da area de ensino de
ci^encias geralmente imputam grande import^ancia ao
espaco de aprendizado de fsica ocorrido nas disciplinas
experimentais [1,2,3]. Aparentemente tambem existe
um consenso entre docentes e estudantes de que deve
haver mudancas na maneira tradicional de se enfocar estas disciplinas [1,4-7]. Apesar disto ha relatos
de que esta import^ancia declarada algumas vezes e
mais quest~ao de postura que de pratica efetiva [5,8],
e tambem ha observac~oes crticas a respeito do real benefcio que estas disciplinas possam trazer ao processo
de aprendizado dos estudantes [9].
A disciplina Laboratorio de Eletromagnetismo e a
primeira disciplina experimental da parte espec ca que
os estudantes dos cursos de Fsica realizam. Nela, em
princpio, os estudantes t^em espaco propcio para realizar experimentos um pouco mais detalhados sobre
o Eletromagnetismo Classico. Os experimentos geralmente apresentam um maior grau de complexidade em
relac~ao aos experimentos das disciplinas basicas. Esta
escolha de maior complexidade neste ponto do curso
baseia-se na hipotese, nem sempre correta, de que o
grau de maturidade e entendimento do estudante podem alcancar esta complexidade [6,10]. Esta e uma
quest~ao que merece cuidado, pois corremos o risco de
colocar o estudante em um emaranhado experimental,
contribuindo para afastar dele o entendimento mais
profundo sobre os conceitos que queremos trabalhar.
Isto e especialmente valido quando tratamos das turmas dos cursos de Licenciatura em Fsica, pois alem
de se defrontarem com quest~oes relativas ao conteudo
espec co, estes estudantes tambem est~ao vivenciando
procedimentos praticos de ensino em disciplinas experimentais. A tend^encia de reproduzir estes procedimentos praticos posteriormente com seus futuros alunos e
grande.
Baseados por um lado em avancos conceituais recentes das pesquisas em ensino de ci^encias, e por outro em nossa experi^encia docente junto aos estudantes
do Curso de Licenciatura em Fsica da Unesp - Bauru,
convidamos docentes e estudantes de Licenciatura em
Fsica, para re etirmos sobre alguns aspectos relacionados aos processos de ensino e aprendizagem na disciplina Laboratorio de Eletromagnetismo, tentando analisar o que poderia ser mudado nestas praticas para que
haja melhor aproximac~ao entre seus objetivos de aprendizagem e os resultados praticos observados.
II Repensando alguns aspectos
Nas pesquisas realizadas durante as tr^es ultimas
decadas sobre o aprendizado de ci^encias pode-se notar
consenso sobre pontos importantes pertinentes ao processo. Um destes pontos de consenso e a import^ancia
de entender o aprendizado em termos de desenvolvi-
472
mento conceitual, em vez de simples acrescimo de novas informac~oes [11], ou de simples substituic~oes de
concepc~oes existentes por novas [12]. Desenvolvimentos mais recentes [2,5] consistem em resgatar algumas
propostas pioneiras sobre o tema [3] dando um enfoque
investigativo as praticas de laboratorio.
Estas pesquisas apresentam fortes implicaco~es para
as praticas em sala de aula por mudarem a concepc~ao,
adotada muitas vezes, de que as praticas de laboratorio
devam ser meras ilustrac~oes da teoria. Quando abordados por este prisma os experimentos realizados adquirem uma orientac~ao rgida e dogmatica [2], na qual
o criterio de verdade e por excel^encia o da autoridade
da teoria, do docente, e do livro-texto. Segundo este
ponto de vista os experimentos s~ao usados para veri car se existe ou n~ao uma concord^ancia dos resultados
com a teoria, e, n~ao raro, quando existe a negativa,
a rma-se que o experimento deve ser repetido ate que
se consiga o resultado \adequado".
Em contraposic~ao a algumas propostas de que os
experimentos neste nvel devam necessariamente implicar em um alto grau de complexidade, observamos
que, neste caso, a probabilidade de termos um enfoque tecnicista do problema apresentado e grande, conforme apontado por Salinas [2], Sebastia [4] e White [9].
Os objetivos desta disciplina n~ao s~ao prioritariamente
tecnicos. Preferimos a proposta de Nedelsky [3] de que
a func~ao central dos laboratorios de fsica e dar ao estudante a oportunidade de explorar os varios aspectos da
relac~ao entre a descric~ao fsica da realidade e a propria
realidade, acrescida dos aspectos motivacionais a nvel
de experi^encia pessoal e de trabalho em grupos [8,9,10].
Quando os experimentos s~ao demasiadamente complexos n~ao ha tempo para o estudante re etir e analisar em detalhe o problema proposto. Nestes casos
os grupos de estudantes acabam passando diretamente
a execuc~ao do experimento, a qual e geralmente descrita em etapas bem de nidas pelo professor, sem antes
ter uma noca~o clara do seu proposito. Os aspectos negativos deste tipo de pratica tambem s~ao observados
por outros pesquisadores [4,6,9]. Neste caso jarg~oes do
tipo \o experimento mostrou-se de acordo com a teoria", sem que nenhum espaco para levantar hipoteses
tenha sido propiciado, muito pouco acrescentam ao entendimento dos estudantes sobre os aspectos fsicos e
tambem sobre o aspecto da natureza da ci^encia que
esta sendo \testada".
Desta maneira, podemos considerar que os principais quesitos para escolher os problemas experimentais
a serem abordados s~ao aqueles de proporcionar o adequado tema e o adequado nvel de hipoteses a serem
testadas, de acordo com o estagio de desenvolvimento
cognitivo, e com o interesse dos estudantes.
J. Humberto Dias da Silva
III Uma proposta baseada no
processo investigativo
A partir das considerac~oes sobre os problemas levantados acima, foi possvel elaborar uma proposta para
a disciplina Laboratorio de Eletromagnetismo do curso
de Licenciatura em Fsica da Unesp-Bauru. Nesta proposta cada experimento foi dividido em tr^es etapas. Na
primeira etapa um problema aberto, tema do experimento, e apresentado aos estudantes a m de promover
sua re ex~ao sobre o tema. O resultado das ideias que
surgem durante esta re ex~ao e analisado, em classe e em
re ex~ao do professor apos a aula. No segundo momento
uma proposta de experimento, elaborada pelo professor sobre o mesmo tema, e apresentada aos estudantes.
Nesta proposta levamos em considerac~ao, e tentamos
propor como hipoteses, algumas ideias levantadas pelos
estudantes no momento anterior. A analise dos resultados, a exposic~ao das discuss~oes, e o fechamento de
algumas conclus~oes sobre o experimento proposto pelo
professor, e realizada em sala, na ultima etapa.
Nesta proposta, os aspectos de investigac~ao do trabalho de laboratorio no sentido proposto por Gil Perez
[5] s~ao enfatizados. Resumimos os principais pontos da
proposta em:
i) Propor situac~oes-problema em vez de experimentos
fechados.
ii) Analisar, aproveitar, e valorizar as propostas dos estudantes, colocando-as quando possvel como hipoteses
a serem testadas.
iii) N~ao menosprezar nesta disciplina o potencial
didatico de experimentos semi-quantitativos que envolvam a aplicac~ao pratica direta de conceitos, propiciando
terreno adequado para o desenvolvimento conceitual e
das habilidades dos estudantes.
iv) Permitir que os estudantes tenham propostas sobre
a maneira de encarar o problema fsico e de executar os
experimentos.
v) Propiciar cooperac~ao e integrac~ao dos grupos na
soluc~ao dos problemas apresentados e na analise coletiva dos resultados obtidos.
IV Exemplos de aplicac~ao
Para ilustrar, vamos apresentar aqui dois experimentos realizados na disciplina durante o primeiro semestre de 2002. Apresentamos os experimentos com pequeno grau de orientac~ao propositalmente, para n~ao
incorrer no erro de propor experimentos totalmente
direcionados. Sob este ponto de vista vamos analisar que benefcios experimentos quantitativos ou semiquantitativos podem apresentar aos estudantes.
No primeiro exemplo propusemos um experimento
para medir o momento magnetico de uma barra imantada. Alguns pontos sobre a aplicabilidade que o conhecimento do momento magnetico possa ter no cotidiano
dos estudantes foram salientados. Como ilustrac~ao citamos a quest~ao do dimensionamento dos alto falantes
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de equipamentos sonoros, e as cabecas de gravac~ao de
tas e cart~oes magneticos. Nenhum equipamento e trazido ao laboratorio, apenas a pequena barra cilndrica,
objeto da proposta. Assim os estudantes s~ao confrontados com o problema, e apos um intervalo geralmente
pequeno de maturac~ao, algumas propostas comecam a
surgir, e desencadeiam um interessante processo de propostas onde os estudantes acabam mostrando muitas de
suas concepc~oes a respeito do tema. Estas concepc~oes
podem ser trabalhadas no momento em que s~ao expressas e tambem na aula seguinte. Nesta etapa a interac~ao
que ocorre nos grupos e fundamental para o processo.
A partir das quest~oes levantadas pelos grupos, o professor atento pode expor em sala ricos aspectos associados a historia da ci^encia, ao conhecimento formal,
e a aplicac~oes praticas da disciplina. Algumas vezes
isto e difcil de realizar no instante em que as quest~oes
importante que os pontos levantados peaparecem. E
los grupos tambem sejam considerados pelo docente
durante a semana que precede a realizac~ao do experimento, para serem retomados em melhores condic~oes
na aula seguinte.
As propostas dos estudantes para realizac~ao de experimentos para medir o momento magnetico de uma
barra foram interessantes. Por exemplo, podemos citar
a proposta de uma equipe que sugeriu a colocac~ao da
barrinha em ^angulo com o campo de um grande im~a em
forma de U, em um eixo perpendicular ao campo, para
realizar movimento oscilatorio. O momento magnetico
seria determinado a partir da frequ^encia de oscilac~ao.
No esquema, reservamos o segundo par de aulas
para tentar promover coletivamente uma evoluc~ao das
representac~oes anteriormente apresentadas. Isto foi
feito partindo dos conceitos apresentados pelos estudantes, enfatizando na proposta de experimento os pontos
que apresentaram conceitos a serem trabalhados. Uma
das possibilidades e introduzirmos alguns dos conceitos como hipoteses de veri cac~ao na proposta dos experimentos [5], de maneira que a mudanca conceitual
possa ser trabalhada de forma mais natural, sem necessariamente enfocarmos o con ito entre a representac~ao
previa do estudante e o conceito aceito dentro da teoria
estudada. Isto propicia um carater de investigac~ao ao
processo de aprendizagem, no sentido mais diretamente
proposto por Gil Perez [5], mas que tambem e apontado
como altamente bene co por outros autores [2-4,6,9].
Propusemos aos estudantes o experimento quantitativo classico da medida do momento magnetico de uma
barra usando uma balanca de torc~ao e um solenoide.
Neste experimento um campo magnetico causado por
corrente contnua que percorre o solenoide provoca um
pequeno torque na barra, o qual e equilibrado pelo
torque mec^anico de uma balanca de torc~ao. Com a
devida calibrac~ao e a considerac~ao de fatores como o
campo magnetico terrestre, o comprimento ( nito) do
solenoide, e a correta triangulac~ao para obtenc~ao do
^angulo de equilbrio, o experimento pode ser realizado
com um erro em torno de 2%.
Neste experimento ocorreram di culdades de visu-
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alizac~ao por parte dos estudantes. A principal di culdade foi identi car corretamente os varios ^angulos
que devem ser usados na execuc~ao do experimento. Ha
^angulos envolvidos na calibrac~ao da balanca, entre o
eixo do solenoide e o eixo do im~a, o ^angulo de deex~ao da balanca ao aplicar o campo magnetico, e o
^angulo interno do solenoide, quando consideramos a
aproximac~ao de solenoide nito. Houve di culdades
tambem na determinac~ao do a^ngulo de de ex~ao a partir de triangulac~ao do feixe de laser. Com esta complexidade o tempo de duas horas foi escasso para a
realizac~ao do experimento e algumas equipes optaram
por retornar em horarios diversos para complementarem ou tomarem novos dados. Um detalhe importante,
quando o tempo permite, e numerar algumas barras
e pedir para que os varios grupos mecam cada uma
delas, identi cando-as com as medidas. Isto e importante por possibilitar a comparac~ao entre os dados dos
varios grupos, e permitir que o teste das hipoteses seja
feito com troca de informac~oes e debates intermediados
entre grupos. Assim, os estudantes encontram espaco,
inexistente na maioria das propostas, para explicar suas
hipoteses e debater suas ideias coletivamente. Este processo e rico e contribui de maneira signi cativa para o
sucesso da proposta.
Apesar das propostas e discuss~oes interessantes
ocorridas, o resultado da analise neste experimento
muitas vezes se perdeu na complexidade tecnica dos
dados. Por este motivo optamos aqui por analisar em
maior detalhe os resultados obtidos no experimento
semi-quantitativo, apresentado a seguir. N~ao propomos com isto o abandono de propostas quantitativas.
Porem e importante n~ao enfatizar demasiadamente os
aspectos quantitativos dos experimentos em detrimento
de outros aspectos igualmente importantes relativos ao
aprendizado.
Para citar um experimento semi-quantitativo utilizado na disciplina, recorremos a determinac~ao de superfcies equipotenciais e campos eletricos em geometrias diversas.
O problema colocado foi: Como os campos eletricos
podem ser medidos na pratica? Que experimento voc^e
faria para medir os campos eletricos e mapear as supercies equipotenciais?
Vejamos inicialmente algumas propostas feitas pelos
estudantes:
Grupo A - \colocamos um dipolo eletrico conhe-
cido num campo eletrico cujo modulo desejamos saber.
Atraves do torque provocado no dipolo, podemos calcular o modulo de E . Isto pode ser feito usando uma
balanca de torc~ao, de maneira analoga ao experimento
anterior".
Comentario: esta proposta indica que o experimento sobre momento magnetico fez parte da bagagem
dos estudantes no experimento seguinte. Uma interessante associac~ao entre o momento magnetico da barra
e o momento de dipolo eletrico foi realizada, sem que
nenhuma menc~ao formal tivesse sido feita pelo professor. Embora a proposta seja correta do ponto de vista
474
conceitual, os estudantes n~ao perceberam que ela seria
de difcil realizac~ao pratica, pois n~ao se pode manter facilmente dipolos eletricos para servirem de prova, contrariamente ao que ocorre com os dipolos magneticos.
Alem disso a proposta aparentemente n~ao leva em considerac~ao a variac~ao espacial do campo, e o consequente
esforco de tomada de dados que teria que ser empregado
para um mapeamento adequado do campo. Estes aspectos praticos negativos n~ao diminuem entretanto o
interesse da proposta.
Grupo B - \criar um campo eletrico uniforme em
uma determinada regi~ao. Isso pode ser feito atraves de
placas paralelas" (apresentam gura de geometria de
placas paralelas, e duas cargas negativas colocadas em
plano paralelo as placas, com linhas de campo corretamente desenhadas). \Se colocarmos duas cargas pun-
tiformes iguais a uma dist^ancia l da placa carregada
positivamente, havera uma forca exercida pelo campo
eletrico E nas duas cargas e estas receber~ao uma certa
acelerac~ao e velocidade como mostra a gura". Ilustra-
ram com vetores F e v colocados em uma das cargas
negativas e dirigidos para a placa positiva.
Comentario: notamos que duas cargas foram utilizadas, provavelmente para denotar a const^ancia do
campo. Observamos que aqui tambem a const^ancia do
campo foi assumida. N~ao explicitaram como as grandezas mencionadas seriam medidas.
Grupo C - Apresentam uma gura onde a parte externa da esfera de um gerador Van de Graa e ligada a
uma placa plana que se encontra numa cuba com oleo e
gr~aos de farinha. Existe outra placa paralela na cuba,
porem nada esta conectado a esta. As placas est~ao a
uma dist^ancia maior que seu comprimento. Linhas de
campo (n~ao uniforme) foram desenhadas corretamente,
porem as equipotenciais foram desenhadas como linhas
paralelas as placas. \liga-se o positivo do Van de Gra-
a em um dos eletrodos. Basicamente com isto pode-se
ver as linhas de campo, e assim, ja e possvel imaginar
as superfcies equipotenciais sabendo-se que as mesmas
s~ao perpendiculares as linhas do campo eletrico. Podemos perceber que o campo eletrico e mais intenso na
regi~ao onde ha maior concentrac~ao dos gr~aos de farinha, ou seja, proximo aos eletrodos. Nisso tudo considerando que os gr~aos de farinha n~ao adquirem carga
no contato com o lquido (oleo). Uma possvel medida
para o campo eletrico seria tirar uma foto da formac~ao
das linhas de campo e contar os gr~aos de farinha numa
determinada regi~ao, e com isso calcular a densidade por
centmetro cubico. A mesma devera diminuir a medida
que nos afastamos das placas".
Comentario: os estudantes utilizaram um experimento demonstrativo apresentado a eles no laboratorio
basico (Laboratorio de Fsica Geral IV), e acrescentaram o interessante processo de fotografar e medir a densidade de gr~aos para estimar o valor do campo. A proposta e interessante o su ciente para possvel utilizac~ao
como hipotese a ser investigada no experimento proposto pelo professor. Infelizmente n~ao dispomos dos
equipamentos fotogra cos necessarios para promover a
J. Humberto Dias da Silva
comparaca~o entre os valores de campo determinados
desta maneira e os valores medidos no experimento.
Nota-se a contradic~ao entre a a rmac~ao de que as equipotenciais deveriam ser perpendiculares ao campo com
o desenho das equipotenciais paralelas as placas. Esta
foi uma das raras propostas que levaram em conta variac~oes espaciais do campo, enquanto nos outros grupos
foi observada uma tend^encia clara de n~ao se levar em
conta estas possveis variac~oes espaciais.
Na aula seguinte, correspondente a segunda etapa
do experimento, propusemos um experimento para o
mapeamento das superfcies equipotenciais utilizando
uma cuba com agua, dois eletrodos de formas a escolher (pelo menos tr^es pares), uma fonte para polarizar,
e um voltmetro digital para medir as diferencas de potencial. Algumas instruc~oes foram dadas para garantir
um mapeamento adequado das equipotenciais. O intuito era propiciar condic~oes para que os estudantes
\vissem" o campo a partir das equipotenciais, tendo
a possibilidade de testar varios aspectos presentes nas
suas concepco~es. Na sequ^encia apresentamos alguns resultados das interpretac~oes dos grupos:
Grupo A - Relataram que o campo deveria ser paralelo ao eixo que une uma placa a uma carga circular
pequena (identi cada por eles como puntiforme), apesar de terem calculado o campo utilizando as dist^ancias
perpendiculares as equipotenciais.
Grupo B - Esperavam que as superfcies equipotenciais para a simulac~ao de um dipolo fossem esferas
conc^entricas com os centros dos polos.
Grupo C - Utilizaram na determinac~ao dos campos
apenas as linhas centrais de simetria nas tr^es geometrias
escolhidas (duas placas, placa e ponto, dois pontos).
Em nossa opini~ao o principal ponto relacionado ao
conhecimento espec co deste experimento foi tratar
com os estudantes o conceito de gradiente, e a consequente ligac~ao entre potencial e campo eletrico. Analogias com situac~oes de mapeamento topogra co de relevos, levou-os a um melhor entendimento das curvas
equipotenciais, permitindo que calculassem o modulo e
indicassem corretamente a direc~ao e o sentido do
campo usando as equipotenciais tracadas. Antes
desta fase, os estudantes citam a relac~ao E= grad (V) no corpo do relatorio, porem pouqussimas
vezes conseguiram indicar, a partir das equipotenciais
obtidas na pratica, uma maneira de calcular E usando
esta relaca~o. Isto quer dizer que o docente que proponha que o campo eletrico seja calculado usando esta
relac~ao em um roteiro de experimento, por melhor intencionado que seja, esta perdendo uma grande oportunidade de deixar que o estudante aprofunde seus conhecimentos, pensando ele proprio esta maneira de encarar
o problema.
Este ultimo experimento n~ao apresenta as vantagens
quantitativas do primeiro, e talvez alguns professores
possam acha-lo simples demais para um Laboratorio de
Eletromagnetismo do terceiro ano. Porem em troca do
aspecto quantitativo podemos citar algumas qualidades. Em primeiro lugar o unico equipamento necessario
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a sua realizac~ao e um voltmetro, que atualmente pode
ser adquirido a baixo custo por uma escola ou que talvez possa ser encontrado entre as ferramentas de trabalho de um estudante com pai eletricista. O experimento pode ser realizado utilizando-se como fonte de
tens~ao uma bateria de 9V, ou um conjunto de pilhas de
lanterna. Atraves das linhas equipotenciais bidimensionais tracadas o estudante pode descobrir a relac~ao entre
potencial e campo eletrico, exempli cando este ultimo
com uma maneira concreta, ate ent~ao provavelmente
desconhecida. Colocados em quest~ao antes do experimento se o campo eletrico tinha uma exist^encia real
ou se era mera abstrac~ao, os estudantes, mesmo neste
grau, caram em sua maioria indecisos, e optaram muitas vezes por considerar que o campo era apenas uma
entidade \teorica" que n~ao conseguiramos expressa-lo
de uma maneira pratica. Entre os que responderam que
o campo possua uma exist^encia real, muitos descreveram o experimento com cargas para testa-lo \soltase uma carga com massa conhecida e mede-se a acelerac~ao". Porem quando indagados a respeito de como
seria possvel saber se o campo seria constante ou n~ao
naquela regi~ao do espaco, nenhum fez menc~ao a alguma
maneira de mapear o campo. Segundo nosso entendimento este experimento, embora semi quantitativo,
apresenta um real valor no contexto da disciplina por
ilustrar de maneira metodologicamente simples uma
grandeza fsica muito importante para a formac~ao do
estudante.
V Comentarios dos estudantes
sobre a proposta
Durante o desenvolvimento da disciplina os estudantes
zeram varios comentarios sobre as modi cac~oes propostas para a disciplina. Com o intuito de entender
como a proposta foi recebida pelos estudantes, apresentamos aqui alguns pontos que consideramos importantes em seus comentarios.
Ao conhecer a proposta da disciplina e nos primeiros
experimentos houve duvidas quanto ao que se pretendia que fosse relatado, e quanto ao criterio que seria
usado nas correc~oes dos trabalhos, em alus~ao clara a
preocupac~ao com relac~ao aos criterios de avaliac~ao a
serem adotados.
Durante a realizac~ao dos primeiros experimentos
houve um desconforto inicial em relac~ao aos procedimentos a serem utilizados nos experimentos propostos
pelo professor, pois apesar de que os objetivos, e ate
certo ponto os mecanismos principais a serem considerados foram estabelecidos, outros pontos foram deixados em aberto no experimento, entre eles a metodologia
de aquisic~ao e interpretac~ao dos dados.
Estas di culdades iniciais foram superadas durante
o decorrer do semestre, e em nosso entender contriburam para desenvolver uma maior autonomia dos
estudantes frente a situac~oes experimentais. Conforme
apontado por Barolli [8], a superac~ao das di culdades
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iniciais e os avancos da autonomia observados, podem
ser atribudos em parte a melhor estruturac~ao dos grupos com o decorrer do tempo de interac~ao. Esta melhor
estruturac~ao pode auxiliar no estabelecimento de ac~oes
cooperativas que contribuem para a superaca~o das diculdades praticas.
A possibilidade de aproveitar melhor suas proprias
ideias foram pontos importantes sobre os quais os estudantes zeram comentarios em varias oportunidades
manifestando clara aprovac~ao. Em nossa avaliac~ao este
e um dos principais pontos favoraveis da proposta: propicia motivac~ao e envolvimento dos estudantes no sentido de utilizar os conhecimentos disponveis para abordar situac~oes praticas. Trocas de informac~oes entre os
membros de cada grupo e entre os grupos da turma, e
a intermediac~ao do professor foram favorecidas de maneira natural nesta din^amica.
Uma di culdade n~ao claramente percebida pelos estudantes, mas externada em seus comentarios em varias
ocasi~oes, foi a de considerar a proposta do professor
como sendo a unica \correta" e de tentar avaliar a validade ou n~ao de sua proposta pela semelhanca com a
proposta do professor. Tal postura dos estudantes e um
problema real deste tipo de proposta e necessita uma
atenc~ao especial do professor que se prop~oe a implementa-las. Esta e uma consequ^encia negativa de metodologias tradicionalmente colocadas para o estudante,
e que merece intervenc~oes cuidadosas do professor.
Apesar da grande import^ancia imputada por nos
as propostas dos estudantes, o envolvimento posterior
dos grupos com suas proprias propostas n~ao foi grande.
Atribumos isto em parte a di culdade mencionada no
paragrafo anterior e tambem a facilidade dos estudantes em modi carem suas propostas a partir do conhecimento de novos dados, ou de objec~oes consistentes que
possam ser levantadas sobre seus argumentos.
VI Considerac~oes nais
Nesta abordagem do Laboratorio de Eletromagnetismo
os estudantes tiveram a oportunidade de fazer uma
proposta de experimento, de analisar os resultados de
experimento proposto pelo professor, e de fazer uma
analise comparativa entre sua proposta de experimento
e aquele proposto pelo professor. Entretanto, o experimento realizado ainda n~ao e verdadeiramente uma investigac~ao proposta pelo grupo de estudantes, e sim
uma investigac~ao dirigida. Apesar disto existe espaco
para que alguns aspectos propostos diretamente pelos
estudantes sejam enfocados: se o experimento proposto
pelo professor contiver hipoteses de veri caca~o relacionadas as propostas as representac~oes previas dos estudantes, muitas das quais manifestaram-se na etapa
inicial de proposic~ao, um dos importantes objetivos da
disciplina sera atingido [3,5].
Nesta disciplina e importante termos em conta
que, em muitas situac~oes, a inadequada complexidade dos experimentos propostos associada ao pequeno
476
tempo dedicado a re ex~ao sobre os conceitos e interpretac~ao dos resultados, pode infelizmente levar a poucos avancos na direc~ao desejada, ou seja ao efetivo aprofundamento do entendimento dos conceitos por parte
dos estudantes. Este e um aspecto negativo, pois alem
do entendimento espec co da disciplina os estudantes
de licenciatura est~ao vivenciando exemplos dos processo
de ensino e aprendizagem e dos relacionamentos em
grupo [8,9,10] aplicados a soluc~ao de situac~oes praticas
O papel dos grupos, o qual ja tem sua import^ancia
em abordagens tradicionais de disciplinas de laboratorio, e fundamental para o sucesso deste tipo de
proposta. A interac~ao entre os membros de cada grupo
e entre grupos foi especialmente produtiva durante as
proposic~oes dos estudantes. Neste momento houve uma
forte interac~ao entre os membros de cada grupo, e uma
abertura muito maior que a normalmente observada
para a mediac~ao buscada junto ao professor. A mediac~ao do professor ocorreu principalmente quando algum membro propunha um experimento ao grupo e tentava explicar baseado em seu entendimento dos conceitos fsicos. As propostas e justi cativas geralmente causavam questionamentos, troca e confronto de ideias com
os colegas. Isto tambem promoveu de maneira natural
o papel do professor como mediador das discuss~oes.
A di culdade rotineira de contarmos com menos
tempo do que gostaramos foi potencializada nesta proposta. Para tratar da profus~ao de ideias, que ocorre
quando deixamos um problema aberto para considerac~ao dos grupos, e necessario um intervalo de tempo
geralmente n~ao disponvel nas propostas tradicionais.
Para tentar superar esta di culdade foi adotado o procedimento de deixar uma semana para cada uma das
tr^es etapas: propostas, realizac~ao, e discuss~oes (a disciplina e oferecida em duas horas semanais). Isto implica que um menor numero de experimentos pode ser
tratado (em geral 4 a 5 por semestre!). Porem o fato
de termos realizado menos experimentos n~ao signi ca
que tenhamos abordado um numero menor de situac~oes
praticas e tratado menos temas experimentais, alias,
tem-se a impress~ao clara de que ocorre justamente o
oposto. Pelas proprias respostas e atitudes dos estudantes, nos parece que com esta abordagem estamos mais
proximos dos objetivo do laboratorio didatico: promover a oportunidade ao estudante de confrontar seus conceitos sobre a fsica com o resultado dos experimentos,
e de repartir suas concepc~oes com os outros estudantes
e com o professor [5,8,9,10].
O fato de podermos abranger os itens principais
da proposta em maior ou menor grau em cada experimento, justi ca a diminuic~ao do numero de experimentos que podem ser trabalhados na disciplina pelo
aumento da efetividade em que cada topico e trabalhado. Alem disso, pela riqueza de troca de experi^encia
J. Humberto Dias da Silva
que proporcionam, podemos dizer que ao inves de reduzir o numero de temas abordados (com a reduc~ao
do numero de experimentos) o numero de temas abordados na disciplina e ampliado, pois ha espaco para
se discutir aspectos historicos, aplicaco~es e abrang^encia
dos conceitos.
Longe de representar uma proposta fechada, este
conjunto de ideias e um convite a docentes e estudantes de licenciatura a re etirem sobre o aprendizado no
Laboratorio de Eletromagnetismo.
Agradecimentos
Agradeco aos estudantes do curso de Licenciatura
em Fsica da Unesp - Bauru, e aos Professores Elizabeth Barolli e Fernando Bastos, pelas interessantes
discuss~oes, ao Mestre Julio Cesar Ribeiro, cujo trabalho de orientac~ao me levou a repensar minhas proprias
praticas de ensino, e a dois estimados professores de
fsica experimental Jorge Ivan Cisneros e Curt Egon
Hennies, do IFGW - Unicamp, que me propiciaram,
enquanto estudante, o gosto pelas disciplinas experimentais.
Refer^encias
[1] D. Gil Perez e M. G. Ozamis, Ensen~anza de Las Ciencias y la Matematica, Editorial Popular S.A / Ministerio de Educacion y Ciencia, Espanha, 1992.
[2] J. S. Salinas, L.C. de Cudmani, Revista de Ense~nanza
de la Fsica, 5, 10 (1992).
[3] L. Nedelsky, Am. J. Phys. 26, 51 (1958).
[4] J.M. Sebastia, Ense~nanza de Las Ciencias, 5, 196
(1987).
[5] D. Gil Perez, P. Valdes Castro. Ense~nanza de las
Ci^encias, 14, 155 (1996).
[6] M. E. Gonzales, Ense~nanza de las Ci^encias, 10, 206
(1992).
[7] J.C. Ribeiro, O Papel do Professor no Laboratorio de
Fsica Basica: Um Exemplo Ilustrativo, Dissertac~ao de
Mestrado, Unesp-Bauru (2000).
[8] E. Barolli, Re ex~oes sobre o Trabalho dos Estudantes
no Laboratorio Didatico, Tese de Doutoramento, USPSP, (1998).
[9] R.T. White, Int. J. Sci. Educ., 18, 76 (1996).
[10] E. Barolli e A. Villani, Ci^encia e Educac~ao, 6, 1 (2000).
[11] A Villani, Revista Brasileira de Ensino de Fsica, 6, 76
(1984).
[12] E.F. Mortimer, Investigac~oes em Ensino de Ci^encias,
1, 22 (1996).
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Vitiligo: a complex disease and a complex approach
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Molecular cytogenetics
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Vitiligo: a complex disease and a complex
approach Rasheedunnisa Begum1*, Yogesh S. Marfatia2, Naresh C Laddha1, Mitesh Dwivedi1, Mohmmad Shoab Mansuri1,
Mala Singh1 From International Conference on Human Genetics and 39th Annual Meeting of the Indian Society of
Human Genetics (ISHG)
Ahmadabad, India. 23-25 January 2013 IL4, CTLA4, SOD2, SOD3, NALP1, MYG1, TNFA, TNFB,
IFNG, IL10 and ICAM1 genes suggest that these genes are
strongly associated with vitiligo susceptibility. We are also
addressing the role of immune-regulatory genes with
respect to their expression in skin along with the effect of
selected cytokines on in vitro cultured melanocytes
derived from healthy and vitiliginous human skin to have
an insight towards vitiligo pathogenesis. We are also
exploring the potential microRNAs involved in pathogen-
esis of vitiligo. This integrated study will provide a better
understanding of the role played by oxidative stress and
autoimmunity in the pathogenesis of vitiligo in Gujarat
population and also to develop selective therapy and the
genetic marker/s for vitiligo. Vitiligo is an acquired, circumscribed hypomelanotic
skin disorder, characterized by milky white patches due
to loss of functional melanocytes from the epidermis. Prevalence of vitiligo is found to be very high in Gujarat
i.e., ~8.8%. Vitiligo is a multifactorial polygenic disorder
with a complex pathogenesis, linked with both genetic
and non-genetic factors. Several theories have been
proposed to explain the etiopathogenesis of vitiligo, but
none of the hypotheses explains the entire spectrum of
this disorder. We are addressing this complex disease in
our Gujarat population with various approaches. Our
study mainly deals with the evaluation of oxidative
stress, autoimmune, genetic and neurochemical hypoth-
eses in Gujarat vitiligo patients. We have shown that
our vitiligo patients exhibit significant oxidative stress
and thus, systemic oxidative stress could play a patho-
physiological role in precipitation of vitiligo in Gujarat
population. Our studies revealed that presence of
increased antimelanocyte antibodies and the imbalance of
T-cell (CD4+/CD8+ and Tregs) subsets along with their
functional defects might result in melanocyte destruction
in vitiligo patients. Our results on selected candidate genes
in conferring oxidative stress and autoimmunity suggest
that HLA-A*33:01, HLA-A*02:01,HLA-B*44:03, HLA-
DRB1*07:01 and a few studied polymorphisms in IL4,
CTLA4, SOD2, SOD3, GPX1, NALP1, MYG1, TNFA,
TNFB, IFNG and IL10 genes are strongly associated with
vitiligo susceptibility, whereas a few studied polymorph-
isms in PTPN22, MBL2, ACE, CAT, G6PD and SOD1
genes are not found to be significantly associated with
Gujarat vitiligo patients. * Correspondence: rasheedunnisab@yahoo.co.in
1Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao
University of Baroda, Vadodara, India
Full list of author information is available at the end of the article Begum et al. Molecular Cytogenetics 2014, 7(Suppl 1):I57
http://www.molecularcytogenetics.org/content/7/S1/I57 SPEAKER PRESENTATION Open Access Vitiligo: a complex disease and a complex
approach Gene expression studies of the Various factors such as the antioxidant status, LPO
(oxidative stress) levels and antimelanocyte antibody
titer decide the selective therapy for our vitiligo patients. The pathogenesis of vitiligo though partially understood
still remains complex and enigmatic to a greater extent. Though the condition may be precipitated by multiple
etiologies, the interaction of oxidative stress and
immune system clearly appears to be the key convergent
pathway that initiates and/or amplifies the enigmatic
loss of melanocytes in vitiligo. 1Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao
University of Baroda, Vadodara, India. 2Department of Skin & V.D., Faculty of
Medicine, The Maharaja Sayajirao University of Baroda, Vadodara, India. Authors’ details
1D
f B 1Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao
University of Baroda, Vadodara, India. 2Department of Skin & V.D., Faculty of
Medicine, The Maharaja Sayajirao University of Baroda, Vadodara, India. Published: 21 January 2014 Published: 21 January 2014 doi:10.1186/1755-8166-7-S1-I57
Cite this article as: Begum et al.: Vitiligo: a complex disease and a
complex approach. Molecular Cytogenetics 2014 7(Suppl 1):I57. doi:10.1186/1755-8166-7-S1-I57
Cite this article as: Begum et al.: Vitiligo: a complex disease and a
complex approach. Molecular Cytogenetics 2014 7(Suppl 1):I57. © 2014 Begum 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. 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.
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Novel Type of References for BMI Aligned for Onset of Puberty - Using the QEPS Growth Model
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Research Square (Research Square)
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cc-by
| 8,162
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Research Article Posted Date: February 11th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1152091/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 Novel type of references for BMI aligned for onset of puberty – using the QEPS growth
model Kerstin Albertsson-Wikland1*, Aimon Niklasson2, Lars Gelander1, Anton Holmgren2,3,
Andreas F.M. Nierop1,4 p
1Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology,
Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
2Göteborg Pediatric Growth Research Center, Department of Pediatrics, Institute of Clinical
Sciences, Sahlgrenska Academy, University of Gothenburg, Sweden
3Department of Pediatrics, Halmstad Hospital, Halmstad, Sweden
4Muvara bv, Multivariate Analysis of Research Data, Leiderdorp, The Netherland, Short title: Pubertal BMI reference Short title: Pubertal BMI reference Keywords: pubertal growth, personalized growth, biological age, SDS, reference population 1 1 Abstract Objectives: Despite inter-individual variations in pubertal timing, growth references are
conventionally constructed relative to chronological age (C-age). Thus, they are based on
reference populations containing a mix of prepubertal and pubertal individuals, making them
of limited use for detecting abnormal growth during adolescence. Recently we developed
new types of height and weight references, with growth aligned to age at onset of the pubertal
growth spurt (P-age). Here, we aim to develop a corresponding reference for pubertal BMI. Methods: The QEPS-height and weight models were used to define a corresponding QEPS-
BMI model. QEPS-BMI was modified by the same individual, constitutional weight–height-
factor (WHF) as computed for QEPS-weight. QEPS-BMI functions were computed with
QEPS weight and height functions fitted on longitudinal measurements from 1418
individuals (698 girls) from GrowUp1990Gothenburg cohort. These individual BMI functions
were used to develop BMI references aligned for height at AgeP5; when 5% of specific
puberty-related (P-function) height had been attained. Pubertal timing, stature at pubertal
onset, and childhood BMI, were investigated in subgroups of children from the cohort
GrowUp1974Gothenburg using the new references. Methods: The QEPS-height and weight models were used to define a corresponding QEPS-
BMI model. QEPS-BMI was modified by the same individual, constitutional weight–height-
factor (WHF) as computed for QEPS-weight. QEPS-BMI functions were computed with
QEPS weight and height functions fitted on longitudinal measurements from 1418
individuals (698 girls) from GrowUp1990Gothenburg cohort. These individual BMI functions
were used to develop BMI references aligned for height at AgeP5; when 5% of specific
puberty-related (P-function) height had been attained. Pubertal timing, stature at pubertal
onset, and childhood BMI, were investigated in subgroups of children from the cohort
GrowUp1974Gothenburg using the new references. Results: References (median, standard deviation score (SDS)) were generated for total BMI
(QEPS-functions), for ongoing prepubertal growth (QE-function) vs C-age, and for total BMI
and separated into BMI specific to puberty (P-function) and BMI gain from ongoing basic
growth (QES-functions), allowing individual growth to be aligned based on P-age. Growth in
basic BMI was greater than average for children categorized as tall and/or with high-BMI at
puberty-start. In children categorized as short at puberty-start, P-function-related-BMI was
greater than average. 2 2 Conclusions: Use of these new pubertal BMI references will make it possible for the first
time to consider individual variations owing to pubertal timing when evaluating BMI. Abstract This
will improve the detection of abnormal changes in body composition when used in
combination with pubertal height and weight references also abnormal growth. Other benefits
in the clinic will include improved growth monitoring during treatment for children who are
overweight/obese or underweight. Furthermore, in research settings these new references
represent a novel tool for exploring human growth. Conclusions: Use of these new pubertal BMI references will make it possible for the first
time to consider individual variations owing to pubertal timing when evaluating BMI. This
will improve the detection of abnormal changes in body composition when used in combination with pubertal height and weight references also abnormal growth. Other benefits
in the clinic will include improved growth monitoring during treatment for children who are
overweight/obese or underweight. Furthermore, in research settings these new references
represent a novel tool for exploring human growth. 3 3 Background Unlike the previous reference developed around 2000 in
Sweden that have used estimates of ongoing, childhood growth from the Infancy–Childhood–
Puberty (ICP) growth model (11-14), we constructed prepubertal growth using the QE-
functions of the QEPS-model (15, 16). Individual growth is described by the QEPS-model
using four mathematical functions: a Q (Quadratic) and E (Exponential) function arising
before birth and resulting in the prepubertal growth, to which a specific pubertal growth
function, P, is added; the QE-growth is during puberty transformed and ended by a S (stop)
function; resulting in basic, QES-function-growth during puberty (15). Thereafter, we also
developed a QEPS-model for weight and constructed a reference for prepubertal weight gain
during adolescence (17). of adolescence which is characterized by great inter-individual variations in growth because
of differences between individuals in the C-age at which they enter puberty (8, 9). When in
2020 developing the most recent Swedish height references, to account for this we included a
‘prepubertal’ reference showing the growth that during adolescence is continuing
independently of puberty (10). Unlike the previous reference developed around 2000 in
Sweden that have used estimates of ongoing, childhood growth from the Infancy–Childhood–
Puberty (ICP) growth model (11-14), we constructed prepubertal growth using the QE-
functions of the QEPS-model (15, 16). Individual growth is described by the QEPS-model
using four mathematical functions: a Q (Quadratic) and E (Exponential) function arising
before birth and resulting in the prepubertal growth, to which a specific pubertal growth
function, P, is added; the QE-growth is during puberty transformed and ended by a S (stop)
function; resulting in basic, QES-function-growth during puberty (15). Thereafter, we also
developed a QEPS-model for weight and constructed a reference for prepubertal weight gain
during adolescence (17). of adolescence which is characterized by great inter-individual variations in growth because
of differences between individuals in the C-age at which they enter puberty (8, 9). When in
2020 developing the most recent Swedish height references, to account for this we included a
‘prepubertal’ reference showing the growth that during adolescence is continuing of adolescence which is characterized by great inter-individual variations in growth because
of differences between individuals in the C-age at which they enter puberty (8, 9). Background For many decades, body mass index (BMI) has been the most commonly used variable to
define and evaluate body composition and weight status in children. In both research and
clinical practice, BMI, which is expressed as kg/m2, has been widely used to define whether a
child is of normal weight, underweight, overweight, or obese (1). While both height and
weight increase with chronological age (C-age), this is not the case for BMI. BMI rises in
infancy, then falls during childhood before rising again (2-4). The second rise in BMI is
usually seen some years before puberty and continues during the adolescent years, with BMI
only reaching adult levels sometime after adult height has been attained. In 2012, the
international obesity task force (IOTF) used merged BMI data from different countries to
create a worldwide-accepted reference with iso-lines defining overweight, obesity, and
thinness (5), which has been especially valuable for international comparison of research
studies. Beside this, there are national BMI references, such as those in Sweden which
include risk estimation curves for obesity (3). The ongoing obesity epidemic complicates the construction of new weight and BMI
references. Although the trend over time toward higher BMI seems partly to have plateaued
in some high-income countries, BMI is still rising in many parts of the world (6). Recently in
Sweden, for example, an increasing prevalence of overweight and obesity with age in both
girls and boys has been reported (7). Such changes in BMI must not be incorporated in
references reflecting optimal body composition. We addressed this issue in the recently
updated Swedish references for weight and BMI by omitting individuals with obesity from
the longitudinal BMI dataset used to develop the references (4). 4
It has been convention for growth references for height, weight, and BMI to be presented in
relation to C-age, making them suboptimal for detecting abnormal growth during the period of adolescence which is characterized by great inter-individual variations in growth because
of differences between individuals in the C-age at which they enter puberty (8, 9). When in
2020 developing the most recent Swedish height references, to account for this we included a
‘prepubertal’ reference showing the growth that during adolescence is continuing
independently of puberty (10). Ethical approval Ethical approval was obtained from the Regional Ethics Review Board in Gothenburg (Ad
91-92/131 -93 and Ad 444-08 T062-09). Informed consent was given by participants and
parents of individuals <18 years of age. Studies were conducted in accordance with the
principles of the Declaration of Helsinki. Background When in
2020 developing the most recent Swedish height references, to account for this we included a
‘prepubertal’ reference showing the growth that during adolescence is continuing Recently, we have developed a new type of pubertal height reference describing growth
aligned for onset of the specific P-function in the QEPS-model responsible for pubertal
height gain; this reference provides information on total growth, as well as separating out the
specific growth related to puberty from basic growth continuing during the pubertal years
(10, 18). Subsequently, we developed a QEPS model for weight to construct corresponding
references for pubertal weight gain (17). It now remains to develop a puberty-adjusted growth
model for BMI and to use it to develop references that enable the separation of prepubertal
and pubertal changes in BMI. 5
The aim of this study was to use the QEPS-models for height and weight to develop a growth
model for BMI, and to develop new BMI references that take biological maturation of the 5 5 individual during adolescence into account. Like the previous references, data used to
construct the new BMI reference will be obtained from longitudinally followed, healthy
children born at term to non-smoking mothers and Nordic parents, selected from the
GrowUp1990Gothenburg cohort born in Sweden (4). Separate references will be constructed
for (a) prepubertal BMI (Q- and E-functions), (b) total BMI (modeled by the Q, E, P and S-
functions), (c) BMI gain specific to puberty (by the P-function), and (d) BMI gain that is not
specific to puberty (basic BMI, by the Q, E, and S-functions). Subgroups of children from the
GrowUp1974Gothenburg cohort categorized based on the timing of puberty (early, average,
late), height at start of puberty (tall, short) or body composition during childhood (high BMI,
low BMI) will be used to explore the utility of the new references for monitoring BMI. reference Evaluation of the utility to research of the new pubertal BMI reference was made using data
from healthy children from the GrowUp1974Gothenburg cohort (2177 subjects: 1081 girls). The impact of grouping children according to P-age, for age at onset of puberty, for height,
and for childhood BMI was explored (see Supplemental Table S3 of Albertsson-Wikland et
al (10)). 1. Reference population from GrowUp1990Gothenburg cohort 6
In total, 1418 individuals (698 girls) from the GrowUp1990Gothenburg cohort were included
in the cohort used to construct the QEPS-BMI reference. This was the same population as
used to develop earlier C-age references for total weight and BMI, and those for weight-for-
height (see Supplemental Table S1 in Albertsson-Wikland et al (4)). The cohort included only
healthy children (see Table 1 in Albertsson-Wikland et al (4)) who had Nordic parents and 6 6 were born in Sweden around 1990 at full term (gestational age (GA) 37–43 weeks) to non-
smoking mothers. Information was available on longitudinal growth until adult height for all
participants. For more information see Albertsson-Wikland et al. (4, 18). QEPS-BMI method The QEPS-height (15) and QEPS-weight models (17), were used to define a corresponding
QEPS-BMI model. The QEPS-BMI model is expressed in kg0.5/m to be consistent with the
QEPS-weight model, which was expressed in kg0.5 to enable the additive property of the
QEPS weight functions. Where appropriate, we show BMI in kg/m2 in figures on the left axis
and BMI in kg0.5/m on the right axis. For detailed information about the QEPS-BMI model
see Supplement, including Supplemental Figures S1, S2, and S3. Like the QEPS-weight
model, QEPS-BMI was modified by an individual ‘constitutional factor’, a weight–height
factor (WHF): WHF=0, a ‘normal body constitution’, WHF>0, a heavy, and WHF<0, a lean
body constitution. Traditional references according to C-age for total and prepubertal BMI
were computed in two steps comparable to the ‘QEPS method used for the references’ section
in Supplement of Albertsson-Wikland et al. (18), but with QEPS height functions replaced by
corresponding QEPS-BMI functions. QEPS-BMI functions were computed with QEPS- 7 7 height functions fitted on height measurements and QEPS-weight functions fitted on weight
measurements while including information from fitted QEPS-height functions. height functions fitted on height measurements and QEPS-weight functions fitted on weight
measurements while including information from fitted QEPS-height functions. Comparison was made between the total BMI reference obtained using the QEPS-BMI
method and the previously published BMI reference obtained by applying the LMS method
(4). Both references had been created using the same data from the same population, see
Supplemental Figure S4. height functions fitted on height measurements and QEPS-weight functions fitted on weight
measurements while including information from fitted QEPS-height functions. Comparison was made between the total BMI reference obtained using the QEPS-BMI
method and the previously published BMI reference obtained by applying the LMS method
(4). Both references had been created using the same data from the same population, see
Supplemental Figure S4. Median curves for the QEPS-derived and LMS-derived references were similar; however, as
expected, variance was smaller for the QEPS-derived reference owing to computation with
fitted functions that excluded residual variation. A new reference for pubertal BMI was then
generated. The onset of puberty was identified based on height-specific P-function growth;
the point at which 5% of height Pmax had been obtained (AgeP5) was used to define the
onset of puberty (Supplemental Figure S2 in Albertsson-Wikland et al. (10))(16). Height for
each individual was then aligned according to age at the onset of puberty. QEPS-BMI method To achieve this for
BMI, all individual longitudinal QEPS-BMI-functions were aligned according to height at
AgeP5. For more information see Supplement. Statistical evaluations Longitudinal growth data for each individual were exported to Matlab® (version 9.3 R2017b,
The MathWorks). Estimation of individual QEPS height and weight parameters by nonlinear
fitting was conducted using the Matlab Curve Fitting Toolbox. The fitting procedure for
height is described in (15), and for weight in the Supplement (17). QEPS-BMI functions were
computed with resulting QEPS height and weight functions. To simplify general
mathematical formulae, age was assumed to be equal to age corrected for GA, here 37–43
weeks. All figures were prepared in Matlab; Figure 7 was finalized in Photoshop. 8 8 2. BMI references aligned for onset of puberty (P-age) The reference shown in Figure 2 is for total (QEPS) and basic (QES-function) BMI in kg/m2
(left axis) and kg0.5/m (right axis) for girls and boys aligned according to the onset of puberty,
estimated as AgeP5 for height, P-age. Specific P-function growth has been included in the
lower panel. BMI references in both figures are depicted from 4 years before to 10 years after
the onset of the pubertal growth spurt to capture changes in BMI relative to the acceleration
in height and weight that occurs during puberty. General differences in the timing of pubertal
height and weight functions resulted in undulations of the aligned BMI functions
(Supplemental Figure S3). 3. Rational for a puberty-aligned BMI reference Total BMI relative to C-age for girls and boys with an early, average, and late onset of
puberty is shown in Figure 3. As expected, BMI gain occurred sooner than average in
children with an early and later than average in children with a late onset of puberty. This
highlights the inadequacy for many adolescents of using a C-age-based BMI reference. 1. Total and prepubertal BMI references vs chronological age (C-age) The novel BMI references in kg/m2 (left axis) and in kg0.5/m (right axis) according to C-age
4–20 years for girls and boys, Figure 1. Total BMI is shown in color (red/blue), and BMI
gained independently of puberty, the prepubertal, (QE-function) BMI reference, in black. 4. Exploring pubertal BMI in subgroups from the GrowUp1974Gothenburg cohort Timing of puberty: Total (QEPS), basic (QES), and pubertal (P-function) BMI relative to the
onset of puberty for subgroups of girls and boys with an early, average, and late onset of 9 9 puberty is shown in Figure 4. For girls in the early and late puberty onset groups, the total
BMI gain and the gain in the basic component of BMI were somewhat higher than average;
for boys in the early and late puberty onset groups, gains in total and basic BMI were similar
to average. Puberty-specific BMI gain was greater than average in the early-puberty-onset
group and lower than average in the late-puberty-onset group. Stature: The total (QEPS-functions), basic (QES-functions), and pubertal (P-function) BMI
for girls and boys according to height at P-age, the age at the onset of puberty is shown in
Figure 5. For girls and boys who were tall at the onset of puberty, the basic component of
BMI was higher than average; for girls and boys who were short at the onset of puberty, this
component was lower than average. In contrast, puberty-specific BMI gain was lower than
average for the tall group and higher than average for the short group. Degree of childhood BMI: Total (QEPS-functions), basic (QES-functions), and pubertal (P-
function) BMI for girls and boys according to the highest BMI during childhood (girls:3.5–
7yrs; boys: 3.5–8yrs), Figure 6. Gains in the basic component of BMI were higher than
average in children in the high BMI group at the onset of puberty and lower than average in
the low BMI group. In the low BMI groups, puberty-specific BMI gain was lower than
average for girls and somewhat lower than average for boys. In the high BMI groups, puberty-specific BMI gain was somewhat higher than average for girls and similar to the
average for boys. 5. Using the new BMI pubertal growth charts prospectively for an individual child In order to monitor pubertal BMI changes prospectively for an individual child using the new
pubertal-age-BMI charts a manual procedure can be undertaken. Early identification of the
‘take-off’ in growth at the start of puberty can be achieved using the growth charts for total 10 and prepubertal height references (18). Figure 7 describes the prospective use of the pubertal-
age-adjusted reference to assess total BMI. For corresponding changes in weight, see Figure
7 in Albertsson-Wikland et al. (17). and prepubertal height references (18). Figure 7 describes the prospective use of the pubertal-
age-adjusted reference to assess total BMI. For corresponding changes in weight, see Figure
7 in Albertsson-Wikland et al. (17). New prepubertal and pubertal BMI references Here, we present the first pubertal BMI references adjusted for biological maturation based
on age at onset of the pubertal growth spurt in the individual child. Thereby, the growth of
any individual will be related only to peers with similar biological age and maturation. Here, we present the first pubertal BMI references adjusted for biological maturation based
on age at onset of the pubertal growth spurt in the individual child. Thereby, the growth of
any individual will be related only to peers with similar biological age and maturation. Another novel benefit of these BMI references is that they allow the separation of prepubertal
and pubertal components of BMI gain. During adolescence this makes it possible to look in
detail at the ongoing BMI gain that is unrelated to puberty in relation to the growth specific to
puberty. As such, we expect these references to be of utility in the clinic and in research-
based investigations of both puberty-independent and puberty-dependent changes in BMI. Moreover, a prepubertal BMI reference was also created, vs C-age, which will be particularly
important when considering body composition as BMI changes in children in whom puberty
is early or delayed. These new references complete a set of three growth references – for pubertal and prepubertal
height, weight, and BMI – developed using growth functions based on QEPS growth models
for height, weight, and BMI. In all cases, these references make it possible to align growth in
the individual relative to the onset of pubertal growth in the individual (10, 17) and as a set,
they will be valuable in pubertal and prepubertal individuals for evaluating growth in terms of
height, weight, and BMI, as well as for evaluating the relationship between weight and
height. To use growth references such as these effectively in healthcare and clinical settings, 11 11 estimation of the stage of pubertal maturation will be required alongside construction of the
growth chart for the individual; this includes investigation of the genitalia and breast
maturation in girls or testicular size in boys (14, 19). Using these three references alongside
estimates of pubertal maturation will help to decide which reference for each variable to use,
and will improve the precision with which we can evaluate changes in BMI and growth
during adolescence (10). BMI changes preceding height changes The occurrence of characteristic sex-specific changes in weight and body fat mass before
changes in pubertal growth suggest a relationship between energy storage in adipose tissue
and pubertal maturation (20). By aligning BMI in relation to the onset of puberty, the new
BMI references will allow us to explore in detail the changes in body composition (BMI and
weight) that occur some years before the increase in height associated with puberty
(Supplemental Figure S3). It is interesting to note that the BMI reference curves were observed to be undulating in
places. This appears to be due to differences in the timing of puberty-related changes in
height and weight, and to the type of tissue growth responsible for weight gain (e.g. fat vs
muscle). Thus, residual variation around individual predicted BMI for normal body
constitution might be related, not only to measuring errors, but also to individual variations in
the relationship of fat and muscle distributions as estimated by body compartment analyses
(dual-energy X-ray absorptiometry (DEXA)) or other comparable measurements (21). 12
Previously, we found sex differences in both the timing and amount of two types of weight:
earlier gain of type A weight in girls and greater gain of type B weight in boys (Supplemental
Figure S3). At present, we speculate that type A weight represents fat tissue because it
correlates with the sex difference in the timing of early pubertal fat gain, as described in the 12 12 study by Vizmanos & Marti-Henneberg, and that type B weight represents muscle tissue;
however, this needs to be confirmed in future studies (20, 22). Puberty-aligned BMI
represents a relative balance between weight and height, making interpretation of these BMI
results quite complex. Interestingly, the total BMI typical mean function for individual boys
and girls became quite similar over time, whereas the P-function-related curve showed sex
differences in both the timing and proportion of pubertal type A BMI gain and pubertal type
B BMI gain (Supplemental Figure S3). DEXA studies will be needed to explore this
observation in greater detail. Usefulness of a reference separating BMI growth functions during puberty The benefits of using a growth model to explore underlying growth regulating mechanisms
have been discussed previously (17, 23, 24). A novel finding from our analysis of subgroups
of children with different characteristics prior to entering puberty was that gain in basic BMI
was greater in tall boys and girls, when compared to either the reference, or to boys and girls
of average pubertal timing or stature. In contrast, lower-than-average basic BMI gain was
observed for children who were short at the start of puberty, although their P-function-related
BMI gain was greater than average. This is consistent with our previous investigations in
children with different BMIs in which we found a positive relationship between BMI and
greater than average gains in basic height and lower than average gains in puberty-specific
height (25). We also found that a high BMI was associated with greater than average gains in
basic BMI and that low BMI was associated with lower-than-average increases in basic BMI. 13
From a research perspective, the new BMI reference will be useful in the exploration of
correlates for predicting future health. It is already well known that childhood obesity is an
important factor associated with increased risk for reduced life expectancy. For example,
increased BMI during adolescence is associated with elevated cardiovascular morbidity, the 13 development of type 2 diabetes and different types of cancer during adult life (26-28). It is
also known that both prenatal and early postnatal nutrition have epigenetic effects on
developmental programming resulting in cardiovascular diseases, diabetes, overweight, and
obesity (29-32). As such, the ability to look at the different components of BMI during
childhood will be useful for exploring which variables help us to predict the development of
obesity. National growth screening and improved BMI monitoring during treatment Longitudinal follow up of children’s growth provides an important way of evaluating present
and future population health and should be conducted at a national level (33). National
monitoring of BMI-related child health will be more precise with the incorporation of
insights from the BMI references described here. We anticipate that investigations using the
new references will lead to re-evaluation of currently used BMI iso-line cutoffs especially
during the pubertal years. With the exception of surgical treatment during late adolescence (34, 35), interventions to
treat obesity during childhood are still generally unsuccessful (34). There is, therefore, a clear
need for more clarity on the underlying causes of childhood obesity so that appropriate
treatments can be developed. Gaining a greater understanding of the relationship of BMI to
puberty will be an important step in this journey. Another condition where more in-depth
understanding of BMI will be useful, is anorexia nervosa. Anorexia nervosa often occurs
during the adolescent period, and monitoring severity of the disease and its treatment will
benefit from using the new puberty onset aligned references for height, weight, and BMI (36-
38). The tool that we present here for investigating BMI changes in new ways and with
higher precision than previously possible will be of great utility in these situations. 14 14 Limitations with using BMI One of the limitations associated with using BMI as a measure of body composition in
growing individuals is that the correlation between BMI (w/h2) and height has been shown to
change with C-age, especially during adolescence. Similarly, other relationships between
weight and height vary with age, see Figure 2 in Karlberg & Albertsson-Wikland, where a
beta-factor was introduced (2). When developing the current QEPS-BMI model we therefore
implicitly applied the nonlinear regression coefficients within the QEPS weight model to
allow a similar correlation to be maintained across different age ranges. This indicates that
the standard formula for calculating BMI is not optimal for describing weight status during
childhood and puberty (2). Human weight represents the sum of fat, muscle, body water, visceral organs, and bones in an
individual. Despite the common use of BMI as a measure of adiposity or thinness, BMI
cannot differentiate between these different tissues. A further limitation of using BMI as a
measure of adiposity is that, while BMI correlates with fat mass in children on a group level,
there is not always a correlation at the individual level (39, 40). Conclusion We present new BMI references that can be used to evaluate prepubertal BMI in relation to
chronological age, as well as assessing pubertal and prepubertal changes in BMI relative to
the onset of the pubertal growth spurt in the individual. When used together with comparable
references developed using the QEPS-models for height and weight (10, 18), these novel
BMI references will facilitate improved detection and monitoring of growth abnormalities
during adolescence within the health care system. Furthermore, in research settings these new
references represent a novel tool for exploring human growth. The availability of this type of 15 reference is an important step towards more meaningful and informative clinical monitoring
of BMI development of the individual during the adolescent years. Declarations: Ethical approval: Ethical approval was obtained from the Regional Ethics Review Board in
Gothenburg (Ad 91-92/131 -93 and Ad 444-08 T062-09). Informed consent was given by
participants and parents of individuals <18 years of age. Studies were conducted in
accordance with the principles of the Declaration of Helsinki. Acknowledgements: The authors are grateful for the contributions of the students of the 12th
grade 1998/9 from the Gothenburg area schools, and their school nurses; the study team led
by C Pfeiffer-Mosesson and A Olsson; for Data support given by L Ljungberg and B
Bengtsson and for careful editing and language revision by H Crofts. Research funding: The authors acknowledge financial support from the Governmental
grants under the ALF agreement, ALFGBG-719041, ALFGBG-812951, ALFGBG-965451,
the Region Halland Grants, and the Foundation Växthuset for children. Competing interest: AFMN works for Muvara, Multivariate Analysis of Research Data,
Statistical Consultation, the Netherlands. KAW, AN, AH, and LG declare that they have no
competing interests. 16 Availability of data and materials: the data generated and analyzed during the current study
are not publicly available due to that these data are part of several ongoing studies and will be
publicly available at a later point in time but are available from the corresponding author on
reasonable request. Author contributions: KAW is the principal investigator of the study populations used. AFMN performed the modeling work for the QEPS model described here and made the
QEPS function estimations, reference values, and Figures 1–6 and Supplemental Figures S1–
S4. AN made Figure 7. KAW, AN, AH, LG, and AFMN have all made substantial
contributions to the conception, design, analysis, and interpretation of these data. KAW wrote
the first draft and AFMN the methods part. All authors revised the manuscript critically for
intellectual content and approved the final version to be submitted for publication. Consent of publication: Not applicable. REFERENCES Holmgren A, Niklasson A, Gelander L, Aronson AS, Nierop AFM, Albertsson-Wikland K. Insight into human pubertal growth by applying the QEPS growth model. BMC pediatrics. 2017; 17:107
17. Albertsson-Wikland K, Niklasson A, Gelander L, Holmgren A, Nierop AFM. Novel type of
references for weight aligned for onset of puberty - using the QEPS growth model. BMC
pediatrics. 2021; 21:507
18. Albertsson-Wikland K, Niklasson A, Holmgren A, Gelander L, Nierop AFM. A new Swedish
reference for total and prepubertal height. Acta Paediatr. 2020; 109:754-63
19. Rollof L, Elfving M. Evaluation of self-assessment of pubertal maturation in boys and girls
using drawings and orchidometer. J Pediatr Endocrinol Metab. 2012; 25:125-9
20. Vizmanos B, Marti-Henneberg C. Puberty begins with a characteristic subcutaneous body f
mass in each sex. European journal of clinical nutrition. 2000; 54:203-8 1. Holmgren A. The Quadratic-Exponential-Pubertal-Stop model is valid for analysing human
growth patterns and developing novel growth references. Acta Paediatr. 2021; Karlberg J, Luo ZC, Albertsson-Wikland K. Body mass index reference values (mean and SD) 4. Albertsson-Wikland K, Niklasson A, Gelander L, Holmgren A, Sjoberg A, Aronson AS, et al. Swedish references for weight, weight-for-height and body mass index: The GrowUp 1990
Gothenburg study. Acta Paediatr. 2021; 110:537-48 5. Cole TJ, Lobstein T. Extended international (IOTF) body mass index cut-offs for thinness,
overweight and obesity. Pediatric obesity. 2012; 7:284-94 6. Collaboration NCDRF. Worldwide trends in body-mass index, underweight, overweight, and
obesity from 1975 to 2016: a pooled analysis of 2416 population-based measurement
studies in 128.9 million children, adolescents, and adults. Lancet. 2017; 390:2627-42 7. Bygdell M, Celind J, Lilja L, Martikainen J, Simonson L, Sjogren L, et al. Prevalence of
overweight and obesity from 5 to 19 years of age in Gothenburg, Sweden. Acta Paediatr. 2021; 110:3349-55 8. Cole TJ. The development of growth references and growth charts. Annals of human biology. 2012; 39:382-94 9. Tanner JM, Whitehouse RH, Takaishi M. Standards from birth to maturity for height, weight,
height velocity, and weight velocity: British children, 1965. I. Archives of disease in
childhood. 1966; 41:454-71 10. Albertsson-Wikland KG, Niklasson A, Holmgren A, Gelander L, Nierop AFM. A new type of
pubertal height reference based on growth aligned for onset of pubertal growth. J Pediatr
Endocrinol Metab. 2020; 33:1173-82
11
K
lb
J O
h
d lli
f h
h S
i i
i
di i
1987 6 185 92 11. Karlberg J. On the modelling of human growth. REFERENCES 17 1. Holmgren A. The Quadratic-Exponential-Pubertal-Stop model is valid for analysing human
growth patterns and developing novel growth references. Acta Paediatr. 2021;
2. Karlberg J, Albertsson-Wikland K. Nutrition and linear growth in childhood. Recent
Developments in Infant Nutrition1996
3. Karlberg J, Luo ZC, Albertsson-Wikland K. Body mass index reference values (mean and SD
for Swedish children. Acta Paediatr. 2001; 90:1427-34
4. Albertsson-Wikland K, Niklasson A, Gelander L, Holmgren A, Sjoberg A, Aronson AS, et al. Swedish references for weight, weight-for-height and body mass index: The GrowUp 1990
Gothenburg study. Acta Paediatr. 2021; 110:537-48
5. Cole TJ, Lobstein T. Extended international (IOTF) body mass index cut-offs for thinness,
overweight and obesity. Pediatric obesity. 2012; 7:284-94
6. Collaboration NCDRF. Worldwide trends in body-mass index, underweight, overweight, an
obesity from 1975 to 2016: a pooled analysis of 2416 population-based measurement
studies in 128.9 million children, adolescents, and adults. Lancet. 2017; 390:2627-42
7. Bygdell M, Celind J, Lilja L, Martikainen J, Simonson L, Sjogren L, et al. Prevalence of
overweight and obesity from 5 to 19 years of age in Gothenburg, Sweden. Acta Paediatr. 2021; 110:3349-55
8. Cole TJ. The development of growth references and growth charts. Annals of human biolog
2012; 39:382-94
9. Tanner JM, Whitehouse RH, Takaishi M. Standards from birth to maturity for height, weigh
height velocity, and weight velocity: British children, 1965. I. Archives of disease in
childhood. 1966; 41:454-71
10. Albertsson-Wikland KG, Niklasson A, Holmgren A, Gelander L, Nierop AFM. A new type of
pubertal height reference based on growth aligned for onset of pubertal growth. J Pediatr
Endocrinol Metab. 2020; 33:1173-82
11. Karlberg J. On the modelling of human growth. Statistics in medicine. 1987; 6:185-92
12. Karlberg J, Fryer JG, Engstrom I, Karlberg P. Analysis of linear growth using a mathematical
model. II. From 3 to 21 years of age. Acta paediatrica Scandinavica. 1987; 337:12-29
13. Albertsson-Wikland K, Luo ZC, Niklasson A, Karlberg J. Swedish population-based
longitudinal reference values from birth to 18 years of age for height, weight and head
circumference. Acta Paediatr. 2002; 91:739-54
14. Gelander L, Hagenäs L, Albertsson-Wikland K. Tillväxtkurvan i skolhälsovården. Tillväxt och
kroppslig mognad 6-18 år. Manual och Lilla tillväxtskolan. Stockholm: Kommentus; 2003
15. Nierop AF, Niklasson A, Holmgren A, Gelander L, Rosberg S, Albertsson-Wikland K. Modelli
individual longitudinal human growth from fetal to adult life - QEPS I. J Theor Biol. 2016;
406:143-65
16. REFERENCES Statistics in medicine. 1987; 6:185-92 12. Karlberg J, Fryer JG, Engstrom I, Karlberg P. Analysis of linear growth using a mathematical
model. II. From 3 to 21 years of age. Acta paediatrica Scandinavica. 1987; 337:12-29 13. Albertsson-Wikland K, Luo ZC, Niklasson A, Karlberg J. Swedish population-based
longitudinal reference values from birth to 18 years of age for height, weight and head
circumference. Acta Paediatr. 2002; 91:739-54 14. Gelander L, Hagenäs L, Albertsson-Wikland K. Tillväxtkurvan i skolhälsovården. Tillväxt och
kroppslig mognad 6-18 år. Manual och Lilla tillväxtskolan. Stockholm: Kommentus; 2003 15. Nierop AF, Niklasson A, Holmgren A, Gelander L, Rosberg S, Albertsson-Wikland K. Modelling
individual longitudinal human growth from fetal to adult life - QEPS I. J Theor Biol. 2016;
406:143-65 16. Holmgren A, Niklasson A, Gelander L, Aronson AS, Nierop AFM, Albertsson-Wikland K. Insight into human pubertal growth by applying the QEPS growth model. BMC pediatrics. 2017; 17:107 17. Albertsson-Wikland K, Niklasson A, Gelander L, Holmgren A, Nierop AFM. Novel type of
references for weight aligned for onset of puberty - using the QEPS growth model. BMC
pediatrics. 2021; 21:507 18. Albertsson-Wikland K, Niklasson A, Holmgren A, Gelander L, Nierop AFM. A new Swedish
reference for total and prepubertal height. Acta Paediatr. 2020; 109:754-63
19. Rollof L, Elfving M. Evaluation of self-assessment of pubertal maturation in boys and girls
using drawings and orchidometer. J Pediatr Endocrinol Metab. 2012; 25:125-9
20. Vizmanos B, Marti-Henneberg C. Puberty begins with a characteristic subcutaneous body fat
mass in each sex. European journal of clinical nutrition. 2000; 54:203-8 18 21. Karlsson AK, Kullberg J, Stokland E, Allvin K, Gronowitz E, Svensson PA, et al. Measurements
of total and regional body composition in preschool children: A comparison of MRI, DXA, and
anthropometric data. Obesity (Silver Spring). 2013; 21:1018-24 22. Backman G. Gewichtswachstum des Mannes. Wilhelm Roux' Archiv für
Entwicklungsmechanik der Organismen 1940; 140:285–314 23. Hochberg Z, Albertsson-Wikland K. Evo-Devo of Infantile and Childhood Growth. Pediatric
research. 2008; 64:2-7 24. Hochberg Z. Evo-Devo of Child Growth: Treatise on Child Growth and Human Evolution, First
Edition.: Wiley-Blackwell, Publishers; 2012 25. Holmgren A, Niklasson A, Nierop AF, Gelander L, Aronson AS, Sjoberg A, et al. Pubertal
height gain is inversely related to peak BMI in childhood. Pediatric research. 2017; 26. Kindblom JM, Bygdell M, Sonden A, Celind J, Rosengren A, Ohlsson C. BMI change during
puberty and the risk of heart failure. Journal of internal medicine. 2018; 283:558-67 27. REFERENCES Celind J, Ohlsson C, Bygdell M, Nethander M, Kindblom JM. Childhood Body Mass Index Is
Associated with Risk of Adult Colon Cancer in Men: An Association Modulated by Pubertal
Change in Body Mass Index. Cancer epidemiology, biomarkers & prevention : a publication
of the American Association for Cancer Research, cosponsored by the American Society of
Preventive Oncology. 2019; 28:974-9 28. Ohlsson C, Bygdell M, Nethander M, Rosengren A, Kindblom JM. BMI Change During Puberty
Is an Important Determinant of Adult Type 2 Diabetes Risk in Men. J Clin Endocrinol Metab. 2019; 104:1823-32 29. Barker DJ, Osmond C, Kajantie E, Eriksson JG. Growth and chronic disease: findings in the
Helsinki Birth Cohort. Annals of human biology. 2009; 36:445-58 30. Eriksson JG, Forsen TJ, Kajantie E, Osmond C, Barker DJ. Childhood growth and hypertension
in later life. Hypertension. 2007; 49:1415-21 31. Forsdahl A. Living conditions in childhood and subsequent development of risk factors for
arteriosclerotic heart disease. The cardiovascular survey in Finnmark 1974-75. Journal of
epidemiology and community health. 1978; 32:34-7 32. Roseboom T, de Rooij S, Painter R. The Dutch famine and its long-term consequences for
adult health. Early human development. 2006; 82:485-91
h
h f
' h ld
d 33. Janson A. The growth of nations' children. Acta Paediatr. 2021; 110:3151-2 34. Hagman E, Danielsson P, Lindberg L, Marcus C, Committee BS. Paediatric obesity treatment
during 14 years in Sweden: Lessons from the Swedish Childhood Obesity Treatment Register-
BORIS. Pediatric obesity. 2020; 15:e12626 35. Olbers T, Beamish AJ, Gronowitz E, Flodmark CE, Dahlgren J, Bruze G, et al. Laparoscopic
Roux-en-Y gastric bypass in adolescents with severe obesity (AMOS): a prospective, 5-year,
Swedish nationwide study. Lancet Diabetes Endocrinol. 2017; 5:174-83 36. Modan-Moses D, Yaroslavsky A, Pinhas-Hamiel O, Levy-Shraga Y, Kochavi B, Iron-Segev S, et
al. Prospective Longitudinal Assessment of Linear Growth and Adult Height in Female
Adolescents With Anorexia Nervosa. J Clin Endocrinol Metab. 2021; 106:e1-e10 37. Leger J, Fjellestad-Paulsen A, Bargiacchi A, Pages J, Chevenne D, Alison M, et al. One Year of
GH Treatment for Growth Failure in Children With Anorexia Nervosa: A Randomized 38. Berkowitz SA, Witt AA, Gillberg C, Rastam M, Wentz E, Lowe MR. Childhood body mass index
in adolescent-onset anorexia nervosa. Int J Eat Disord. 2016; 49:1002-9 39. Mei Z, Grummer-Strawn LM, Pietrobelli A, Goulding A, Goran MI, Dietz WH. 40.
Freedman DS, Wang J, Maynard LM, Thornton JC, Mei Z, Pierson RN, et al. Relation of BMI to
fat and fat-free mass among children and adolescents. Int J Obes (Lond). 2005; 29:1-8 Freedman DS, Wang J, Maynard LM, Thornton JC, Mei Z, Pierson RN, et al. Relation of BMI t fat and fat-free mass among children and adolescents. Int J Obes (Lond). 2005; 29:1-8 REFERENCES Validity of body
mass index compared with other body-composition screening indexes for the assessment of
body fatness in children and adolescents. The American journal of clinical nutrition. 2002;
75:978-85 19 40. 20 Figure 1: Chronological age reference for total BMI and prepubertal BMI (QE-function)) for girls
(left) and boys (right) aged 4–20 years. Median total BMI (red/blue solid line, ±1SDS (red/blue dotted
line)), and ±2SDS (red/blue dashed line) and median prepubertal BMI (black). Figure 1: Chronological age reference for total BMI and prepubertal BMI (QE-function)) for girls
(left) and boys (right) aged 4–20 years. Median total BMI (red/blue solid line, ±1SDS (red/blue dotted
line)), and ±2SDS (red/blue dashed line) and median prepubertal BMI (black). 21 21 Figure 2 upper panel: References in kg/m2 (left axis) and in kg0.5/m (right axis) for total BMI and the
basic BMI (BMI gained independently of puberty (QES-function) for girls (red, left) and boys (blue,
right). Curves are aligned for age at onset of pubertal height spurt (AgeP5). Median total BMI
(red/blue solid line, ±1SDS (red/blue dotted line), and ±2SDS (red/blue dashed line)) and median
basic BMI (black). General differences in timing between pubertal height and weight functions result
in an undulation of aligned BMI functions. Figure 2 upper panel: References in kg/m2 (left axis) and in kg0.5/m (right axis) for total BMI and the
basic BMI (BMI gained independently of puberty (QES-function) for girls (red, left) and boys (blue,
right). Curves are aligned for age at onset of pubertal height spurt (AgeP5). Median total BMI
(red/blue solid line, ±1SDS (red/blue dotted line), and ±2SDS (red/blue dashed line)) and median
basic BMI (black). General differences in timing between pubertal height and weight functions result
in an undulation of aligned BMI functions. Figure 2 upper panel: References in kg/m2 (left axis) and in kg0.5/m (right axis) for total BMI and the
basic BMI (BMI gained independently of puberty (QES-function) for girls (red, left) and boys (blue,
right). Curves are aligned for age at onset of pubertal height spurt (AgeP5). Median total BMI
(red/blue solid line, ±1SDS (red/blue dotted line), and ±2SDS (red/blue dashed line)) and median
basic BMI (black). General differences in timing between pubertal height and weight functions result
in an undulation of aligned BMI functions. 22 Figure 2 lower panel: BMI gain resulting from the P-function of the QEPS (P-BMI) provides a
puberty-specific BMI reference. REFERENCES Specific P-function-derived BMI as median, ±1SDS (dotted line), and
±2SDS (dashed line). The individual onset of puberty was identified and aligned based on the age at
which 5% (AgeP5) of the total specific P-function growth (Pmax) for height had occurred. General
differences in timing between pubertal height and weight functions result in an undulation of aligned
BMI functions. Figure 2 lower panel: BMI gain resulting from the P-function of the QEPS (P-BMI) provides a
puberty-specific BMI reference. Specific P-function-derived BMI as median, ±1SDS (dotted line), and
±2SDS (dashed line). The individual onset of puberty was identified and aligned based on the age at
which 5% (AgeP5) of the total specific P-function growth (Pmax) for height had occurred. General
differences in timing between pubertal height and weight functions result in an undulation of aligned
BMI functions. 23 23 23 Figure 3: Median total BMI (in kg/m2, top; in SDS with 95% CI, bottom) according to chronological
age for girls (left) and boys (right) from the GrowUp1974 Gothenburg cohort grouped according to
onset of puberty: early, <−1.5yrs (- - -), average, ±0.25yrs (• • •), and late, >+1.5yrs (- ― -). Data are
visualized alongside the reference for median total BMI (thick solid lines) and ±2SDS (dotted red
(left) and blue (right) lines). Figure 3: Median total BMI (in kg/m2, top; in SDS with 95% CI, bottom) according to chronological
age for girls (left) and boys (right) from the GrowUp1974 Gothenburg cohort grouped according to
onset of puberty: early, <−1.5yrs (- - -), average, ±0.25yrs (• • •), and late, >+1.5yrs (- ― -). Data are
visualized alongside the reference for median total BMI (thick solid lines) and ±2SDS (dotted red
(left) and blue (right) lines). 24 igure 4. Median total, basic, and puberty-specific BMI gain (in kg/m2, top; in SDS with 95% CI bottom) relative to onset of puberty for girls (red at the top) and boys (blue at the bottom) grouped
according to pubertal onset: early, <−1.5yrs (- - -), average, ±0.25yrs (• • •), and late, >+1.5yrs (- ― -
). Median total BMI (QEPS-functions, left panels), basic BMI growth (QES-functions, middle
panels), and specific pubertal BMI growth (P-function, right panels) from the GrowUp1974Gothenburg
cohort. Curves are aligned for age at onset of pubertal growth spurt. Data are visualized alongside the
new reference for median total BMI (thick red or blue solid lines) and ±2SDS (red/blue dotted lines). REFERENCES bottom) relative to onset of puberty for girls (red at the top) and boys (blue at the bottom) grouped
according to pubertal onset: early, <−1.5yrs (- - -), average, ±0.25yrs (• • •), and late, >+1.5yrs (- ― -
). Median total BMI (QEPS-functions, left panels), basic BMI growth (QES-functions, middle
panels), and specific pubertal BMI growth (P-function, right panels) from the GrowUp1974Gothenburg
cohort. Curves are aligned for age at onset of pubertal growth spurt. Data are visualized alongside the
new reference for median total BMI (thick red or blue solid lines) and ±2SDS (red/blue dotted lines). 25 Figure 5: Median total, basic, and puberty-specific BMI gain (in kg/m2, top; in SDS with 95% CI,
bottom) relative to the onset of puberty for girls (red at the top) and boys (blue at the bottom)
according to height at the onset of puberty: tall, >+1.5 SDS (- ― -) and short, <−1.5 SDS (- - -). Median total BMI (QEPS-functions, left panels), basic BMI growth (QES-functions, middle panels),
and specific pubertal BMI growth (P-function, right panels) from the GrowUp1974Gothenburg cohort. Curves are aligned for age at onset of pubertal growth spurt. Data are visualized alongside the new
reference for median total BMI (thick red or blue solid lines) and ±2SDS (red/blue dotted lines). 26 Figure 6: Total, basic, and puberty-specific BMI gain (in kg/m2, top; in SDS with 95%CI, bottom)
relative to the onset of puberty for girls (red at the top) and boys (blue at the bottom) according to
BMI in childhood: high BMI, >+1.5SDS (- ― -) and low BMI, <−1.5SDS (- - -). Median total BMI
(QEPS-functions, left panels), basic growth (QES-functions, middle panels), and specific pubertal
growth (P-function, right panels) in girls and boys from the GrowUp1974Gothenburg cohort. Curves
are aligned for age at onset of pubertal growth spurt. Data are visualized alongside the new reference
for median total BMI (thick red or blue solid lines) and ±2SDS (red/blue dotted lines). 27 Figure 7: Guide for prospective use of the pubertal-age-adjusted reference for total BMI (kg/m2 and
kg0.5/m) and total height (cm) for girls (♀) and boys (♂). Figure 7: Guide for prospective use of the pubertal-age-adjusted reference for total BMI (kg/m2 and
kg0.5/m) and total height (cm) for girls (♀) and boys (♂). REFERENCES Figure 7: Guide for prospective use of the pubertal-age-adjusted reference for total BMI (kg/m2 and
kg0.5/m) and total height (cm) for girls (♀) and boys (♂). Individual age adjustment is made using only the height measurement at the chronological age (C-
age) at which puberty started (see upper left traditional C-age-reference for girls and boys) (14), by
using the height increase from the individual prepubertal growth curve (as drawn in upper left panel)
through the individual measuring points. This measurement is usually corresponding to the time when
secondary sex characteristics develop; early breast development stage, B2 in girls (red, upper left
panel), or testicular volume increase to 4-6 ml in boys (blue, upper left panel). Individual growth curves are shown for a girl aged 8 years and a boy aged 13-years at the start of
puberty (15). Height and BMI according to C-age and P-age, ie the age adjusted to reflect the start of
puberty, are shown for an 8-year-old girl and a 13-year-old boy (15). Height (cm) and BMI (in kg/m2
or kg0.5/m) according to C-age at the onset of puberty, and to P-age, after adjustment for age at onset
of the pubertal growth spurt, are depicted as red dots (all panels), based only on age in the height
references for each sex, respectively. Corresponding heights and BMIs are then moved to puberty-
adjusted age = zero, in the P-age-references shown in the lower panels (left panels for height and right
for BMI). Thereafter, all measured heights and BMIs are depicted at ages/times recalculated in
relation to the specific onset of puberty in that individual, labelled on the x-axis as ‘Time from onset
of growth spurt (years)’. Thus, changes in BMI and height in the years preceding the pubertal growth
spurt can be evaluated using the novel references. Note, the BMI increase precedes the height increase
in relation to puberty. For corresponding values for weight, see Figure 7 in Albertsson-Wikland et al. (17). 28 Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. 20220107aBMIpubSuppl.pdf
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https://openalex.org/W4375921168
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https://zenodo.org/record/3365363/files/cva-solution-2.pdf
|
English
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An Accurate Solution for Credit Valuation Adjustment (CVA) and Wrong Way Risk
| null | 2,019
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public-domain
| 11,721
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1 Email: tim_yxiao@yahoo.com Url: https://finpricing.com/ Tim Xiao1 Tim Xiao1 Journal of Fixed Income, 25(1) 84-95 Summer 2015 ABSTRACT This paper presents a Least Square Monte Carlo approach for accurately calculating credit value
adjustment (CVA). In contrast to previous studies, the model relies on the probability distribution
of a default time/jump rather than the default time itself, as the default time is usually inaccessible. As such, the model can achieve a high order of accuracy with a relatively easy implementation. We find that the valuation of a defaultable derivative is normally determined via backward
induction when their payoffs could be positive or negative. Moreover, the model can naturally
capture wrong or right way risk. Key Words: credit value adjustment (CVA), wrong way risk, right way risk, credit risk modeling,
least square Monte Carlo, default time approach (DTA), default probability approach (DPA),
collateralization, margin and netting. 1 1 For years, a widespread practice in the industry has been to mark derivative portfolios to market
without taking counterparty risk into account. All cash flows are discounted using the LIBOR
curve. But the real parties, in many cases, happen to be of lower credit quality than the
hypothetical LIBOR party and have a chance of default. As a consequence, the International Accounting Standard (IAS) 39 requires banks to
provide a fair-value adjustment due to counterparty risk. Although credit value adjustment (CVA)
became mandatory in 2000, it received a little attention until the recent financial crises in which
the profit and loss (P&L) swings due to CVA changes were measured in billons of dollars. Interest in CVA began to grow. Now CVA has become the first line of defense and the central
part of counterparty risk management. CVA not only allows institutions to move beyond the traditional control mindset of credit
risk limits and to quantify counterparty risk as a single measurable P&L number, but also offers
an opportunity for banks to dynamically manage, price and hedge counterparty risk. The benefits
of CVA are widely acknowledged. Many banks have set up internal credit risk trading desks to
manage counterparty risk on derivatives. The earlier works on CVA are mainly focused on unilateral CVA that assumes that only
one counterparty is defaultable and the other one is default-free. The unilateral treatment neglects
the fact that both counterparties may default, i.e., counterparty risk can be bilateral. A trend that
has become increasingly relevant and popular has been to consider the bilateral nature of
counterparty credit risk. ABSTRACT Although most institutions view bilateral considerations as important in
order to agree on new transactions, Hull and White (2013) argue that bilateral CVA is more
controversial than unilateral CVA as the possibility that a dealer might default is in theory a
benefit to the dealer. 2
CVA, by definition, is the difference between the risk-free portfolio value and the true (or
risky or defaultable) portfolio value that takes into account the possibility of a counterparty’s
default. The risk-free portfolio value is what brokers quote or what trading systems or models 2 2 normally report. The risky portfolio value, however, is a relatively less explored and less
transparent area, which is the main challenge and core theme for CVA. In other words, central to
CVA is risky valuation. In general, risky valuation can be classified into two categories: the default time
approach (DTA) and the default probability approach (DPA). The DTA involves the default time
explicitly. Most CVA models in the literature (Brigo and Capponi (2008), Lipton and Sepp
(2009), Pykhtin and Zhu (2006) and Gregory (2009), etc.) are based on this approach. Although the DTA is very intuitive, it has the disadvantage that it explicitly involves the
default time. We are very unlikely to have complete information about a firm’s default point,
which is often inaccessible (see Duffie and Huang (1996), Jarrow and Protter (2004), etc.). Usually, valuation under the DTA is performed via Monte Carlo simulation. On the other hand,
however, the DPA relies on the probability distribution of the default time rather than the default
time itself. Sometimes the DPA yields simple closed form solutions. The current popular CVA methodology (Pykhtin and Zhu (2006) and Gregory (2009),
etc.) is first derived using DTA and then discretized over a time grid in order to yield a feasible
solution. The discretization, however, is inaccurate. In fact, this model has never been rigorously
proved. Since CVA is used for financial accounting and pricing, its accuracy is essential. Moreover, this current model is based on a well-known assumption, in which credit exposure and
counterparty’s credit quality are independent. Obviously, it can not capture wrong/right way risk
properly. In this paper, we present a framework for risky valuation and CVA. In contrast to
previous studies, the model relies on the DPA rather than the DTA. ABSTRACT Our study shows that the
pricing process of a defaultable contract normally has a backward recursive nature if its payoff
could be positive or negative. 3
An intuitive way of understanding these backward recursive behaviours is that we can
think of that any contingent claim embeds two default options. In other words, when entering an 3 3 OTC derivatives transaction, one party grants the other party an option to default and, at the same
time, also receives an option to default itself. In theory, default may occur at any time. Therefore,
the default options are American style options that normally require a backward induction
valuation. Wrong way risk occurs when exposure to a counterparty is adversely correlated with the
credit quality of that counterparty, while right way risk occurs when exposure to a counterparty is
positively correlated with the credit quality of that counterparty. For example, in wrong way risk
exposure tends to increase when counterparty credit quality worsens, while in right way risk
exposure tends to decrease when counterparty credit quality declines. Wrong/right way risk, as an
additional source of risk, is rightly of concern to banks and regulators. Since this new model
allows us to incorporate correlated and potentially simultaneous defaults into risky valuation, it
can naturally capture wrong/right way risk. The rest of this paper is organized as follows: Section 2 discusses unilateral risky
valuation and unilateral CVA. Section 2 elaborates bilateral risky valuation and bilateral CVA. Section 3 presents numerical results. The conclusions are given in Section 4. . All proofs and a
practical framework that embraces netting agreements, margining agreements and wrong/right
way risk are contained in the appendices. 1. Unilateral Risky Valuation and Unilateral CVA We consider a filtered probability space ( , F ,
0
t
t
F
, P ) satisfying the usual
conditions, where denotes a sample space; F denotes a -algebra; P
denotes a
probability measure;
0
t
t
F
denotes a filtration. The default model is based on the reduced-form approach proposed by Duffie and
Singleton (1999) and Jarrow and Turnbell (1994), which does not explain the event of default
endogenously, but characterizes it exogenously by a jump process. The stopping (or default) time 4 4 of a firm is modeled as a Cox arrival process (also known as a doubly stochastic Poisson
process) whose first jump occurs at default and is defined as, of a firm is modeled as a Cox arrival process (also known as a doubly stochastic Poisson
process) whose first jump occurs at default and is defined as,
t
s ds
s
h
t
0
)
,
(
:
inf
(1) (1) where
)
(t
h
or
)
,
(
t
t
h
denotes the stochastic hazard rate or arrival intensity dependent on an
exogenous common state
t, and is a unit exponential random variable independent of
t
. where
)
(t
h
or
)
,
(
t
t
h
denotes the stochastic hazard rate or arrival intensity dependent on an
exogenous common state
t, and is a unit exponential random variable independent of
t
. It is well-known that the survival probability from time t to s in this framework is defined It is well-known that the survival probability from time t to s in this framework is defined
by by
s
t
du
u
h
Z
t
s
P
s
t
p
)
(
exp
)
,
|
(
:)
,
(
(2a) (2a) The default probability for the period (t, s) in this framework is defined by
s
t
du
u
h
s
t
p
Z
t
s
P
s
t
q
)
(
exp
1
)
,
(
1
)
,
|
(
:)
,
(
(2b) (2b) Two counterparties are denoted as A and B. Let valuation date be t. 2 Here we use the recovery of market value (RMV) assumption. 1. Unilateral Risky Valuation and Unilateral CVA Under a risk-neutral measure, the value of this defaultable contract is the discounted expectation
of all the payoffs and is given by
t
T
T
T
V
t
D
X
T
t
D
E
t
V
F
|
1)
(
)
,
(
1
)
,
(
)
(
(4)
t
T
T
T
V
t
D
X
T
t
D
E
t
V
F
|
1)
(
)
,
(
1
)
,
(
)
(
(4) (4) where
Y is an indicator function that is equal to one if Y is true and zero otherwise. n indicator function that is equal to one if Y is true and zero otherwise. where
Y is an indicator function that is equal to one if Y is true and zero otherwise. Although the DTA is very intuitive, it has the disadvantage that it explicitly involves the
default time/jump. We are very unlikely to have complete information about a firm’s default
point, which is often inaccessible. Usually, valuation under the DTA is performed via Monte
Carlo simulation. The DPA relies on the probability distribution of the default time rather than the default
time itself. We divide the time period (t, T) into n very small time intervals ( t
) and assume that
a default may occur only at the end of each very small period. In our derivation, we use the
approximation
y
y
1
exp
for very small y. The survival and the default probabilities for the
period ( t ,
t
t
) are given by
t
t
h
t
t
h
t
t
t
p
t
p
)
(
1
)
(
exp
)
,
(
:)
(ˆ
(5a)
t
t
h
t
t
h
t
t
t
q
t
q
)
(
)
(
exp
1
)
,
(
:)
(ˆ
(5b) (5a) (5b) The binomial default rule considers only two possible states: default or survival. 1. Unilateral Risky Valuation and Unilateral CVA Consider a financial
contract that promises to pay a
0
T
X
from party B to party A at maturity date T, and nothing
before date T. All calculations in the paper are from the perspective of party A. The risk free value
of the financial contract is given by
t
F
T
F
X
T
t
D
E
t
V
)
,
(
)
(
(3a)
t
F
T
F
X
T
t
D
E
t
V
)
,
(
)
(
(3a)
where
du
u
r
T
t
D
T
t
)
(
exp
)
,
(
(3b) (3a) where
du
u
r
T
t
D
T
t
)
(
exp
)
,
(
(3b)
du
u
r
T
t
D
T
t
)
(
exp
)
,
(
(3b) (3b) where
t
E
F
denotes the expectation conditional on the
t
F ,
)
,
( T
t
D
denotes the risk-free
discount factor at time t for the maturity T and
)
(u
r
denotes the risk-free short rate at time u
(
T
u
t
). Next, we turn to risky valuation. In a unilateral credit risk case, we assume that party A is
default-free and party B is defaultable. Risky valuation can be generally classified into two 5 5 categories: the default time approach (DTA) and the default probability (intensity) approach
(DPA). The DTA involves the default time explicitly. If there has been no default before time T
(i.e.,
T
), the value of the contract at T is the payoff
T
X . If a default happens before T (i.e.,
T
t
), a recovery payoff is made at the default time as a fraction of the market value2
given by
)
(
V
where is the default recovery rate and
)
(
V
is the market value at default. 1. Unilateral Risky Valuation and Unilateral CVA
t
t
t
t
V
t
t
y
E
t
t
V
t
q
t
t
p
t
t
r
E
t
V
F
F
)
(
)
(
exp
)
(
)
(ˆ
)
(
)
(ˆ
)
(
exp
)
(
(6)
where
)
(
)
(
)
(
1
)
(
)
(
)
(
t
c
t
r
t
t
h
t
r
t
y
denotes the risky rate and
)
(
1
)
(
)
(
t
t
h
t
c
is called
the (short) credit spread. (6) Similarly, we have Similarly, we have
t
t
t
t
V
t
t
t
y
E
t
t
V
F
)
2
(
)
(
exp
)
(
(7) (7) Note that
t
t
y
)
(
exp
is
t
t
F
-measurable. By definition, an
t
t
F
-measurable
random variable is a random variable whose value is known at time
t
t
. 1. Unilateral Risky Valuation and Unilateral CVA For the
one-period
)
,
(
t
t
t
economy, at time
t
t
the asset either defaults with the default 2 Here we use the recovery of market value (RMV) assumption. 6 probability
)
,
(
t
t
t
q
or survives with the survival probability
)
,
(
t
t
t
p
. The survival payoff
is equal to the market value
)
(
t
t
V
and the default payoff is a fraction of the market value:
)
(
)
(
t
t
V
t
t
. Under a risk-neutral measure, the value of the asset at t is the expectation of
all the payoffs discounted at the risk-free rate and is given by
t
t
t
t
V
t
t
y
E
t
t
V
t
q
t
t
p
t
t
r
E
t
V
F
F
)
(
)
(
exp
)
(
)
(ˆ
)
(
)
(ˆ
)
(
exp
)
(
(6)
where
)
(
)
(
)
(
1
)
(
)
(
)
(
t
c
t
r
t
t
h
t
r
t
y
denotes the risky rate and
)
(
1
)
(
)
(
t
t
h
t
c
is called
the (short) credit spread.
t
t
t
t
V
t
t
y
E
t
t
V
t
q
t
t
p
t
t
r
E
t
V
F
F
)
(
)
(
exp
)
(
)
(ˆ
)
(
)
(ˆ
)
(
exp
)
(
(6)
where
)
(
)
(
)
(
1
)
(
)
(
)
(
t
c
t
r
t
t
h
t
r
t
y
denotes the risky rate and
)
(
1
)
(
)
(
t
t
h
t
c
is called
the (short) credit spread. 1. Unilateral Risky Valuation and Unilateral CVA Based on the taking
out what is known and tower properties of conditional expectation, we have
t
i
t
t
t
t
t
t
V
t
t
i
t
y
E
t
t
V
t
t
t
y
E
t
t
y
E
t
t
V
t
t
y
E
t
V
F
F
F
F
)
2
(
)
)
(
exp
)
2
(
)
(
exp
)
(
exp
)
(
)
(
exp
)
(
1
0
(8) (8) By recursively deriving from t forward over T and taking the limit as t
approaches zero,
the risky value of the asset can be expressed as
t
T
t
T
V
du
u
y
E
t
V
F
)
(
)
(
exp
)
(
(9) (9) We may think of
)
(u
y
as the risk-adjusted short rate. Equation (9) is the same as
Equation (10) in Duffie and Singleton [1999], which is the market model for pricing risky bonds. Using the DPA, we obtain a closed-form solution for pricing an asset subject to credit risk. Other
good examples of the DPA are the CDS model proposed by J.P. Morgan (1999) and a more
generic risky model presented by Xiao (2013a). 7 7 In theory, a default may happen at any time, i.e., a risky contract is continuously
defaultable. This Continuous Time Risky Valuation Model is accurate but sometimes complex
and expensive. For simplicity, people sometimes prefer the Discrete Time Risky Valuation Model
that assumes that a default may only happen at some discrete times. A natural selection is to
assume that a default may occur only on the payment dates. Fortunately, the level of accuracy for
this discrete approximation is well inside the typical bid-ask spread for most applications (see
O’Kane and Turnbull (2003)). From now on, we will focus on the discrete setting only, but many
of the points we make are equally applicable to the continuous setting. For a derivative contract, usually its payoff may be either an asset or a liability to each
party. 1. Unilateral Risky Valuation and Unilateral CVA Proposition 2: The unilateral risky value of the multiple-payment contract is given by Proposition 2: The unilateral risky value of the multiple-payment contract is given by Proposition 2: The unilateral risky value of the multiple-payment contract is given by
m
i
t
i
i
j
j
j
X
T
T
F
E
t
V
1
1
0
1)
,
(
)
(
F
(11a) (11a) where
0T
t
and
)
(
1
)
,
(
1
1
)
,
(
)
,
(
1
1
0
))
(
(
1
1
1
1
j
j
j
T
V
X
j
j
j
j
T
T
T
q
T
T
D
T
T
F
j
j
(11b)
)
(
1
)
,
(
1
1
)
,
(
)
,
(
1
1
0
))
(
(
1
1
1
1
j
j
j
T
V
X
j
j
j
j
T
T
T
q
T
T
D
T
T
F
j
j
(11b)
e appendix (11b) Proof: See the appendix. Proof: See the appendix. The risky valuation in Proposition 2 has a backward nature. The intermediate values are
vital to determine the final price. For a discrete time interval, the current risky value has a
dependence on the future risky value. Only on the final payment date
m
T
, the value of the
contract and the maximum amount of information needed to determine the risk-adjusted discount
factor are revealed. The coupled valuation behavior allows us to capture wrong/right way risk
properly where counterparty credit quality and market prices may be correlated. This type of
problem can be best solved by working backwards in time, with the later risky value feeding into
the earlier ones, so that the process builds on itself in a recursive fashion, which is referred to as
backward induction. The most popular backward induction valuation algorithms are lattice/tree
and least square Monte Carlo. For an intuitive explanation, we can posit that a defaultable contract under the unilateral
credit risk assumption has an embedded default option (see Sorensen and Bollier (1994)). 1. Unilateral Risky Valuation and Unilateral CVA Thus, we further relax the assumption and suppose that
T
X may be positive or negative. In the case of
0
T
X
, the survival value is equal to the payoff
T
X and the default payoff
is a fraction of the payoff
T
X
. Whereas in the case of
0
T
X
, the contract value is the payoff
itself, because the default risk of party B is irrelevant for unilateral risky valuation in this case. Therefore, we have Proposition 1: The unilateral risky value of the single-payment contract in a discrete-time setting
is given by Proposition 1: The unilateral risky value of the single-payment contract in a discrete-time setting
is given by
t
F
T
X
T
t
F
E
t
V
)
,
(
)
(
(10a) (10a) where
)
(
1
)
,
(
1
1
)
,
(
)
,
(
0
T
T
t
q
T
t
D
T
t
F
T
X
(10b) (10b) Proof: See the appendix. Here
)
,
( T
t
F
can be regarded as a risk-adjusted discount factor. Proposition 1 says that
the unilateral risky valuation of the single payoff contract has a dependence on the sign of the
payoff. If the payoff is positive, the risky value is equal to the risk-free value minus the
discounted potential loss. Otherwise, the risky value is equal to the risk-free value. 8 8 Proposition 1 can be easily extended from one-period to multiple-periods. Suppose that a
defaultable contract has m cash flows. Let the m cash flows be represented as
1
X ,…,
m
X with
payment dates 1T ,…,
m
T . Each cash flow may be positive or negative. We have the following
proposition. 1. Unilateral Risky Valuation and Unilateral CVA In other
words, one party entering a defaultable financial transaction actually grants the other party an
option to default. If we assume that a default may occur at any time, the default option is an 9 American style option. American options normally have backward recursive natures and require
backward induction valuations. The similarity between American style financial options and American style default
options is that both require a backward recursive valuation procedure. The difference between
them is in the optimal strategy. The American financial option seeks an optimal value by
comparing the exercise value with the continuation value, whereas the American default option
seeks an optimal discount factor based on the option value in time. The unilateral CVA, by definition, can be expressed as The unilateral CVA, by definition, can be expressed as
m
i
t
i
i
j
j
j
i
F
X
T
T
F
T
t
D
E
t
V
t
V
t
CVA
1
1
0
1)
,
(
)
,
(
)
(
)
(
)
(
F
(12) (12) Proposition 2 provides a general form for pricing a unilateral defaultable contract. Applying it to a particular situation in which we assume that all the payoffs are nonnegative, we
derive the following corollary: Corollary 1: If all the payoffs are nonnegative, the risky value of the multiple-payments contract
is given by
m
i
t
i
i
j
j
j
X
T
T
F
E
t
V
1
1
0
1)
,
(
)
(
F
(13a) (13a) where
0T
t
and where
0T
t
and
)
(
1
)
,
(
1
)
,
(
)
,
(
1
1
1
1
j
j
j
j
j
j
j
T
T
T
q
T
T
D
T
T
F
(13b) (13b) The proof of this corollary is easily obtained according to Proposition 2 by setting
0
)
(
1
1
j
j
T
V
X
, since the value of the contract at any time is also nonnegative. 1. Unilateral Risky Valuation and Unilateral CVA e CVA in this case is given by The CVA in this case is given by The CVA in this case is given by
m
i
t
i
i
j
j
j
j
i
F
X
T
T
T
q
T
t
D
E
t
V
t
V
t
CVA
1
1
0
1
1
))
(
1
)(
,
(
1
1
)
,
(
)
(
)
(
)
(
F
(14) (14) The current popular CVA model (e.g., equation (17) in Pykhtin and Zhu (2007) and
equation (3) in Gregory (2009)) is quite different from above either equation (12) or equation (14). As a matter of fact, the current CVA model has never been rigorously proved. In order to reflect 10 the economic value of counterparty credit risk, to measure the profit and loss of a bank and to
provide proper incentives to traders, a good CVA model must be not only rigorous and accurate
but also feasible to implement. 2. Bilateral Risky Valuation and Bilateral CVA There is ample evidence that corporate defaults are correlated. The default of a firm’s
counterparty might affect its own default probability. Thus, default correlation and dependence
arise due to the counterparty relations. Default correlation can be positive or negative. The effect
of positive correlation is usually called contagion, whereas the latter is referred to as competition
effect. Two counterparties are denoted as A and B. The binomial default rule considers only two
possible states: default or survival. Therefore, the default indicator
j
Y for party j (j=A, B) follows
a Bernoulli distribution, which takes value 1 with default probability
j
q and value 0 with survival
probability
j
p , i.e.,
j
j
p
Y
P
}
0
{
and
j
j
q
Y
P
}
1
{
. The marginal default distributions can be
determined by the reduced-form models. The joint distributions of a bivariate Bernoulli variable
can be easily obtained via the marginal distributions by introducing extra correlations. Consider a pair of random variables (
A
Y ,
B
Y ) that has a bivariate Bernoulli distribution. The joint probability representations are given by Consider a pair of random variables (
A
Y ,
B
Y ) that has a bivariate Bernoulli distribution. 3 There are two default settlement rules in the market. The one-way payment rule was specified
by the early ISDA master agreement. The non-defaulting party is not obligated to compensate the
defaulting party if the remaining market value of the instrument is positive for the defaulting
party. The two-way payment rule is based on current ISDA documentation. The non-defaulting
party will pay the full market value of the instrument to the defaulting party if the contract has
positive value to the defaulting party. 2. Bilateral Risky Valuation and Bilateral CVA The joint probability representations are given by (15a) AB
B
A
B
A
p
p
Y
Y
P
p
)
0
,0
(
:
00
(15a)
AB
B
A
B
A
q
p
Y
Y
P
p
)1
,0
(
:
01
(15b)
AB
B
A
B
A
p
q
Y
Y
P
p
)
0
,1
(
:
10
(15c)
AB
B
A
B
A
q
q
Y
Y
P
p
)1
,1
(
:
11
(15d)
2
Y
Y
E
)
)(
(
h (15b) (15d) e
j
j
q
Y
E
)
(
,
j
j
j
q
p
2
,
B
B
A
A
AB
B
A
AB
B
B
A
A
AB
p
q
p
q
q
Y
q
Y
E
)
)(
(
:
where where
j
j
q
Y
E
)
(
,
j
j
j
q
p
2
,
B
B
A
A
AB
B
A
AB
B
B
A
A
AB
p
q
p
q
q
Y
q
Y
E
)
)(
(
:
where
AB
denotes the default correlation coefficient and
AB
denotes the default covariance. 11 Table 1. Payoffs of a bilaterally defaultable contract This table displays all possible payoffs at time T. In the case of
0
T
X
, there are a total of four
possible states at time T: i) Both A and B survive with probability
00
p
. The contract value is
equal to the payoff
T
X . ii) A defaults but B survives with probability
10
p
. The contract value is
T
BX
, where
B
represents the non-default recovery rate 3. B
=0 represents the one-way
settlement rule, while
B
=1 represents the two-way settlement rule. iii) A survives but B defaults
with probability
01
p
. The contract value is
T
BX
, where
B
represents the default recovery rate. iv) Both A and B default with probability
11
p . The contract value is
T
ABX
, where
AB
denotes
the joint recovery rate when both parties A and B default simultaneously. A similar logic applies
to the case of
0
T
X
. State
0
,0
B
A
Y
Y
0
,1
B
A
Y
Y
1
,0
B
A
Y
Y
1
,1
B
A
Y
Y
Comments
A & B survive
A defaults, B survives
A survives, B defaults
A & B default
Probability
00
p
10
p
01
p
11
p
Payoff
0
T
X
T
X
T
BX
T
BX
T
ABX
0
T
X
T
X
T
AX
T
AX
T
ABX
12 12 Suppose that a financial contract that promises to pay a
T
X from party B to party A at
maturity date T, and nothing before date T where
t
T . The payoff
T
X may be positive or
negative, i.e. the contract may be either an asset or a liability to each party. All calculations are
from the perspective of party A. At time T, there are a total of four (
4
22
) possible states shown in Table 1. The risky
value of the contract is the discounted expectation of the payoffs and is given by the following
proposition. Table 1. Payoffs of a bilaterally defaultable contract oposition 3: The bilateral risky value of the single-payment contract is given by oposition 3: The bilateral risky value of the single-payment contract is given by
t
t
F
F
T
A
X
B
X
T
X
T
t
k
T
t
k
T
t
D
E
X
T
t
K
E
t
V
T
T
)
,
(
1
)
,
(
1
)
,
(
)
,
(
)
(
0
0
(16a)
t
t
F
F
T
A
X
B
X
T
X
T
t
k
T
t
k
T
t
D
E
X
T
t
K
E
t
V
T
T
)
,
(
1
)
,
(
1
)
,
(
)
,
(
)
(
0
0
(16a)
where
t
t
F
F
T
A
X
B
X
T
X
T
t
k
T
t
k
T
t
D
E
X
T
t
K
E
t
V
T
T
)
,
(
1
)
,
(
1
)
,
(
)
,
(
)
(
0
0
(16a)
where (16a) where
)
(
)
(
)
(
1
)
,
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
,
(
T
T
T
T
t
T
t
q
T
t
q
T
T
t
q
T
t
p
T
T
t
p
T
t
q
T
T
t
p
T
t
p
T
t
k
AB
B
B
AB
A
B
AB
A
B
B
A
B
B
A
B
B
(16b)
)
(
)
(
)
(
1
)
,
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
,
(
T
T
T
T
t
T
t
q
T
t
q
T
T
t
q
T
t
p
T
T
t
p
T
t
q
T
T
t
p
T
t
p
T
t
k
AB
A
A
AB
A
B
AB
B
A
A
B
A
A
A
B
A
(16c)
)
(
)
(
)
(
1
)
,
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
,
(
T
T
T
T
t
T
t
q
T
t
q
T
T
t
q
T
t
p
T
T
t
p
T
t
q
T
T
t
p
T
t
p
T
t
k
AB
B
B
AB
A
B
AB
A
B
B
A
B
B
A
B
B
(16b) (16b)
)
(
)
(
)
(
1
)
,
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
,
(
T
T
T
T
t
T
t
q
T
t
q
T
T
t
q
T
t
p
T
T
t
p
T
t
q
T
T
t
p
T
t
p
T
t
k
AB
A
A
AB
A
B
AB
B
A
A
B
A
A
A
B
A
(16c)
)
(
)
(
)
(
1
)
,
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
,
(
T
T
T
T
t
T
t
q
T
t
q
T
T
t
q
T
t
p
T
T
t
p
T
t
q
T
T
t
p
T
t
p
T
t
k
AB
A
A
AB
A
B
AB
B
A
A
B
A
A
A
B
A
(16c) (16c) Proof: See the appendix. Table 1. Payoffs of a bilaterally defaultable contract The bilateral CVA of the multiple-payment contract can be expressed as
m
i
t
i
i
j
j
j
t
i
i
F
X
T
T
K
E
X
T
t
D
E
t
V
t
V
t
CVA
1
1
0
1)
,
(
)
,
(
)
(
)
(
)
(
F
F
(18)
m
i
t
i
i
j
j
j
t
i
i
F
X
T
T
K
E
X
T
t
D
E
t
V
t
V
t
CVA
1
1
0
1)
,
(
)
,
(
)
(
)
(
)
(
F
F
(18)
m
i
t
i
i
j
j
j
t
i
i
F
X
T
T
K
E
X
T
t
D
E
t
V
t
V
t
CVA
1
1
0
1)
,
(
)
,
(
)
(
)
(
)
(
F
F
(18) (18) 3. Numerical Results Table 1. Payoffs of a bilaterally defaultable contract
)
,
(
1
)
,
(
1
)
,
(
)
,
(
1
0
))
(
(
1
0
))
(
(
1
1
1
1
1
1
j
j
A
T
V
X
j
j
B
T
V
X
j
j
j
j
T
T
k
T
T
k
T
T
D
T
T
K
j
j
j
j
(17b) (17b) where
)
,
(
1
j
j
A
T
T
k
and
)
,
(
1
j
j
B
T
T
k
are defined in Proposition 3. Proof: The proof is similar to Proposition 2 by replacing
)
,
(
1
j
j T
T
F
with
)
,
(
1
j
j T
T
K
. Proposition 4 says that the pricing process of a multiple-payment contract has a backward
nature since there is no way of knowing which risk-adjusted discounting rate should be used
without knowledge of the future value. Only on the maturity date, the value of the contract and
the decision strategy are clear. Therefore, the evaluation must be done in a backward fashion,
working from the final payment date towards the present. This type of valuation process is
referred to as backward induction. There is a common misconception in the market. Many people believe that the cash flows
of a defaultable financial contract can be priced independently and then be summed up to give the
final risky price of the contract. We emphasize here that this conclusion is only true of the
financial contracts whose payoffs are always positive. In the cases where the promised payoffs
could be positive or negative, the valuation requires not only a backward recursive induction
procedure, but also a strategic selection of different discount factors according to the market
value in time. This coupled valuation process allows us to capture correlation between
counterparties and market factors. Table 1. Payoffs of a bilaterally defaultable contract Proof: See the appendix. Proof: See the appendix. We may think of
)
,
( T
t
K
as the risk-adjusted discount factor. Proposition 3 tells us that
the bilateral risky price of a single-payment contract can be expressed as the present value of the
payoff discounted by a risk-adjusted discount factor that has a switching-type dependence on the
sign of the payoff. Using a similar derivation as in Proposition 2, we can easily extend Proposition 3 from
one-period to multiple-periods. Suppose that a defaultable contract has m cash flows. Let the m
cash flows be represented as
i
X with payment dates
iT , where i = 1,…,m. Each cash flow may
be positive or negative. The bilateral risky value of the multiple-payment contract is given by
Proposition 4: The bilateral risky value of the multiple-payment contract is given by
m
i
t
i
i
j
j
j
X
T
T
K
E
t
V
1
1
0
1)
,
(
)
(
F (17a) 13 13 where
0T
t
and where
0T
t
and
)
,
(
1
)
,
(
1
)
,
(
)
,
(
1
0
))
(
(
1
0
))
(
(
1
1
1
1
1
1
j
j
A
T
V
X
j
j
B
T
V
X
j
j
j
j
T
T
k
T
T
k
T
T
D
T
T
K
j
j
j
j
(17b)
where
)
,
(
1
j
j
A
T
T
k
and
)
,
(
1
j
j
B
T
T
k
are defined in Proposition 3. 3. Numerical Results In this section, we present some numerical results for CVA calculation based on the
theory described above. First, we study the impact of margin agreements on CVA. The testing 14 portfolio consists of a number of interest rate and equity derivatives. The number of simulation
scenarios (or paths) is 20,000. The time buckets are set weekly. If the computational requirements
exceed the system limit, one can reduce both the number of scenarios and the number of time
buckets. The time buckets can be designed fine-granularity at the short end (e.g., daily and then
weekly) and coarse-granularity at the far end (e.g. monthly and then yearly). The rationale is that
the calculation becomes less accurate due to the accumulated error from simulation discretization,
and inherited errors from calibration of the underlying models, such as those due to the change of
macro-economic climate. The collateral margin period of risk is assumed to be 14 days (2 weeks). For risk-neutral simulation, we use a Hull-White model for interest rate and a CIR (Cox-
Ingersoll-Ross) model for hazard rate scenario generations a modified GBM (Geometric
Brownian Motion) model for equity and collateral evolution. The results are presented in the
following tables. Table 2 illustrates that if party A has an infinite collateral threshold
A
H
i.e., no collateral requirement on A, the CVA value increases while the threshold
B
H
increases. Table 3 shows that if party B has an infinite collateral threshold
B
H
, the CVA value actually
decreases while the threshold
A
H increases. This reflects the bilateral impact of the collaterals
on the CVA. The impact is mixed in Table 4 when both parties have finite collateral thresholds. Table 2. The impact of collateral threshold
B
H
on the CVA This table shows that given an infinite
A
H , the CVA increases while
B
H increases, where
B
H
denotes the collateral threshold of party B and
A
H denotes the collateral threshold of party A. Collateral Threshold
B
H
10.1 Mil
15.1 Mil
20.1 Mil
Infinite ( )
CVA
19,550.91
20,528.65
21,368.44
22,059.30
Table 3. The impact of collateral threshold
A
H
on the CVA This table shows that given an infinite
A
H , the CVA increases while
B
H increases, where
B
H
denotes the collateral threshold of party B and
A
H denotes the collateral threshold of party A. Collateral Threshold
B
H
10.1 Mil
15.1 Mil
20.1 Mil
Infinite ( )
CVA
19,550.91
20,528.65
21,368.44
22,059.30 This table shows that given an infinite
A
H , the CVA increases while
B
H increases, where
B
H
denotes the collateral threshold of party B and
A
H denotes the collateral threshold of party A. Collateral Threshold
B
H
10.1 Mil
15.1 Mil
20.1 Mil
Infinite ( )
CVA
19,550.91
20,528.65
21,368.44
22,059.30 Table 3. The impact of collateral threshold
A
H
on the CVA Table 3. The impact of collateral threshold
A
H
on the CVA 15 This table shows that given an infinite
B
H , the CVA decreases while
A
H increases, where
B
H
denotes the collateral threshold of party B and
A
H denotes the collateral threshold of party A. Collateral Threshold
A
H
10.1 Mil
15.1 Mil
20.1 Mil
Infinite ( )
CVA
28,283.64
25,608.92
23,979.11
22,059.30 This table shows that given an infinite
B
H , the CVA decreases while
A
H increases, where
B
H
denotes the collateral threshold of party B and
A
H denotes the collateral threshold of party A. Collateral Threshold
A
H
10.1 Mil
15.1 Mil
20.1 Mil
Infinite ( )
CVA
28,283.64
25,608.92
23,979.11
22,059.30 Table 4. The impact of the both collateral thresholds on the CVA
The CVA may increase or decrease while both collateral thresholds change, where
B
H denotes
the collateral threshold of party B and
A
H
denotes the collateral threshold of party A. This
reflects the fact that the collaterals have bilateral impacts on the CVA. Collateral Threshold
B
H
10.1 Mil
15.1 Mil
20.1 Mil
Infinite ( )
Collateral Threshold
A
H
10.1 Mil
15.1 Mil
20.1 Mil
Infinite ( )
CVA
25,752.98
22,448.45
23,288.24
22,059.30 Table 5. The impact of wrong way risk on the CVA This table shows that the CVA increases while the negative correlation increases in the
absolute value. We use an equity swap as an example and assume that there is a negative
correlation between the equity price and the credit quality of party B. Correlation
0
-50%
-100%
CVA
165.15
205.95
236.99 Table 4. The impact of the both collateral thresholds on the CVA The CVA may increase or decrease while both collateral thresholds change, where
B
H denotes
the collateral threshold of party B and
A
H
denotes the collateral threshold of party A. This
reflects the fact that the collaterals have bilateral impacts on the CVA. reflects the fact that the collaterals have bilateral impacts on the CVA. Collateral Threshold
B
H
10.1 Mil
15.1 Mil
20.1 Mil
Infinite ( )
Collateral Threshold
A
H
10.1 Mil
15.1 Mil
20.1 Mil
Infinite ( )
CVA
25,752.98
22,448.45
23,288.24
22,059.30 Next, we examine the impact of wrong way risk. Wrong way risk occurs when exposure
to a counterparty is adversely correlated with the credit quality of that counterparty, while right
way risk occurs when exposure to a counterparty is positively correlated with the credit quality of
that counterparty. Wrong/right way risk, as an additional source of risk, is rightly of concern to
banks and regulators. Some financial markets are closely interlinked, while others are not. For example, CDS
price movements have a feedback effect on the equity market, as a trading strategy commonly
employed by banks and other market participants consists of selling a CDS on a reference entity
and hedging the resulting credit exposure by shorting the stock. On the other hand, Moody’s
Investor’s Service (2000) presents statistics that suggest that the correlations between interest
rates and CDS spreads are very small. 16 16 To capture wrong/right way risk, we need to determine the dependency between
counterparties and to correlate the credit spreads or hazard rates with the other market risk factors,
e.g. equities, commodities, etc., in the scenario generation. We use an equity swap as an example. Assume the correlation between the underlying
equity price and the credit quality (hazard rate) of party B is . The impact of the correlation on
the CVA is show in Table 5. The results say that the CVA increases when the absolute value of
the negative correlation increases. 4. Conclusion This article presents a framework for pricing risky contracts and their CVAs. The model
relies on the probability distribution of the default jump rather than the default jump itself,
because the default jump is normally inaccessible. We find that the valuation of risky assets and
their CVAs, in most situations, has a backward recursive nature and requires a backward
induction valuation. An intuitive explanation is that two counterparties implicitly sell each other
an option to default when entering into an OTC derivative transaction. If we assume that a default
may occur at any time, the default options are American style options. If we assume that a default
may only happen on the payment dates, the default options are Bermudan style options. Both
Bermudan and American options require backward induction valuations. 17 Based on our theory, we propose a novel cash-flow-based framework (see appendix) for
calculating bilateral CVA at the counterparty portfolio level. This framework can easily
incorporate various credit mitigation techniques, such as netting agreements and margin
agreements, and can capture wrong/right way risk. Numerical results show that these credit
mitigation techniques and wrong/right way risk have significant impacts on CVA. A. Proofs Proof of Proposition 1: Under the unilateral credit risk assumption, we only consider the
default risk when the asset is in the money. Assume that a default may only occur on the payment
date. Therefore, the risky value of the asset at t is the discounted expectation of all possible
payoffs and is given by
t
t
t
F
F
F
T
T
X
T
X
X
X
T
t
F
E
X
T
t
q
T
T
t
D
E
X
T
t
q
T
T
t
p
T
t
D
E
t
V
T
T
T
)
,
(
)
,
(
)
(
1
1
1
)
,
(
1
)
,
(
)
(
)
,
(
1
)
,
(
)
(
0
0
0
(A1a) (A1a) where
)
(
1
)
,
(
1
1
)
,
(
)
,
(
0
T
T
t
q
T
t
D
T
t
F
T
X
(A1b) (A1b) Proof of Proposition 2: Let
0
T
t
. On the first payment day, let
)
( 1T
V
denote the risky Proof of Proposition 2: Let
0
T
t
. On the first payment day, let
)
( 1T
V
denote the risky value of the asset excluding the current cash flow
1
X . According to Proposition 1, the risky value
of the asset at t is given by the asset excluding the current cash flow
1
X . According to Proposition 1, the risky value
i
i
b value of the asset excluding the current cash flow
1
X . A. Proofs According to Proposition 1, the risky value
of the asset at t is given by
t
F
)
(
)
,
(
)
(
1
1
1
0
T
V
X
T
T
F
E
t
V
(A2a) (A2a) where
)
(
1
)
,
(
1
1
)
,
(
)
,
(
0
)
(
1
0
1
0
1
1
T
T
t
q
T
T
D
T
T
F
X
T
V
(A2b)
Similarly, we have
1
)
(
)
,
(
)
(
2
2
2
1
1
T
T
V
X
T
T
F
E
T
V
F
(A3)
)
(
1
)
,
(
1
1
)
,
(
)
,
(
0
)
(
1
0
1
0
1
1
T
T
t
q
T
T
D
T
T
F
X
T
V
(A2b)
)
(
1
)
,
(
1
1
)
,
(
)
,
(
0
)
(
1
0
1
0
1
1
T
T
t
q
T
T
D
T
T
F
X
T
V
(A2b) (A2b)
1
)
(
)
,
(
)
(
2
2
2
1
1
T
T
V
X
T
T
F
E
T
V
F
(A3) (A3) 18 Note that
)
,
(
1
0 T
T
F
is
1
T
F
-measurable. According to the taking out what is known and Note that
)
,
(
1
0 T
T
F
is
1
T
F
-measurable. A. Proofs Depending on whether the payoff is in the money or out of the money at T, we
have
t
T
A
X
B
X
t
T
t
T
AB
A
A
X
t
T
AB
B
B
X
X
T
t
k
T
t
k
T
t
D
E
X
T
t
K
E
X
T
t
p
T
T
t
p
T
T
t
p
T
T
t
p
X
T
t
p
T
T
t
p
T
T
t
p
T
T
t
p
T
t
D
E
t
V
T
T
T
T
F
F
F
F
)
,
(
1
)
,
(
1
)
,
(
)
,
(
)
,
(
)
(
)
,
(
)
(
)
,
(
)
(
)
,
(
1
)
,
(
)
(
)
,
(
)
(
)
,
(
)
(
)
,
(
1
)
,
(
)
(
0
0
11
10
01
00
0
11
10
01
00
0
(A6a)
h
t
T
A
X
B
X
t
T
t
T
AB
A
A
X
t
T
AB
B
B
X
X
T
t
k
T
t
k
T
t
D
E
X
T
t
K
E
X
T
t
p
T
T
t
p
T
T
t
p
T
T
t
p
X
T
t
p
T
T
t
p
T
T
t
p
T
T
t
p
T
t
D
E
t
V
T
T
T
T
F
F
F
F
)
,
(
1
)
,
(
1
)
,
(
)
,
(
)
,
(
)
(
)
,
(
)
(
)
,
(
)
(
)
,
(
1
)
,
(
)
(
)
,
(
)
(
)
,
(
)
(
)
,
(
1
)
,
(
)
(
0
0
11
10
01
00
0
11
10
01
00
0
(A6a) (A6a) where
)
(
)
(
)
(
1
)
,
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
,
(
T
T
T
T
t
T
t
q
T
t
q
T
T
t
q
T
t
p
T
T
t
p
T
t
q
T
T
t
p
T
t
p
T
t
k
AB
B
B
AB
A
B
AB
A
B
B
A
B
B
A
B
B
(A6b) (A6b)
)
(
)
(
)
(
1
)
,
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
,
(
T
T
T
T
t
T
t
q
T
t
q
T
T
t
q
T
t
p
T
T
t
p
T
t
q
T
T
t
p
T
t
p
T
t
k
AB
A
A
AB
A
B
AB
B
A
A
B
A
A
A
B
A
(A6c) A. Proofs According to the taking out what is known and tower properties of conditional expectation, we have tower properties of conditional expectation, we have
t
t
t
t
t
F
F
F
F
F
F
F
)
(
)
,
(
)
,
(
)
(
)
,
(
)
,
(
)
,
(
)
,
(
)
(
)
,
(
)
(
2
1
0
1
2
1
1
0
1
2
2
1
2
2
1
1
0
1
1
0
1
1
1
0
1
1
T
V
T
T
F
E
X
T
T
F
E
T
V
T
T
F
E
X
T
T
F
E
T
T
F
E
X
T
T
F
E
T
V
X
T
T
F
E
t
V
j
j
j
i
i
i
j
j
j
T
T
(A4) (A4) iving from
2
T forward over
m
T , where
m
m
X
T
V
)
(
, we have By recursively deriving from
2
T forward over
m
T , where
m
m
X
T
V
)
(
, we have By recursively deriving from
2
T forward over
m
T , where
m
m
X
T
V
)
(
, we have
m
i
t
i
i
j
j
j
X
T
T
F
E
t
V
1
1
0
1)
,
(
)
(
F
(A5) (A5) Proof of Proposition 3: We assume that a default may only occur on the payment date. Proof of Proposition 3: We assume that a default may only occur on the payment date. Proof of Proposition 3: We assume that a default may only occur on the payment date. p
y
y
p y
At time T, there are four possible states: 1) both A and B survive, 2) A defaults but B survives, 3)
A survives but B defaults, and 4) both A and B default. The joint distributions of A and B are
given by (15). B. A practical framework for calculating bilateral CVA We develop a practical framework for calculating bilateral CVA at counterparty portfolio
level based on the theory described above. The framework incorporates netting and margin
agreements, and captures right/wrong way risk. 19 Two parties are denoted as A and B. All calculations are from the perspective of party A. Let the valuation date be t. The CVA computation procedure consists of the following steps. B.1. One core element of the trading credit risk modeling is the Monte Carlo scenario
generation (market evolution). This must be able to run a large number of scenarios for each risk
factor with flexibility over parameterization of processes and treatment of correlation between
underlying factors. Credit exposure may be calculated under real probability measure, while CVA
or pricing counterparty credit risk should be conducted under risk-neutral probability measure. Due to the extensive computational intensity of pricing counterparty risk, there will
inevitably be some compromise of limiting the number of market scenarios (paths) and the
number of simulation dates (also called “time buckets” or “time nodes”). The time buckets are
normally designed fine-granularity at the short end and coarse-granularity at the far end. The
details of scenario generation are beyond the scope of this paper. B.2. For ease of illustration, we choose a vanilla interest rate swap, as interest rate swaps
collectively account for around two-thirds of both the notional and market value of all
outstanding derivatives (FinPricing (2015)) Assume that party A pays a fixed rate, while party B pays a floating-rate. Assume that
there are M time buckets (
M
T
T
T
,...,
,
1
0
) in each scenario and N cash flows in the sample swap. Let consider scenario j first. For swaplet i, there are four important dates: the fixing date
f
it , , the starting date
s
it , ,
the ending date
e
it , and the payment date
p
it , . In general, these dates are not coincidently at the
simulation time buckets. The time relationship between swaplet i and the simulation time buckets
is illustrated in Figure B1. 20 Figure B1: An interest rate swaplet
j
T
1
jT
k
T
1
kT
f
it ,
s
it ,
e
it ,
p
it ,
Terms
Rates
Interest rate curve simulated at
f
it , Interest rate curve simulated at
f
it , Interest rate curve simulated at
f
it , Rates Figure B1: An interest rate swaplet This figure illustrates the time relationship between an interest rate swaplet and the simulation
time buckets. The floating leg of the swaplet is reset at the fixing date
f
it , with the starting date
s
it , , the ending date
e
it , , and the payment date
p
it ,
. The simulation time buckets are
1
1,...,
,
k
i
i
T
T
T
. The simulated interest rate curve is starting at
f
it , . Both fixed rate payments and
floating-rate payments occur on the same payment dates. The cash flow of swaplet i is determined at the fixing date
f
it , that is assumed to be
between the simulation time buckets
jT and
1
j
T
. First, we need to create an interest rate curve
observed at
f
it ,
by interpolating the interest rate curves simulated at
jT and
1
j
T
via either
Brownian Bridge or linear interpolation. The linear interpolation is the expectation of the
Brownian Bridge. B.2. Then we can calculate the payoff of swaplet i at scenario j as
)
,
(
)
,
;
(
,
,
,
,
,
,
e
i
s
i
e
i
s
i
f
i
i
j
t
t
R
t
t
t
F
N
(B1) (B1) where N denotes the notional;
)
,
;
(
,
,
,
e
i
s
i
f
i
t
t
t
F
denotes the simply compounded forward rate reset
at
f
it ,
for the forward period (
s
it , ,
e
it , );
)
,
(
,
,
e
i
s
i
t
t
denotes the accrual factor or day count where N denotes the notional;
)
,
;
(
,
,
,
e
i
s
i
f
i
t
t
t
F
denotes the simply compounded forward rate reset
at
f
it ,
for the forward period (
s
it , ,
e
it , );
)
,
(
,
,
e
i
s
i
t
t
denotes the accrual factor or day count fraction for the period (
s
it , ,
e
it , ) and R denotes the fixed rate. fraction for the period (
s
it , ,
e
it , ) and R denotes the fixed rate. 21 The cash flow amount calculated by (B1) is paid on the payment date
p
it , . This value
should be allocated into the nearest previous time bucket
k
T as: The cash flow amount calculated by (B1) is paid on the payment date
p
it , . This value
should be allocated into the nearest previous time bucket
k
T as: e allocated into the nearest previous time bucket
k
T as: )
,
(
~
,
,
,
,
p
i
k
i
j
i
k
j
t
T
D
(B2) (B2) where
)
,
(
,p
i
k t
T
D
denotes the risk-free discount factor based on the interest rate curve simulated
at
k
T . where
)
,
(
,p
i
k t
T
D
denotes the risk-free discount factor based on the interest rate curve simulated
at
k
T . at
k
T . Cash flow generation for products without early-exercise provision is quite
straightforward. B.2. For early-exercise products, one can use the approach proposed by Longstaff and
Schwartz (2001) to obtain the optimal exercise boundaries and then the payoffs. B3. After generating cash flows for each deal, we need to aggregate them at counterparty
portfolio level at each scenario and each time bucket. The cash flows are aggregated by either
netting or nonnetting based on the netting agreements. A netting agreement is a provision that
allows the offset of settlement payments and receipts on all contracts between two counterparties. Another important use of netting is the close-out netting that allows the offset of close-out values. For netting, we add all cash flows together at the same scenario and the same time bucket
to recognize offsetting. The aggregated cash flow under netting at scenario j and time bucket k is
given by
i
i
k
j
k
j
,
,
,
~
~
(B3) (B3) For nonnetting, we divided cash flows into positive and negative groups and add them
separately. In other words, the offsetting is not recognized. The aggregated cash flows under
nonnetting at scenario j and time bucket k are given by
0
~
0
~
~
,
,
,
,
,
,
,
,
,
m
k
j
m
m
k
j
l
k
j
l
l
k
j
k
j
if
if
(B4) (B4) 22 Margin (or collateral) agreements For a more detailed discussion on pricing collateralized contract/portfolio, see Xiao
(2013b). B5. After aggregating all cash flows via netting, one can price a portfolio in the same manner
as pricing a single deal. We assume that the reader is familiar with the least square Monte Carlo
valuation model proposed by Longstaff and Schwartz (2001) and thus do not repeat some well-
known procedures for brevity. If the counterparty portfolio is collateralized, we can calculate the risky value based on
equation (21) of Xiao (2013b). If there is no collateral agreement, we can price the portfolio
according to Proposition 4 in this paper. CVA is by definition the difference between the risk-free portfolio value and the true (or
risky or defaultable) portfolio value. FinPricing, 2015, Financial Market Solution, https://finpricing.com/product.html Pykhtin, Michael, and Steven Zhu, 2007, A guide to modeling counterparty credit risk, GARP
Risk Review, July/August, 16-22. Reference Brigo, D., and Capponi, A., 2008, Bilateral counterparty risk valuation with stochastic dynamical
models and application to Credit Default Swaps, Working paper. Duffie, Darrell, and Ming Huang, 1996, Swap rates and credit quality, Journal of Finance, 51,
921-949. Duffie, Darrell, and Kenneth J. Singleton, 1999, Modeling term structure of defaultable bonds,
Review of Financial Studies, 12, 687-720. Duffie, Darrell, and Kenneth J. Singleton, 1999, Modeling term structure of defaultable bonds,
Review of Financial Studies, 12, 687-720. Duffie, Darrell, and Kenneth J. Singleton, 1999, Modeling term structure of defaultable bonds,
Review of Financial Studies, 12, 687-720. 23 23 Gregory, Jon, 2009, Being two-faced over counterparty credit risk, RISK, 22, 86-90. Hull, J. and White, A., 2013, CVA and wrong way risk, forthcoming, Financial Analysts Journal. Jarrow, R. A., and Protter, P., 2004, Structural versus reduced form models: a new information
based perspective, Journal of Investment Management, 2, 34-43. Jarrow, Robert A., and Stuart M. Turnbull, 1995, Pricing derivatives on financial
securities subject to credit risk, Journal of Finance, 50, 53-85. Lipton, A., and Sepp, A., 2009, Credit value adjustment for credit default swaps via the structural
default model, Journal of Credit Risk, 5(2), 123-146. Longstaff, Francis A., and Eduardo S. Schwartz, 2001, Valuing American options by simulation:
a simple least-squares approach, The Review of Financial Studies, 14 (1), 113-147. Moody’s Investor’s Service, 2000, Historical default rates of corporate bond issuers, 1920-99. J. P. Morgan, 1999, The J. P. Morgan guide to credit derivatives, Risk Publications. O’Kane, D. and S. Turnbull, 2003, Valuation of credit default swaps, Fixed Income Quantitative
Credit Research, Lehman Brothers, QCR Quarterly, 2003 Q1/Q2, 1-19. Pykhtin, Michael, and Steven Zhu, 2007, A guide to modeling counterparty credit risk, GARP
Risk Review, July/August, 16-22. 24 24 Sorensen, E. and T. Bollier, 1994, Pricing swap default risk, Financial Analysts Journal, 50, 23-
33. 33. Xiao, T., 2013a, The impact of default dependency and collateralization on asset pricing and
credit risk modeling, Working paper. Xiao, T., 2013b, An economic examination of collateralization in different financial market,
Working paper. 33. 33. Xiao, T., 2013a, The impact of default dependency and collateralization on asset pricing and
credit risk modeling, Working paper. Xiao, T., 2013a, The impact of default dependency and collateralization on asset pricing and
credit risk modeling, Working paper. Xiao, T., 2013a, The impact of default dependency and collateralization on asset pricing and
credit risk modeling, Working paper. Reference Xiao, T., 2013b, An economic examination of collateralization in different financial market,
Working paper. 25
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https://openalex.org/W4226183901
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https://zenodo.org/records/5639377/files/1-2%202021%20-%20Benes%20-%20Evolu%C5%A3ia%20jurispruden%C5%A3ei%20CEDO%20cu%20privire%20la%20cererile%20interstatale.pdf
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Romanian, Moldavan
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Evoluţia jurisprudenţei Curţii Europene a Drepturilor Omului cu privire la cererile interstatale
|
Zenodo (CERN European Organization for Nuclear Research)
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cc-by
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1 Convenţia Europeană a Drepturilor Omului din 04.11.1950 https://www.echr.coe.int/Docu
ments/Convention_RON.pdf CZU: 341.231.14 DOI: 10.5281/zenodo.5639377 CZU: 341.231.14 DOI: 10.5281/zenodo.5639377 Olga BENEŞ, master în drept, lector universitar (ORCID: 0000-0001-7853-9557) Olga BENEŞ, master în drept, lector universitar (ORCID: 0000-0001-7853-9557) Evolution of the Jurisprudence of the European Court of Human Rights on
Interstate Requests
Article 33 of the European Convention on Human Rights, entitled Interstate Cases, provides:
„Any High Contracting Party may refer to the Court any violation of the provisions of the
Convention and its Protocols by another High Contracting Party.“ To that end, the requesting
State is acting rather to denounce one or more infringements alleged to have been suffered by
private individuals who have been replaced in a certain way. Keywords: interstate demand, European public order, collective guarantee of rights and freedoms. Articolul 33 al Convenţiei Europene a Drepturilor Omului întitulat Cauze interstatale dispune:
„Orice Înaltă Parte contractantă poate sesiza Curtea asupra oricărei pretinse încălcări a preve
derilor Convenţiei şi ale Protocoalelor sale de către o altă Înaltă Parte Contractantă„. În acest
sens, statul reclamant acţionează mai degrabă pentru a denunţa una sau mai multe încălcări
pretinse a fi suferite de către persoane particulare cărora li se substituie într-un fel anume. Cuvinte-cheie: cerere interstatală, ordine publică europeană, garanţie colectivă a drepturilor
şi libertăţilor. s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 La acest capitol Convenţia Europeană a Drepturilor Omului a suferit o evoluţie
foarte diferită de cea concepută de autorii săi, care la origini au decis instituirea
unui caracter constrângător pentru statele care aveau deficienţe în materia protecţiei
drepturilor omului, impunând prin aceasta obligaţia de a se supune Curţii Europene. „Recursul interstatal, prevăzut ca un instrument cheie de către autorii Convenţiei, în
scopul de a împiedica un stat membru să admită dictatura, trebuia să constituie me
canismul principal. Protecţia drepturilor fundamentale, apărea într-un fel oarecare pe
planul doi. Dar n-a fost cazul, recursul interstatal care la origini trebuia să constituie
pivotul sistemului a fost de fapt abandonat pe parcurs de zece ani“2. Majoritatea plângerilor care au apărut pe parcurs se referă la situaţiile de criză
sau de conflict, printre care metodele de interogare folosite de autorităţile britanice
în anii 1971—1975 pe parcursul tulburărilor din Irlanda de Nord, operaţiunile
militare desfăşurate de Turcia în Ciprul de Nord în anul1974, conflictul armat
dintre Georgia şi Rusia din 2008 şi mai recent, evenimentele din Crimeea şi Estul
Ucrainei din 2014. Încă în cauza Austria c. Italiei din 1963, Comisia a dispus în raportul său că
„Orice Parte contractantă poate sesiza Comisia cu privire la orice încălcare a dispozi
ţiilor Convenţiei care crede că poate fi imputată unei alte Părţi contractante, că prin
acest articol, Înaltele Părţi Contractante au autorizat, în consecinţă, oricare dintre ele
să aducă în faţa Comisiei orice presupusă încălcare a dispoziţiilor Convenţiei, indife
rent dacă victimele acestei încălcări sunt sau nu resortisanţi ai statului solicitant, fie
că presupusa violare prejudiciază sau nu în special interesele acestui stat, că un stat
contractant, atunci când sesizează Comisia în temeiul articolului 24 (33), nu trebuie,
prin urmare, să fie considerat ca acţionând pentru a-şi asigura propriile drepturi, ci
mai degrabă ca adresând Comisiei o întrebare care aduce atingere ordinii publice a
europene“3. În acest caz, statul reclamant acţionează mai puţin în apărarea drepturilor sale
proprii, aşa cum este cazul în faţa Curţii Internaţionale de Justiţie, dar are scopul
de „a denunţa una sau mai multe încălcări pretinse a fi suferite de către persoane
particulare cărora li se substituie într-un fel anume,“4 încălcări care aduc atingere
ordinii publice europene. 2 Petitti L.E., Decaux E., Imbert P.H. La Convention Européenne des Droits de l’Homme. Com
mentaire article par article. Réflexions sur les principes et les mécanismes de la Convention.
Paris : Economica, 1999. p. 27.
3 Rapport de la Com.EDO, Autriche c. Italie, 788/60, 30.03.1963. http://hudoc.echr.coe.int/app/
conversion/pdf/?library=ECHR&id=001-27972&filename=001-27972.pdf
4 CEDO, Georgia c. Rusiei (II), n° 38263/08, dec. din 13.12. 2011. https://hudoc.echr.coe.int/
eng#{%22itemid%22:[%22001-108097%22]} 4 CEDO, Georgia c. Rusiei (II), n° 38263/08, dec. din 13.12. 2011. https://hudoc.echr.coe.int/
eng#{%22itemid%22:[%22001-108097%22]} Evolution of the Jurisprudence of the European Court of Human Rights on
Interstate Requests Article 33 of the European Convention on Human Rights, entitled Interstate Cases, provides:
„Any High Contracting Party may refer to the Court any violation of the provisions of the
Convention and its Protocols by another High Contracting Party.“ To that end, the requesting
State is acting rather to denounce one or more infringements alleged to have been suffered by
private individuals who have been replaced in a certain way. words: interstate demand, European public order, collective guarantee of rights and freedoms. Articolul 33 al Convenţiei Europene a Drepturilor Omului întitulat Cauze interstatale dispune:
„Orice Înaltă Parte contractantă poate sesiza Curtea asupra oricărei pretinse încălcări a preve
derilor Convenţiei şi ale Protocoalelor sale de către o altă Înaltă Parte Contractantă„. În acest
sens, statul reclamant acţionează mai degrabă pentru a denunţa una sau mai multe încălcări
pretinse a fi suferite de către persoane particulare cărora li se substituie într-un fel anume. Cuvinte-cheie: cerere interstatală, ordine publică europeană, garanţie colectivă a drepturilor
şi libertăţilor. Curtea Europeană a Drepturilor Omului nu poate să se sesizeze din oficiu, ea
fiind competentă de a statua asupra plângerilor de încălcare a Convenţiei Europene
a Drepturilor Omului şi în acest scop trebuie să fie sesizată cu cereri individuale sau
interstatale. Convenţia distinge două tipuri de cereri: cererile individuale depuse
conform articolului 34 al Convenţiei şi cererile interstatale conform articolului 33
al Convenţiei. Articolul 33 al Convenţiei Europene a Drepturilor Omului întitulat Cauze inter
statale dispune: „Orice Înaltă Parte contractantă poate sesiza Curtea asupra oricărei
pretinse încălcări a prevederilor Convenţiei şi ale Protocoalelor sale de către o altă
Înaltă Parte Contractantă“1. Astfel, prin această dispoziţie, Convenţia Europeană a
Drepturilor Omului îi acordă Curţii Europene o competenţă în materie de diferende
între state, competenţă exercitată pe parcursul timpului foarte rar. 32 2 Petitti L.E., Decaux E., Imbert P.H. La Convention Européenne des Droits de l’Homme. Com
mentaire article par article. Réflexions sur les principes et les mécanismes de la Convention.
Paris : Economica, 1999. p. 27. 5 Annuaire de la Convention Europeenne des Droits de l’Homme. Commission et Cour Eu
ropeenne des Droits de l’Homme 1958-1959. Springer Science+Business Media, B.V. 1960.
PP.179 https://books.google.md/books?id=7HrwCAAAQBAJ&pg=PA174&lpg=PA174&dq=
Requete+No.+176/56+Par+cette+premiere+requ%C3%8ATe,+introduite+le+7+mai+I956,+l
e+Gouvernement+hellenique&source=bl&ots=GrHVB1gQ9j&sig=ACfU3U0QmZDxdLI8u
X58AE8lL6gT_1oHjg&hl=en&sa=X&ved=2ahUKEwjYjf2l9PDmAhVMCewKHUHRBQ0Q
6AEwAHoECAkQAQ#v=onepage&q=Requete%20No.%20176%2F56%20Par%20cette%20
premiere%20requ%C3%8ATe%2C%20introduite%20le%207%20mai%20I956%2C%20le%20
Gouverne-ment%20hellenique&f=false
b d s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 Pentru prima dată Comisia a fost sesizată cu două cereri interstatale ale guver
nului elen împotriva Regatului Unit. Aceste două cereri făceau referinţă la aplicarea
Convenţiei în insula Cipru. Prin prima cerere nr. 176/56, depusă la 7 mai 1956,
Guvernul elen susţinea că derogarea de la dispoziţiile Convenţiei notificate de către 2 Petitti L.E., Decaux E., Imbert P.H. La Convention Européenne des Droits de l’Homme. Com
mentaire article par article. Réflexions sur les principes et les mécanismes de la Convention. Paris : Economica, 1999. p. 27. 33 s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 Guvernul britanic cu privire la Cipru în temeiul articolului 15 al Convenţiei, nu era
conformă şi că condiţiile de fond cerute de acest articol nu erau îndeplinite în speţă. Prin cererea 299/57 din 17 iulie 1957, guvernul elen sesiza Comisia de 49 de cazuri de
„tortură sau servicii asimilabile torturii“ produse în Cipru şi de care guvernul britanic
se făcea responsabil. De asemenea, el a contestat compatibilitatea unei serii de legi
şi regulamente de urgenţă în vigoare în Cipru cu dispoziţiile Convenţiei. În special,
el a susţinut că legislaţia care prevedea impunerea pedepsei cu biciuirea şi anumite
pedepse colective încălca articolul 3 din Convenţie, de la care articolul 15 interzice
Părţilor contractante derogarea, chiar şi în caz de război sau alt „pericol public care
ameninţă viaţa naţiunii“. La rândul său, Guvernul britanic a negat încălcarea Convenţiei
bazându-se fie pe însăşi definiţia drepturilor şi libertăţilor recunoscute de Convenţie,
alteori pe existenţa în Cipru a unui „pericol public care ameninţă viaţa naţiunii“ în
sensul articolului 15 citat mai sus5. Guvernul britanic cu privire la Cipru în temeiul articolului 15 al Convenţiei, nu era
conformă şi că condiţiile de fond cerute de acest articol nu erau îndeplinite în speţă. Prin cererea 299/57 din 17 iulie 1957, guvernul elen sesiza Comisia de 49 de cazuri de
„tortură sau servicii asimilabile torturii“ produse în Cipru şi de care guvernul britanic
se făcea responsabil. De asemenea, el a contestat compatibilitatea unei serii de legi
şi regulamente de urgenţă în vigoare în Cipru cu dispoziţiile Convenţiei. s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 În special,
el a susţinut că legislaţia care prevedea impunerea pedepsei cu biciuirea şi anumite
pedepse colective încălca articolul 3 din Convenţie, de la care articolul 15 interzice
Părţilor contractante derogarea, chiar şi în caz de război sau alt „pericol public care
ameninţă viaţa naţiunii“. La rândul său, Guvernul britanic a negat încălcarea Convenţiei
bazându-se fie pe însăşi definiţia drepturilor şi libertăţilor recunoscute de Convenţie,
alteori pe existenţa în Cipru a unui „pericol public care ameninţă viaţa naţiunii“ în
sensul articolului 15 citat mai sus5. În procedura din perioada respectivă s-a pus întrebarea de a şti care erau prero
gativele Comisiei atunci când un stat invocând articolul 15, deroga de la obligaţiile
prevăzute de Convenţie. În această situaţie, Comisia a considerat că era „competentă
să se pronunţe cu privire la existenţa unui pericol public, care conform articolului
15 îi acordă statului dreptul de a deroga de la obligaţiile prevăzute de Convenţie“. De asemenea, Comisia a considerat că era „competentă să se pronunţe asupra fap
tului dacă măsurile luate de către o Parte conform articolului 15 al Convenţiei, au
fost în strictă conformitate cu exigenţele situaţiei“. În acest sens, ea a precizat că
„guvernul trebuie să păstreze o anumită marjă de apreciere cu privire la stricteţea
măsurii cerută de situaţie“6. Deoarece în perioada dată nici Grecia, nici Regatul Unit
nu acceptase competenţa obligatorie a Curţii Europene a Drepturilor Omului şi
odată cu reglementarea politică a problemei cipriote, Comisia a decis să pună capăt
procedurii fără a se pronunţa pe fond în cauza dată. În următoarea cauză Austria c. Italiei nr.788/60 din 11 iulie 1960, Austria a
invocat încălcarea de către guvernul italian a dispoziţiilor articolului 6 al Convenţiei
contestând procedurile care au adus la pedepsirea persoanelor în speţă, acestea fiind
considerate ilegale şi contrare Convenţiei Europene a Drepturilor Omului, şi anume
dispoziţiilor care enunţă regulile cu privire la o bună administrare a justiţiei şi a
articolului 14 care interzice orice discriminare în prevalarea drepturilor garantate 34 s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 de Convenţie7. s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 Fiind chemată să se expună asupra obligaţiilor statelor părţi ce re
zultă din textul Convenţiei, Comisia a enunţat că: „…rezultă că obligaţiile asumate
de statele contractante prin Convenţie au, în esenţă un caracter obiectiv deoarece
vizează protejarea drepturilor fundamentale ale indivizilor împotriva abuzurilor
statelor contractante mai degrabă decât crearea unor drepturi subiective şi reci
proce între acestea, că natura obiectivă a angajamentelor menţionate apare şi în
mecanismul instituit de Convenţie, că acest mecanism se bazează pe conceptul de
garanţie colectivă, de către statele contractante, a drepturilor şi libertăţilor definite
în Convenţie“8. Prin decizia Comisiei în această cauză s-a instituit dreptul fiecărei
Părţi contractante de a pune în aplicare mecanismul instituit prin articolul 24 al
Convenţiei chiar şi pentru fapte anterioare ratificării sale de către statul reclamant,
rolul acestui mecanism fiind protecţia „patrimoniului comun, de tradiţii politice,
de idealuri, de libertate şi de preeminenţă a dreptului, astfel asigurând garanţia
colectivă a unor drepturi enunţate în Declaraţia Universală9“. Deci, după cum observăm, în această perioadă se enunţă scopul recursului
interstatal, şi anume, garantarea colectivă a drepturilor şi libertăţilor fundamentale,
acesta fiind consolidat printr-o serie de cauze care au urmat depuse de Danemarca,
Franţa, Germania, Suedia şi Ţările de Jos împotriva Turciei la 1 iulie 1982. Chiar
dacă cetăţenii acestor state nu erau direct ameninţaţi de situaţia stabilită, aceste
state totuşi au contestat în faţa Curţii Europene atingerile aduse drepturilor şi
libertăţilor comise de către autorităţile turce în urma unei lovituri de stat militare
intervenite în septembrie 1980. Se consideră că sub acest aspect cauza Irlanda c. Regatului Unit10 din 10 martie 1976, poate fi considerată un punct de încercare,
anunţând un moment de cotitură deoarece a reprezentat o importanţă şi o semni
ficaţie particulară în măsura în care punea problema recurgerii la anumite metode
de represiune, şi aceasta nu într-un regim dictatorial dar în cadrul unei democraţii. Această cauză este de o importanţă deosebită în definirea anumitor termeni cu
privire la articolul 3 CEDO şi determinarea marjei pentru stabilirea diferenţei
între calificativele tratamentului uman sau inuman, umilitor sau degradant, care
trebuie să fie apreciate în condiţiile şi contextul cauzei. Astfel, regula de principiu
care a fost stabilită enunţă că: „Aplicarea sistematică a metodelor în scopul de a
instiga o persoană să dea informaţii prezintă similitudini clare cu metodele de
tortură sistematică cunoscută pe parcursul secolelor. 7 Annuaire de la Convention Europeenne des Droits de l’Homme. Commission et Cour Eu
ropeenne des Droits de l’Homme 1962. Springer Science+Business Media, B.V. 1963, p.47;
Commission et Cour%20 Europeenne des Droits de l’Homme 1958-1959.
8 Rapport de la Com.EDO, Autriche c. Italie, 788/60, 30.03.1963, p. 9.
9 Ibidem, p.8.
10 CtEDO, cauza Irlanda c. Regatului Unit, hot. din 18.01.1978. https://hudoc.echr.coe.int/eng
#{%22itemid%22:[%22001-62064%22]} 10 CtEDO, cauza Irlanda c. Regatului Unit, hot. din 18.01.1978. https://hudoc.echr.coe.int/eng
#{%22itemid%22:[%22001-62064%22]} 11 Annuaire de la Convention Europeenne des Droits de l’Homme. 1976. La Haye: Martinus
Nijhoff, 1977. p.795
12 CtEDO, cauza Cipru c. Turciei nr. 25781/94, hot. din 12 .05.2014. https://hudoc.echr.coe.int/
fre#{%22itemid%22:[%22001-144153%22]}
13 CtEDO, cauza Cipru c. Turciei nr. 25781/94, hot. din 12 .05.2014. Opinia comună a judecă
torului PINTO DE ALBUQUERQUE şi VUČINIĆ https://hudoc.echr.coe.int/fre#{%22item
id%22:[%22001-144153%22]} s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 Chiar dacă cele cinci metode
cunoscute drept metode de dezorientare sau de privare senzorială nu lasă în mod
necesar urme grave, Comisia vede în aceasta un sistem modern de tortură care se 35 s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 află în aceiaşi categorie cu sistemele aplicate anterior pentru a obţine informaţii
sau mărturii“11. O altă hotărâre care a fost de o importanţă particulară în evoluţia jurispruden
ţei este Cipru c. Turciei12, pronunţată de Marea Cameră la 12 mai 2014 şi vizează
noţiunea de satisfacţie echitabilă în cadrul cererilor interstatale. Pentru prima dată
Curtea impune statului pârât obligaţia să plătească despăgubiri altui stat, şi anume
90 milioane de Euro cu titlu de satisfacţie echitabilă în cadrul unei cauze intersta
tale, statul destinatar fiind şi el obligat să distribuie această sumă victimelor sub
supravegherea Comitetului de Miniştri în termenul fixat de Curte. Referindu-se
la caracterul satisfacţiei echitabile, Curtea reaminteşte că logica articolului 41 cu
privire la satisfacţia echitabilă decurge direct din regulile şi principiile dreptului
internaţional public cu privire la răspunderea statului pentru încălcarea unei obligaţii
ce decurge dintr-un tratat şi trebuie interpretată în acest context. Având în vedere
natura Convenţiei Europene a Drepturilor Omului, anume individul şi nu statul este
direct afectat prin încălcarea unuia sau mai multor drepturi garantate de Convenţie
şi deci, o satisfacţie echitabilă într-o cauză interstatală trebuie să fie întotdeauna în
beneficiul victimelor individuale. i
În urma acestui precedent şi alte state membre ar putea invoca sume importante
în baza încălcărilor repetate, continue sau multiple a drepturilor fundamentale mai
ales având în vedere faptul că cererile interstatale sunt adesea izvorâte dintr-un context
belicos între statele părţi. Astfel, exemplul acestei hotărâri poate aduce la creşterea
numărului cererilor interstatale în cadrul cărora s-ar obţine o indemnizaţie globală
pentru o multitudine de victime. Exprimând opinia lor comună referitor la acest precedent, unii judecători au
atras atenţia că: „Hotărârea Cipru c. Turciei (satisfacţia echitabilă) este cea mai
importantă contribuţie la pacea în Europa în istoria Curţii Europene a Dreptu
rilor Omului. s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 Mesajul adresat satelor membre ale Consiliului Europei este clar:
statele membre care fac război, invadează alte state membre sau susţin interven
ţiile militare în alte state membre trebuie să plătească pentru actele lor ilegale
şi consecinţele acestor acte, în timp ce victimele şi familiile lor cât şi statele ale
căror sunt resortisanţi au un drept dobândit şi executoriu de a fi compensate în
mod corespunzător şi pe deplin de către statul beligerant responsabil. Războiul şi
consecinţele sale tragice nu mai pot fi tolerate în Europa, iar statele membre care
nu respectă acest principiu trebuie să răspundă pentru actele lor în faţa justiţiei,
fără alte consecinţe în plan politic“13. 36 14 CtEDO, cauza Cipru c. Turciei nr. 25781/94, hot. din 12 .05.2014. Opinia comună a judecă
torilor ZUPANČIČ, GYULUMYAN, DAVÍD THÓR BJÖRGVINSSON, NICOLAOU, SAJÓ,
LAZAROVA TRAJKOVSKA, POWER-FORDE, VUČINIĆ ET PINTO DE ALBUQUERQUE.
https://hudoc.echr.coe.int/fre#{%22itemid%22:[%22001-144153%22]} p
p
j
torilor ZUPANČIČ, GYULUMYAN, DAVÍD THÓR BJÖRGVINSSON, NICOLAOU, SAJÓ,
LAZAROVA TRAJKOVSKA, POWER-FORDE, VUČINIĆ ET PINTO DE ALBUQUERQUE.
https://hudoc.echr.coe.int/fre#{%22itemid%22:[%22001-144153%22]}
15 CtEDO, cauza Georgia c. Rusiei (I) nr.13255/07, hot. din 03.07.2014. https://hudoc.echr.coe.
int/fre#{%22itemid%22:[%22001-145552%22]}
16 CtEDO, cauza Georgia c. Rusiei (I) nr.13255/07, hot. sat. echit. din 31.01.2019. https://www.
doctrine.fr/d/CEDH/HFJUD/GRANDCHAMBER/2019/CEDH001-189852
17 CtEDO, cauza Slovenia c. Croatia nr. 5155/16, cerere din 15.09.2016. https://www.google.
com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=2ahUKEwiPk5KDhYXnAhUlsa
QKHRnJDicQFjABegQIBBAC&url=http%3A%2F%2Fhudoc.echr.coe.int%2Fapp%2Fconve
rsion%2Fpdf%2F%3Flibrary%3DECHR%26id%3D003-6427775 8450864%26filename%3D
Grand%2520Chamber%2520hearing%2520Slovenia%2520v.%2520Croatia.pdf&usg=AOvV
aw15y2jR8ia5B23O6XFBC5v8 s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 Atenţionând asupra importanţei acestei hotărâri, 10 din cei 17 judecători ai
Marii Camere au menţionat în opinia lor că: „Această hotărâre anunţă începutul
unei noi ere în implementarea drepturilor omului protejate de Curte şi marchează
o etapă importantă în ceea ce priveşte respectarea statului de drept în Europa. Este
pentru prima dată în istoria Curţii când aceasta face o declaraţie specifică cu privire
la scopul şi efectul uneia dintre hotărârile sale în contextul executării.“14 Altă cauză care consolidează jurisprudenţa Curţii în materie de satisfacţie echita
bilă în cazurile interstatale este Georgia c. Rusiei (I)15, cu privire la expulzarea colectivă
a resortisanţilor georgieni de către autorităţile ruse începând cu luna octombrie 2006
şi până în ianuarie 2007. În hotărârea sa asupra fondului cauzei din 3 iulie 2014,
Curtea a stabilit aplicarea de către Federaţia Rusă a unei a politici coordonate de
arestare, detenţie şi expulzare a resortisanţilor georgieni care a constituit o practică
administrativă, în sensul jurisprudenţei Curţii, astfel încălcându-se articolul 4 din
Protocolul nr.4, articolele 3 şi 13 combinat cu articolele 5§1 şi 3. În această hotărâre
Curtea a enumerat cele trei condiţii necesare de a fi reunite pentru ca să stabilească
dacă se justifică acordarea unei satisfacţii echitabile în cadrul cauzelor interstatale:
capătul de acuzare formulat de guvernul reclamant care trebuie să se refere la încăl
carea drepturilor fundamentale a resortisanţilor săi sau a altor persoane, posibilitatea
de identificare a victimelor şi obiectivul principal al procedurii. Astfel, Curtea a
hotărât în hotărârea cu privire la satisfacţie echitabilă, că Rusia trebuie să plătească
Georgiei 10 milioane de Euro pentru prejudiciul moral, care să fie repartizat unui
grup de victime de circa 1500 de resortisanţi georgieni16. În prezent pe rolul Curţii sunt opt cereri pendinte dintre care o cerere Slovenia
c.Croatiei, două cereri Georgia c. Rusiei şi 5 cereri depuse de Ucraina c. Rusiei. Cauza Slovenia c. Croaţiei17 nr. 54155/16 se referă la lipsa de echitate, la parţialitatea
şi discriminarea din partea instanţelor croate în cadrul procedurilor iniţiate de o bancă
slovenă Ljubljanska banka d.d., pentru încasarea datoriilor de la companiile croate. La rândul său Georgia a depus două plângeri împotriva Rusiei, în anul 2008
cu privire la conflictul armat dintre Georgia şi Rusia din 2008 şi consecinţele sale, 14 CtEDO, cauza Cipru c. 18 CtEDO, cauza Ucraina c. Federaţiei Ruse nr. 20958/14, cerere din 13.03.2014. https://www.
google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=2ahUKEwjBuuuy5ILnA
hUOkxQKHfuJB24QFjABegQIARAC&url=https%3A%2F%2Fhudoc.echr.coe.int%2Fapp%
2Fconversion%2Fpdf%3Flibrary%3DECHR%26id%3D003-6498862-8572168%26filename%
3DAudience%2520de%2520Grande%2520Chambre%2520Ukraine%2520c.%2520Russie%2
520%2528Crim%25E9e%2529.pdf&usg=AOvVaw3cuONJfE0Z8mCUmHUr-Nyi s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 Turciei nr. 25781/94, hot. din 12 .05.2014. Opinia comună a judecă
torilor ZUPANČIČ, GYULUMYAN, DAVÍD THÓR BJÖRGVINSSON, NICOLAOU, SAJÓ,
LAZAROVA TRAJKOVSKA, POWER-FORDE, VUČINIĆ ET PINTO DE ALBUQUERQUE. https://hudoc.echr.coe.int/fre#{%22itemid%22:[%22001-144153%22]} 17 CtEDO, cauza Slovenia c. Croatia nr. 5155/16, cerere din 15.09.2016. https://www.google. com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=2ahUKEwiPk5KDhYXnAhUlsa
QKHRnJDicQFjABegQIBBAC&url=http%3A%2F%2Fhudoc.echr.coe.int%2Fapp%2Fconve
rsion%2Fpdf%2F%3Flibrary%3DECHR%26id%3D003-6427775 8450864%26filename%3D
Grand%2520Chamber%2520hearing%2520Slovenia%2520v.%2520Croatia.pdf&usg=AOvV
aw15y2jR8ia5B23O6XFBC5v8 37 s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 care se află pe rolul Marii Camere, şi în 2018 cu privire la pretinsele deteriorări ale
situaţiei drepturilor omului de-a lungul frontierei administrative dintre teritoriul
controlat de Georgia, Abhazia şi Osetia de Sud. Începând cu anul 2014, Ucraina depune mai multe cereri împotriva Federaţiei
Ruse şi în prezent pe rolul Curţii regăsim cinci cereri pendinte: două în faţa Marii
Camere, una cu privire la situaţia din Crimeea şi alta la cea din partea de Est a Ucrai
nei. Alte trei cereri sunt pendinte în faţa unei camere: una cu privire la presupusa
răpire de copii din estul Ucrainei şi transferul lor temporar în Rusia în 2014, alta
referitor la detenţia şi acuzarea resortisanţilor ucraineni pentru diferite infracţiuni
şi una privind incidentul din strâmtoarea Kerch din noiembrie 2018, care a dus la
capturarea a trei nave de război ucrainene şi a echipajului lor. Este interesantă abordarea Curţii în cererea Ucraina c. Federaţiei Ruse din 13
martie 2014, cu privire la măsurile provizorii cerute de guvernul ucrainean referitor
la obligarea imediată a Federaţiei Ruse să se abţină de la orice intervenţie care ar
constitui ameninţări la viaţa şi sănătatea populaţiilor civile de pe teritoriul lor, în
special în Crimeea. Curtea Europeană a acceptat să ia măsuri provizorii conform
articolului 39 al Regulamentului său, dar în acelaşi timp ea s-a adresat ambelor părţi
contractante, atât Ucrainei cât şi Rusiei „să se abţină de a lua orice măsură şi, în
special de natură militară, care ar putea implica pentru populaţia civilă, încălcări ale
drepturilor garantate de Convenţie, inclusiv cele care ar putea pune în pericol viaţa şi
sănătatea populaţiei civile“18. Pentru o gestionare mai eficientă a cazurilor interstatale
rezultate din cererile formulate de Ucraina împotriva Federaţiei Ruse, Curtea a decis
anul trecut să soluţioneze toate plângerile referitoare la evenimentele din Crimeea
în contextul cauzei nr. 20958/14 şi toate reclamaţiile referitoare la evenimentele din
estul Ucrainei şi la Donbass în contextul cauzei Ucraina v. Rusia (V), nr. s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 8019/16.l Aflându-ne deci în câmpul recursului interstatal, putem spune că respectarea
drepturilor omului este o bază esenţială a existenţei societăţilor democratice care
sunt mai bine plasate decât alte regimuri pentru a evita conflictele în cadrul statelor
Consiliului Europei, acestea având legături strânse între ele prin acorduri de ordin
politic, economic şi social astfel încât să se asigure o veritabilă ordine europeană. 38
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Regulatory and Mechanistic Actions of Glucocorticoids on T and Inflammatory Cells
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Regulatory and Mechanistic Actions
of Glucocorticoids on T and
Inflammatory Cells Ana C. Liberman1†, Maia L. Budziñski1†, Clara Sokn1, Romina Paula Gobbini1,
Anja Steininger1 and Eduardo Arzt1,2* Ana C. Liberman1†, Maia L. Budziñski1†, Clara Sokn1, Romina Paula Gobbini1,
Anja Steininger1 and Eduardo Arzt1,2* 1 Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA) – CONICET – Partner Institute of the Max Planck
Society, Buenos Aires, Argentina, 2 Departamento de Fisiología y Biología Molecular y Celular, Facultad de Ciencias Exactas
y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina Glucocorticoids (GCs) play an important role in regulating the inflammatory and immune
response and have been used since decades to treat various inflammatory and autoim-
mune disorders. Fine-tuning the glucocorticoid receptor (GR) activity is instrumental in
the search for novel therapeutic strategies aimed to reduce pathological signaling and
restoring homeostasis. Despite the primary anti-inflammatory actions of GCs, there are
studies suggesting that under certain conditions GCs may also exert pro-inflammatory
responses. For these reasons the understanding of the GR basic mechanisms of action
on different immune cells in the periphery (e.g., macrophages, dendritic cells, neutro-
phils, and T cells) and in the brain (microglia) contexts, that we review in this chapter, is a
continuous matter of interest and may reveal novel therapeutic targets for the treatment
of immune and inflammatory response. INTRODUCTION Living organisms must sustain a dynamic equilibrium in order to maintain homeostasis and survival
which is constantly challenged by internal or external stressors. In order to appropriately cope with
stressful stimuli, they have developed a highly conserved regulatory system. This neuroendocrine
system consists mainly of the hypothalamic–pituitary–adrenal (HPA) axis and the autonomic nerv-
ous system. Glucocorticoids (GCs), are the end-product of the HPA axis, and play an important role
in the maintenance of both resting and stress-related responses. If the stress response is dysregu-
lated, homeostasis is altered and probably a wide range of adverse effects may appear on many vital
physiological functions, such as growth, development, metabolism, reproduction, immune response,
cognition, and behavior. †These authors have contributed
equally to this work. Specialty section:
This article was submitted to
Neuroendocrine Science,
a section of the journal
Frontiers in Endocrinology Specialty section:
This article was submitted to
Neuroendocrine Science,
a section of the journal
Frontiers in Endocrinology Received: 12 January 2018
Accepted: 25 April 2018
Published: 16 May 2018 GCs act on almost all types of cells and in particular in the immune cells they have been shown
to have powerful immunosuppressive and anti-inflammatory activities (1–5). As a result of their
anti-inflammatory properties, GCs are widely used to help treat many different conditions, such as
allergic, autoimmune, inflammatory, and hematological alterations. Interestingly, an accumulating
body of evidence now strongly suggests that GCs can have both pro- and anti-inflammatory roles
under specific conditions. The pro-inflammatory activity of GCs is most apparent in the central
nervous system (CNS). These opposite effects work together in order to resolve cellular responses to
inflammatory stimuli and also as a protective mechanism “priming” the immune cells to efficiently
respond to the noxa or stressor and then restore homeostasis (6). Keywords: glucocorticoids, inflammation, FKBP51, transactivation, transrepression Edited by:
Ana Rosa Pérez,
CONICET, Argentina
Reviewed by:
Tullio Florio,
Università di Genova, Italy
Luciano David D’Attilio,
CONICET Rosario, Argentina
*Correspondence:
Eduardo Arzt
earzt@iboba-mpsp-conicet.gov.ar
†These authors have contributed
equally to this work. Edited by:
Ana Rosa Pérez,
CONICET, Argentina Reviewed by:
Tullio Florio,
Università di Genova, Italy
Luciano David D’Attilio,
CONICET Rosario, Argentina Keywords: glucocorticoids, inflammation, FKBP51, transactivation, transrepression Review published: 16 May 2018
doi: 10.3389/fendo.2018.00235 Citation: Liberman AC, Budziñski ML, Sokn C,
Gobbini RP, Steininger A and Arzt E
(2018) Regulatory and Mechanistic
Actions of Glucocorticoids on T and
Inflammatory Cells. Front. Endocrinol. 9:235. doi: 10.3389/fendo.2018.00235 May 2018 | Volume 9 | Article 235 Frontiers in Endocrinology | www.frontiersin.org 1 GC-Mediated Immune Regulation Liberman et al. nuclear localization signals, which are important for GR nuclear
translocation. The DBD also contains the nuclear export signal
sequence (NES) which targets it for export from the cell nucleus
to the cytoplasm through the nuclear pore complex. Upon peripheral or cerebral immune stimulation, the HPA axis
is activated. When the immunogenic stress occurs in the brain,
local inflammatory components activate the HPA axis. However,
if the challenge takes place outside the brain, multiple pathways
bring together stimulatory signals from the periphery to the HPA
axis. Mounting evidence suggests that cytokine signals access to
the brain through different pathways. These pathways mainly
include: cytokines passing across the blood–brain barrier; by
specific saturable transport molecules on the brain endothelium;
activation of endothelial cells of brain capillaries that release
second messengers within the brain parenchyma; transmission
of cytokine signals via afferent nerve fibers and finally by periph-
erally activated monocytes that can enter into the brain (7–11). The induction of these different mechanisms modulates cytokine
activity in the brain (12–14).h Some degree in the heterogeneity in GR proteins may result
from alternative splicing (30) (Figure 1B). The specificity and
sensitivity of different target tissues to GCs has been reported
to be related to GR isoforms (30). The GRα is the predominant
isoform, and it is the one that transduces GCs signaling in the
cell (31). There are other four additional splice variants identified:
GRβ, GRγ, GR-A, and GR-P. GRβ differs from GRα in the carboxy
terminal sequence, rendering GRβ non-responsive to GCs (32,
33), with no transcription of target genes. Therefore, GRβ can be
described as a dominant negative inhibitor of GRα activity. GRβ
does not bind GC agonists, however, it does bind to the GR antag-
onist RU-486 (34). GRβ can inhibit GRα transcriptional activity
by different molecular mechanisms including competition for
glucocorticoid response elements (GRE), interference with the
activity of coregulators, and formation of inactive dimers (35, 36). In most tissues, GRβ is expressed at very low levels. Citation: However,
abundant GRβ expression has been described especially in some
inflammatory cells, such as lymphocytes and macrophages, and
have been related to GCs resistance in diseases such as asthma
(37), rheumatoid arthritis (38), ulcerative colitis (39), systemic
lupus erythematosus (40), and acute lymphoblastic leukemia and
chronic lymphocytic leukemia (41, 42). Considering that GRβ
can inhibit GRα activity, the modulation of GRα/GRβ expression
ratios may be an interesting approach to regulate GC sensitiv-
ity (42, 43). In addition, eight alternative translation initiation
sites increase the repertory of GR proteins to almost 40 distinct
isoforms of GR protein (44) (Figure 1B). The accurate regulation of the HPA axis activity is critical,
since GC imbalances can result in many different pathological
conditions (13, 15). Long-term treatment with GCs may result
in a plethora of harmful undesired side effects, such as diabetes,
hypertension, growth retardation, dyslipidemia, osteoporosis,
glaucoma, muscle atrophy, and is also related to many important
behavioral alterations, among others (16, 17). Chronic exposure
to GCs can also be associated with GC insensitivity, reducing the
efficacy of the therapy (18). Also, alterations or deficits in the HPA
axis response are tightly associated with a wide range of autoim-
mune and inflammatory diseases (19–24). l
In this review, we will discuss the role of GCs on the immune
and inflammatory cells in the periphery and also the physiologi-
cal importance and mechanisms implicated in the apparent para-
doxical functions of GCs in the brain in order to appropriately
maintain a coordinated homeostatic response. At the cellular level, GC availability is also modulated by
enzymes of the 11β-hydroxysteroid dehydrogenase (11β-HSD)
family, mainly 11β-HSD1 and 11β-HSD2 which regulate the
conversion of active cortisol into inactive cortisone. 11β-HSD1
favors the conversion of cortisol from cortisone, increasing local
GC activity (45). In contrast, 11β-HSD2 catalyzes cortisol to
cortisone, thereby reducing GC availability. Thus, the balance
in the expression of these two enzymes in a given tissue or cell,
regulates GC-mediated responses. In addition, some studies
show that inflammatory cytokine signaling modulates the relative
expression of 11β-HSD genes, favoring 11β-HSD1 and inhibiting
11β-HSD2 (46, 47), adding another level of regulation of GC
activity.i Frontiers in Endocrinology | www.frontiersin.org THE GLUCOCORTICOID RECEPTOR (GR) As a small lipophilic hormone, GCs can rapidly diffuse into cells
and exert their main actions. These actions are elicited by the
binding of GCs to their intracellular receptor, the GR. The GR is
a hormone-activated transcription factor (TF) that belongs to the
superfamily of nuclear hormone receptors (25). GR is a modular
protein composed of three distinct regions with different func-
tions (Figure 1A). The N-terminal domain (NTD) contains a
transactivation domain called activation function 1 (AF1) that
is responsible for the transcriptional activation and is implicated
in the association with coregulators and the basal transcription
machinery. The DNA-binding domain (DBD) is composed of
two zinc fingers that have been shown to be important for GR
homodimerization and DNA-binding specificity. The hinge
region, which separates the DBD from the ligand binding domain
(LBD), is a flexible linker structure which is implicated in allow-
ing proper DNA binding, dimerization, and nuclear translocation
of the receptor (26). The C-terminal LBD, contains the ligand
binding site and a second transactivation domain (AF2) regulated
by hormone binding (27). The AF2 transactivation domain is
important for the interaction with co-chaperones, coregulators,
and other TFs (28). The LBD also encompasses a dimer interface
which is critical for GR function and the binding of the heat
shock protein (Hsp) 90 (29). The DBD and LBD both contain y
Another important level for fine-tuning the cellular response
to GCs in different environmental situations is the modulation
of GR activity by posttranslational modifications (PTMs). These
PTMs include phosphorylation, acetylation, ubiquitination,
and sumoylation, which may accurately regulate GR activity
in response to diverse external stimuli (48) (Figure 1A). In
particular, SUMO conjugation has been extensively described
to modulate GR transcriptional activity (49–52). GR contains
three consensus sumoylation sites. Two sumoylation sites located
at the NTD have been demonstrated to be part of the synergy
control (SC) motif sequence (50). The SC motifs consist of
short regulatory sequences which are important for inhibiting
the synergistic transactivation. SUMO conjugation to the two May 2018 | Volume 9 | Article 235 Frontiers in Endocrinology | www.frontiersin.org 2 GC-Mediated Immune Regulation Liberman et al. Figure 1 | The glucocorticoid receptor (GR) structure, isoforms, and mechanisms of transcriptional regulation. (A) Full human GRα protein has an N-terminal
domain (NTD), a DNA-binding domain (DBD), a ligand binding domain (LBD) and a hinge region (H) between DBD and LBD. THE GLUCOCORTICOID RECEPTOR (GR) They have different associated
functions, e.g., transactivation, dimerization, nuclear localization, DNA binding, and heat-shock protein 90 binding. The receptor can be post-translationally modified
by phosphorylation (P), ubiquitination (U), acetylation (A) and sumoylation (S). Regions associated with transactivation (activation function 1 and 2: AF1 and AF2) are
shown. (B) The GR has various isoforms which result from alternative splicing and multiple transcriptional start sites at exon 2. The colors indicate NTD (red, exon 2
DBD (green, exons 3–4), H (light blue, exon 5) and LBD (dark blue, exon 5–9). The 5′ and 3′-untranslated regions are colored in gray. There are five patterns of
alternative splicing that result in GR isoforms α, β, P, γ, A. Each of them has eight translational variants (A, B, C1, C2, C3, D1, D2, D3) depending on the
transcriptional start site (“*” denotes an alternative splice donor site in the intron between exons 3 and 4). (C) The GR, carrying GC ligand, translocates to the
nucleus and regulates gene expression. GR can directly activate/inactivate gene expression by interacting with GREs/nGREs, it can bind to GREs and modulate
gene transcription by interacting with neighboring DNA-bound transcription factors (TFs) (composite mechanism) and it can act by attaching itself to DNA-bound
TFs (tethering mechanism). Abbreviations: TF, transcription factor; GRE, glucocorticoid response element; nGRE, negative glucocorticoid response element; TFRE,
transcription factor response element. Liberman et al. GC-Mediated Immune Regulat Figure 1 | The glucocorticoid receptor (GR) structure, isoforms, and mechanisms of transcriptional regulation. (A) Full human GRα protein has an N-terminal
domain (NTD), a DNA-binding domain (DBD), a ligand binding domain (LBD) and a hinge region (H) between DBD and LBD. They have different associated
functions, e.g., transactivation, dimerization, nuclear localization, DNA binding, and heat-shock protein 90 binding. The receptor can be post-translationally modified
by phosphorylation (P), ubiquitination (U), acetylation (A) and sumoylation (S). Regions associated with transactivation (activation function 1 and 2: AF1 and AF2) are
shown. (B) The GR has various isoforms which result from alternative splicing and multiple transcriptional start sites at exon 2. The colors indicate NTD (red, exon 2),
DBD (green, exons 3–4), H (light blue, exon 5) and LBD (dark blue, exon 5–9). The 5′ and 3′-untranslated regions are colored in gray. There are five patterns of
alternative splicing that result in GR isoforms α, β, P, γ, A. GCs ANTI-INFLAMMATORY ACTIONS The GR forms complex with chaperone molecules, such as Hsp90
and 70, and immunophilins, such as FKBP51, FKBP52, Cyp44,
and PP5 (63). FKBP51 binds to the unbound GR and reduces
GR activity mainly by reducing GR hormone binding and its
nuclear translocation. Therefore, FKBP51 is considered as an
inhibitor of GR transcriptional activity. Upon ligand binding,
the GR exchanges FKBP51 for FKBP52, which is able to interact
with the dynein motor protein, facilitating GR translocation to
the nucleus (64). Interestingly, FKBP51 overexpression has been
associated with GC resistance in autoimmune diseases. FKBP51
expression was found to be enhanced in sputum samples from
patients with chronic obstructive pulmonary disease (65). Moreover, in a genome-wide profiling focused on the iden-
tification of epithelial gene markers of asthmatic patients and
response to corticosteroids, GC treatment was found to induce
FKBP51 expression, which in turn was associated with a poor
response to corticosteroids, suggesting a role of FKBP51 in GC
resistance (66, 67). Also, enhanced expression of FKBP51 has
been found in bone marrow cells in patients with rheumatoid
arthritis (68). Evidence also suggests that FKBP51 modulates
NFĸB-dependent gene expression, with possible implications
for various inflammatory and immune pathways (69–73). Considering that GR is a key modulator of immune and inflam-
matory responses, FKBP51 dysregulation may provide the basis
for a role of FKBP51 in these processes (66). Moreover, FKBP51
has recently been shown to be a target of SUMO conjugation and
that sumoylation of FKBP51 is necessary for its association to
Hsp90 and modulates FKBP51-mediated inhibition of GR activ-
ity in neuronal cells (58). In the brain, FKBP51 has been shown
to be important for the development of psychiatric diseases
and the response to antidepressant treatment, suggesting that
regulation of FKBP51 activity might be an interesting approach
for modulating GR outcome in the stress response and also in
the inflammatory context (74–76). l
It has been shown that several of the undesirable metabolic side
effects associated with chronic GC treatment are mediated via
transactivation. However the anti-inflammatory effects of GCs
are mainly mediated via the transrepression elicited by a mono-
meric GR with the activity of TFs, such as NFĸB and AP1 (1–3,
85). These TFs are involved in the activation of pro-inflammatory
and immunoregulatory genes, such as inflammatory cytokines,
cytokine receptors, adhesion molecules, and chemotactic pro-
teins that play a key role for the coordination of the inflamma-
tory response (1, 86–88). THE GLUCOCORTICOID RECEPTOR (GR) A genome-wide analysis of GR sumoylation
impact on gene expression, showed that genes differentially
regulated by this PTM are mostly related to proliferation and
apoptosis pathways and also strongly suggests that sumoylation
can regulate genome-wide chromatin occupancy of the GR (56). Also, GR SUMO conjugation is influenced by other PTMs such as
phosphorylation in order to fine-tune GR transcriptional activity
in a target gene-specific manner (57). Important coregulators of
the GR are also modified by SUMO conjugation, such as Hsp90,
GRIP1, and also FKBP51, further regulating GR activity (58–62). Therefore, PTMs that impact on the GR but also on key molecules
that fine-tune its activity, helps to understand the complexity of
GR-mediated regulation of its target gene expression (2, 48). Once in the nucleus, the activated GR can regulate gene
expression by different mechanisms known as genomic effects
(Figure 1C) (27). The genomic mechanism involves changes
in the levels of specific genes: binding of GR to GREs in the
promoters of its target genes and activation of transcription
(transactivation); DNA binding of the GR with other TFs to
“composite” elements which contain a GRE and an overlapping
response element of another TF (binding can lead to gene activa-
tion or repression); or binding of the GR to a TF (e.g., NFĸB; or
AP1) by means of a “tethering” mechanism without contacting
DNA, to influence the activity of the TF (this mechanism is
considered to be the prevailing mechanism for transrepression)
(2, 77, 78). Furthermore, GR-mediated transcriptional repression
can be exerted via GR binding to a negative GRE (nGRE) (79). Binding to these nGRE prevent receptor dimerization through
a strong negative cooperativity and alters the conformation of
GR residues that are critical for transcriptional activation so that
negative regulation is accomplished (80). A growing body of
evidence shows that GC can also mediate non-genomic actions
that do not require protein synthesis and are implicated in rapid
cellular responses. For example, in the cytoplasm the activated
GR can acutely interact with signaling pathways, such as PI3K,
JNK, 14-3-3 proteins, and components of the T cell receptor
signaling complex (81), modulating pro-inflammatory gene
expression. In thymocytes, the activated GR can translocate to
mitochondria and induce a rapid apoptotic response (82). In
addition, membrane-bound GR on monocytes was reported to
mediate non-genomic effects (82). THE GLUCOCORTICOID RECEPTOR (GR) On the other hand, binding of
GCs to GR can modify the recruitment of different factors such
as the multiprotein chaperone complex that participate in many
signaling pathways, modifying secondary signaling cascades and,
therefore, may further regulate the immune response (78, 83). GCs may also exert anti-inflammatory responses by direct nega-
tive interaction with components of the MAPK pathway, such as
ERK, c-Jun NH2-terminal kinases (JNK), and p38 isoforms
(p38) regulating their activity (84). Further studies are required
to clarify the implications of non-genomic GC-mediated activity
in the immune and inflammatory context. THE GLUCOCORTICOID RECEPTOR (GR) Each of them has eight translational variants (A, B, C1, C2, C3, D1, D2, D3) depending on the
transcriptional start site (“*” denotes an alternative splice donor site in the intron between exons 3 and 4). (C) The GR, carrying GC ligand, translocates to the
nucleus and regulates gene expression. GR can directly activate/inactivate gene expression by interacting with GREs/nGREs, it can bind to GREs and modulate
gene transcription by interacting with neighboring DNA-bound transcription factors (TFs) (composite mechanism) and it can act by attaching itself to DNA-bound
TFs (tethering mechanism). Abbreviations: TF, transcription factor; GRE, glucocorticoid response element; nGRE, negative glucocorticoid response element; TFRE,
transcription factor response element. May 2018 | Volume 9 | Article 235 Frontiers in Endocrinology | www.frontiersin.org Liberman et al. GC-Mediated Immune Regulation NTD sumoylation sites is responsible for the functional effect
of the SC motifs and thereby they inhibit GR activity (50, 53)
(Figure 1A). It has also been demonstrated that in the presence
of the sumoylation enhancer, RSUME (54), a SUMO peptide is
conjugated to the third sumoylation site located in the LBD of
the GR. Sumoylation in the LBD may be important for inducing
GR-mediated transcriptional regulation during stress adaptation
(55) (Figure 1A). A genome-wide analysis of GR sumoylation
impact on gene expression, showed that genes differentially
regulated by this PTM are mostly related to proliferation and
apoptosis pathways and also strongly suggests that sumoylation
can regulate genome-wide chromatin occupancy of the GR (56). Also, GR SUMO conjugation is influenced by other PTMs such as
phosphorylation in order to fine-tune GR transcriptional activity
in a target gene-specific manner (57). Important coregulators of
the GR are also modified by SUMO conjugation, such as Hsp90,
GRIP1, and also FKBP51, further regulating GR activity (58–62). Therefore, PTMs that impact on the GR but also on key molecules
that fine-tune its activity, helps to understand the complexity of
GR-mediated regulation of its target gene expression (2, 48). NTD sumoylation sites is responsible for the functional effect
of the SC motifs and thereby they inhibit GR activity (50, 53)
(Figure 1A). It has also been demonstrated that in the presence
of the sumoylation enhancer, RSUME (54), a SUMO peptide is
conjugated to the third sumoylation site located in the LBD of
the GR. Sumoylation in the LBD may be important for inducing
GR-mediated transcriptional regulation during stress adaptation
(55) (Figure 1A). Frontiers in Endocrinology | www.frontiersin.org GCs ANTI-INFLAMMATORY ACTIONS By all these different
mechanisms, GCs regulate important functions, not only in the
periphery but also in the brain.t GCs can regulate the maturation, survival, and migration
toward the lymph nodes and motility of dendritic cells (DCs),
and also inhibit their immunogenic functions (Figure 2). GCs
were shown to reduce the ability of DCs to stimulate T cells by
inhibiting the upregulation of co-stimulatory molecules and
cytokines, such as IL6, IL12, and TNFα and by inducing the
tolerance-inducing transcription factor GILZ (119–125). The
distinct actions exerted by GCs in immature and mature DCs are
due to differential expression of GR translational isoforms (126). Synthetic analogs of GC are often employed in the clinic in
the therapy of allergic, inflammatory, and autoimmune disorders
(97–99). It is generally accepted that GR-mediated transrepres-
sion holds the beneficial anti-inflammatory action, whereas their
side effects are due mainly to the direct binding of GR to GREs
as depicted before (98–100). However, transactivation is also
necessary for the induction of several anti-inflammatory genes,
such as MAP kinase phosphatase 1 (101), glucocorticoid-induced
leucine zipper (102), and inhibitor kappa B-alpha (IĸBα) (85). Therefore, the ideal GC analogs should be those that have high
repressive activity against inflammatory mediators, but low
transactivation activity, causing minimal side effects. Several
steroidal and nonsteroidal ligands have been reported to have this
dissociated function between transactivation and transrepressive
mechanisms (97–99, 103). These compounds were shown to
repress the activity of key inflammatory and immune TFs in vivo
(104–107). However, GCs can induce gene expression not only
by binding to GRE, but also in combination with other TFs and
also by binding to promoter regions in a mechanism that does
not involve GR dimerization or DNA interaction; therefore,
unexpected secondary side effects might appear (78).lf GCs are important modulators of neutrophilia (Figure 2). Leukocyte extravasation is the movement of leukocytes out of
the circulation and toward the site of tissue damage or infection. Rolling, adhesion, activation, and transmigration are necessary
to arrive to the damaged tissue. GCs can modulate each of these
steps. Rolling and adhesion is mediated by the interaction of the
leukocyte integrins with the endothelial counterparts, which are
inhibited by GCs (127–129). Also, GCs increase the number of
circulating neutrophils in the blood stream by favoring their
egress from the bone marrow and also inhibiting their migration
to inflammatory sites by hindering the expression of adhesion
molecules (32, 129, 130). GCs ANTI-INFLAMMATORY ACTIONS The first example of the transrepressive
mechanism was the inhibitory interaction described between GR
and AP1 (89), which results in the inhibition of IL2 expression
(90). NFĸB is present in almost all immune cells and regulates
the expression of inflammatory cytokines. Thus, inhibition of
NFĸB activity is an important feature for GR-mediated anti-
inflammatory activity (85, 91). It also inhibits NFAT-dependent
IL2 transcription (92). The main mechanism of the GR action
over these TFs is via transrepression: the activated GR acts by
binding proximal to the NFĸB or AP1-binding site and interacts Frontiers in Endocrinology | www.frontiersin.org May 2018 | Volume 9 | Article 235 4 GC-Mediated Immune Regulation Liberman et al. with these TFs inhibiting gene expression (93). The transrepres-
sion mechanism is not restricted to these TFs, but has expanded
including among others, CREB, STAT, and T-bet (1–3, 94). with these TFs inhibiting gene expression (93). The transrepres-
sion mechanism is not restricted to these TFs, but has expanded
including among others, CREB, STAT, and T-bet (1–3, 94). anti-inflammatory macrophages (111). The intrinsic molecular
mechanism involves a prolonged induction of the extracellular
signal-regulated kinase/MAPK (ERK/MAPK) pathway resulting
in inhibition of caspase activities and expression of anti-apoptotic
genes (111). GCs can also improve the phagocytic activity of these
cells and stimulate the clearance of harmful elements, such as
neutrophil clearance (112–114). GCs also suppress immunostim-
ulatory functions of these cells and inhibit the release of various
pro-inflammatory mediators, such as cytokines, chemokines,
and reactive oxygen through different mechanisms (115, 116). Functional clustering of GC-regulated genes by human anti-
inflammatory macrophages by microarray technology indicated
induction of phagocytosis and motility as well as repression of
adhesion, apoptosis, and oxidative burst (117, 118). Alterations in chromatin structure have been reported to be
important for regulating GC actions. The GR can differentially
interact with proteins that have histone acetyltransferase (HAT)
activity, but also with histone deacetylases and kinases that can
influence the chromatin environment modifying chromatin
accessibility and further regulating immune and inflammatory
gene expression (3). In addition, chromatin accessibility has been
reported to pre-determine GR binding patterns and, therefore,
is critical for cell-specific outcome, providing new molecular
basis for the tissue selectivity (95, 96). GCs ANTI-INFLAMMATORY ACTIONS GCs exert distinct immunomodulatory actions on T cells
(Figure 2). GCs decrease the number of circulating T cells by
favoring their migration back to the bone marrow and second-
ary lymphoid tissues or through the induction of chemokine
receptors, adhesion molecules, and matrix metalloproteinases
(131, 132). The steroid hormone also favors T cells apoptosis. GC-induced apoptosis of T cells requires the dimerization of the
GR (133) and is mediated via the induction of Puma and Bim
expression (134–137). The relative expression of distinct GR
isoforms increases the susceptibility of T cells to GC-induced cell
death (138). Helper T (Th) cells are important players of the adap-
tive immunity (1). Upon antigen stimulation, naive Th cells can
differentiate into different subsets: Th1, Th2, Th17, or regulatory
T (Tregs) cells among others, each with specific effector functions. Th1 cells express the lineage-specific TF T-bet and STAT4 and
release pro-inflammatory cytokines, such as IFNγ and IL2 (139). Th1 cells help in the activation of effector T cells, natural killer
(NK) cells, and macrophages at the site of infection, promote
effective immune responses against intracellular pathogens and
are also implicated in autoimmune pathologies. Th2 lymphocytes
selectively express the TF GATA3 and are characterized by the
expression of IL5, IL4, IL10, and IL13 and are important for the GCs may exert acute anti-inflammatory effects through the
release of annexin-A1 (ANXA1) (108). Originally, this protein
was suggested to have anti-inflammatory actions because it was
described to inhibit phospholipase A2 (109). However, ANXA1
has been reported to regulate different cellular processes, such as
migration, growth, differentiation, apoptosis, membrane fusion
during exocytosis, lipid metabolism, and cytokine expression. Importantly, in the HPA axis, ANXA1 has been reported to play
a critical role in the negative feedback exerted by GCs, therefore,
affecting hypothalamic-releasing hormones secretion possibly
via non-genomic mechanisms (110). GCs ACTIVITY ON PERIPHERAL IMMUNE
CELLS They also suppress GATA3 activity in Th2 cells inhibiting the expression of IL-4 and IL-5. The action of GCs toward Th17 and
regulatory T cells is not yet well understood. Figure 2 | Glucocorticoid (GC) activity on periphery immune cells. GCs act upon almost every immune cell type. GCs promote an anti-inflammatory state on both
monocytes and macrophages. GCs prevent monocytes into entering apoptosis and inhibit the liberation of pro-inflammatory mediators by both types of cells. Particularly in macrophages, GCs promote phagocytosis and motility, while they inhibit adhesion, apoptosis and oxidative burst. They also act upon neutrophils
function by inhibiting rolling, adhesion and activation. GCs act toward dendritic cells by promoting their maturation, survival, migration and motility, and at the same
time GCs inhibit their ability to activate T cells by suppressing the production of pro-inflammatory molecules. A naïve helper T (Th) cell can differentiate into different
Th lineages and GCs exert different actions. They act upon Th1 by decreasing T-bet transcriptional activity and suppressing the production of pro-inflammatory
molecules such as IL-2 and IFNγ. They also suppress GATA3 activity in Th2 cells inhibiting the expression of IL-4 and IL-5. The action of GCs toward Th17 and
regulatory T cells is not yet well understood. (94) (Figure 2). Also GCs where shown to reduce mRNA and
protein levels of T-bet (94). The activity of the Th2-specific TF
GATA3 is also suppressed by GCs via two main mechanisms:
first by GR-mediated inhibition of GATA3 translocation into
the nucleus and second by the inhibition of GATA3 phospho-
rylation by GC-induced MKP1 expression (147, 148) (Figure 2). Furthermore, STAT6 activity also involved in Th2 differentiation
is inhibited by GCs (149). How GCs modulate Th17-mediated
responses has not been extensively studied, and the importance
of Th17 modulation by GCs for the suppression of allergic or
autoimmune diseases remains unclear (150). In rheumatoid
arthritis, GC treatment diminished IL17 levels (151). In addition,
in rat lymphocytes methylprednisolone inhibited IL17 expres-
sion due to the inhibition of RORγt expression (152) (Figure 2). However, several studies strongly suggest that GC resistance is
associated with a pathogenic inflammatory Th17 phenotype that
is refractory to GCs (150, 153, 154). Recently, a gene-expression
profiling to characterize the steroid-resistant phenotype showed proper eradication of extracellular pathogens (140). Also, Th2
cells activate B cells to produce antibodies and play a triggering
role in the activation/recruitment of eosinophils and mast cells in
allergic responses. GCs ACTIVITY ON PERIPHERAL IMMUNE
CELLS GCs mediate immunosuppressive functions by acting on almost
all types of immune cells (Figure 2). GCs can regulate the phe-
notype, survival, and functions of monocytes and macrophages
which have crucial roles in tissue homeostasis and innate immu-
nity. GCs exhibit anti-apoptotic effects promoting the survival of May 2018 | Volume 9 | Article 235 Frontiers in Endocrinology | www.frontiersin.org 5 GC-Mediated Immune Regulation Liberman et al. Figure 2 | Glucocorticoid (GC) activity on periphery immune cells. GCs act upon almost every immune cell type. GCs promote an anti-inflammatory state on both
monocytes and macrophages. GCs prevent monocytes into entering apoptosis and inhibit the liberation of pro-inflammatory mediators by both types of cells. Particularly in macrophages, GCs promote phagocytosis and motility, while they inhibit adhesion, apoptosis and oxidative burst. They also act upon neutrophils
function by inhibiting rolling, adhesion and activation. GCs act toward dendritic cells by promoting their maturation, survival, migration and motility, and at the same
time GCs inhibit their ability to activate T cells by suppressing the production of pro-inflammatory molecules. A naïve helper T (Th) cell can differentiate into different
Th lineages and GCs exert different actions. They act upon Th1 by decreasing T-bet transcriptional activity and suppressing the production of pro-inflammatory
molecules such as IL-2 and IFNγ. They also suppress GATA3 activity in Th2 cells inhibiting the expression of IL-4 and IL-5. The action of GCs toward Th17 and
regulatory T cells is not yet well understood. Figure 2 | Glucocorticoid (GC) activity on periphery immune cells. GCs act upon almost every immune cell type. GCs promote an anti-inflammatory state on both
monocytes and macrophages. GCs prevent monocytes into entering apoptosis and inhibit the liberation of pro-inflammatory mediators by both types of cells. Particularly in macrophages, GCs promote phagocytosis and motility, while they inhibit adhesion, apoptosis and oxidative burst. They also act upon neutrophils
function by inhibiting rolling, adhesion and activation. GCs act toward dendritic cells by promoting their maturation, survival, migration and motility, and at the same
time GCs inhibit their ability to activate T cells by suppressing the production of pro-inflammatory molecules. A naïve helper T (Th) cell can differentiate into different
Th lineages and GCs exert different actions. They act upon Th1 by decreasing T-bet transcriptional activity and suppressing the production of pro-inflammatory
molecules such as IL-2 and IFNγ. Frontiers in Endocrinology | www.frontiersin.org GCs ACTIVITY ON PERIPHERAL IMMUNE
CELLS IL17-producing Th17 cells selectively express
RORγt and also RORα (141, 142). Th17 cells play an important
role in autoimmune diseases and in host defense against infec-
tion. Treg cells mainly express the TF Foxp3 and inhibit effector
T-cell differentiation and proliferation and suppress autoimmune
and allergic responses (143). GCs inhibit the expression of many
T cell cytokines (1) and can produce a shift from Th1-mediated
cellular immunity to mediating humoral Th2 responses at physi-
ological doses or chronic treatment (144). Upon acute treatment
with GCs, they inhibit the synthesis of Th1 cytokines like IL2
and IFNγ and reduce STAT4 activity (145) and also reduce
Th2 cytokines expression (146). The molecular mechanism by
which GCs inhibit Th1 responses involves the reduction of T-bet
transcriptional activity by the inhibitory interaction between GR
and T-bet that results in diminished binding of T-bet to DNA May 2018 | Volume 9 | Article 235 Frontiers in Endocrinology | www.frontiersin.org 6 GC-Mediated Immune Regulation Liberman et al. by intraparenchymal endotoxin injection (174). Mice treated with
the GR antagonist RU486 before intracerebral LPS administration
showed an increase in the pro-inflammatory response, which in
turn induced neuronal death. These findings suggest that GCs are
important for protecting the brain during innate immunity (175,
176). Interestingly, when mice lacking GR in microglia were chal-
lenged with an intracerebral administration of LPS, the activation
of the toll-like receptor 4 signaling pathway induced cellular
lesion, and also neuronal and axonal damage (177). In addition,
microglial cell cultures have reduced motility and increased
amoeboid morphology in the absence of GR expression. This
study strongly suggests that microglial GR is the principal media-
tor preventing neuronal degeneration triggered by LPS and that it
also contributes to the protection of other cell types (177), having
an important role in promoting neuronal survival.hl that Th17 cells have restricted genome-wide responses to GCs
and that they are refractory to GC inhibition at this level. In addi-
tion, Th17 cells were sensitive to suppression with the calcineurin
inhibitor, cyclosporine A, suggesting that the clinical efficacy of
cyclosporine A in the treatment of steroid resistance may be due
to its selective inhibition of Th17 cells (155). Another interesting
study has shown that Th17 cells are insensitive to GC-induced
apoptosis and had high levels of BCL-2, knockdown of which sen-
sitized Th17 cells to GC-induced cell death (156). GCs ACTIVITY ON PERIPHERAL IMMUNE
CELLS Also, lung Th17
development in the murine severe asthma model was enhanced
by GCs, supporting a role of Th17 cells in GC-refractory inflam-
matory conditions such as asthma (157).fl In contrast to the inhibitory effect of GCs on pro-inflamma-
tory effector T cells, it has been shown that Treg cells, which
are key suppressors of T cell-dependent immune responses, are
enhanced upon dexamethasone treatment by being more resist-
ant to GC-induced cell death (158) (Figure 2). Also, GCs where
shown to amplify IL2-dependent expansion of Treg cells and
to enhance their capacity to reduce experimental autoimmune
encephalomyelitis (EAE) in mice (159). In addition, GCs increase
the percentage of Treg cells that express Foxp3 in patients with
multiple sclerosis (160). In vivo, T cell-specific targeted GR dele-
tion in pregnant animals undergoing EAE, resulted in a reduction
of Treg population and a loss of pregnancy-induced protection,
suggesting that steroid hormones can shift the immunological
balance in favor of Tregs via differential engagement of the GR
in T cells (161). However, others have found that GC treatment
suppresses the expression of Foxp3 Tregs in an EAE model (162)
and also in lungs of allergic mice (163).l The majority of GC pro-inflammatory activity has been
described in animal models of acute or chronic stress which
occurred previous to peripheral or cerebral immune challenges. For instance, acute stressors were reported to induce the expres-
sion of pro-inflammatory cytokines in specific brain regions,
such as the hippocampus, following LPS peripheral challenge
(178–180). GCs were also found to upregulate microglial activa-
tion markers including the toll-like receptor 2 pro-inflammatory
pathway (178, 181) (Figure 3A). It was also shown that chronic
unpredictable stress was able to potentiate LPS-mediated activa-
tion of NFĸB activity in the frontal cortex and hippocampus via
GC production (182). Also, chronically stressed animals that were Figure 3 | Glucocorticoids (GCs) actions in the brain. Acute stressors
promote an inflammatory phenotype in the brain. (A) In the microglia, GCs
bind to the glucocorticoid receptor (GR) which then promotes the translation
of the toll-like receptor 2 (TLR2) by interacting with STAT5 and NFƙB
response elements. TLR2 then exerts a pro-inflammatory response by
promoting the production of inflammatory cytokines. (B) In macrophages,
GCs promote the expression of the purinergic receptor P2Y2R which then
produces IL-6 in response to ATP. Moreover, GCs enhance the expression of
NLRP3 which in turn promotes the production of pro-inflammatory
cytokines. GCs ACTIVITY ON PERIPHERAL IMMUNE
CELLS In addition to their well-studied anti-inflammatory and
immunosuppressive activity, an increasing body of evidence has
revealed situations in which GCs have the opposite effect. This has
been shown to depend on the dose, timing, duration of exposure,
and cell population or tissue analyzed (164). The paradoxical pro-
inflammatory role of GCs is mostly evident in the brain, where
accumulating evidence show that GCs elicit different immune
responses depending on the affected brain regions. Frontiers in Endocrinology | www.frontiersin.org CONCLUSION GCs are widely used in the clinic to control not only peripheral,
but also CNS inflammatory response. However, the prolonged
administration of this steroid hormone is often ineffective and
can even worsen the outcome of the disease. Considering the
known undesirable metabolic side effect, the induction of pro-
inflammatory responses and the existence of GC resistance, GCs
should be used carefully. Future research should be focused not
only in understanding the molecular basis of GCs side effects
and resistance, but also in dissecting how GCs induce pro-
inflammatory responses in order to avoid serious detrimental
consequences, particularly in the brain. In the future, a combi-
nation of different therapeutic approaches may lead to a more
effective treatment and may help to lower the doses or duration
of GC treatment thus minimizing the risk of toxicity and drug
resistance (196). Finally, taking into account inter-individual
differences in patient responsiveness to GC treatment, where
different molecular mechanisms might be implicated, future
directions should be in support of a customized and personal-
ized treatment to meet individual patient needs. 3. Ratman D, Vanden Berghe W, Dejager L, Libert C, Tavernier J, Beck IM, et al.
How glucocorticoid receptors modulate the activity of other transcription
factors: a scope beyond tethering. Mol Cell Endocrinol (2013) 380:41–54.
doi:10.1016/j.mce.2012.12.014 FUNDING This work was supported by grants from the Max Planck Society,
Germany; the University of Buenos Aires; CONICET; the Agencia
Nacional de Promoción Científica y Tecnológica, Argentina; and
FOCEM-Mercosur (COF 03/11). diseases. Microbiol Mol Biol Rev (2016) 80:495–522. doi:10.1128/MMBR.
00064-15 GCs ACTIONS IN THE BRAIN There is a significant body of evidence indicating that GCs can
suppress the innate immunity in the brain after a peripheral or
cerebral challenge (23). In this way, in adrenalectomized mice,
there is an induction in the levels of pro-inflammatory cytokines
in the brain following LPS injection (165–168). Studies also dem-
onstrated that GCs inhibit the release of pro-inflammatory media-
tors in microglial cells treated with LPS (169, 170). Experiments
performed in vivo support these findings by revealing that dexa-
methasone causes a strong reduction in LPS induction of NFĸB
expression in the brain (171). In addition, COX inhibitors were
demonstrated to increase the expression of pro-inflammatory
genes in the brain during systemic inflammation by reducing
the activation of the HPA axis and the release of GCs (172, 173). This same effect took place when the GR antagonist RU486 was
administrated (172, 173). Also, systemic inflammation, through
the increase in circulating GCs, has been reported to have the
ability to prevent the cerebral innate immune response induced Figure 3 | Glucocorticoids (GCs) actions in the brain. Acute stressors
promote an inflammatory phenotype in the brain. (A) In the microglia, GCs
bind to the glucocorticoid receptor (GR) which then promotes the translation
of the toll-like receptor 2 (TLR2) by interacting with STAT5 and NFƙB
response elements. TLR2 then exerts a pro-inflammatory response by
promoting the production of inflammatory cytokines. (B) In macrophages,
GCs promote the expression of the purinergic receptor P2Y2R which then
produces IL-6 in response to ATP. Moreover, GCs enhance the expression of
NLRP3 which in turn promotes the production of pro-inflammatory
cytokines. May 2018 | Volume 9 | Article 235 Frontiers in Endocrinology | www.frontiersin.org 7 GC-Mediated Immune Regulation Liberman et al. injected with LPS in the prefrontal cortex or the hippocampus,
exhibited microglia activation, an increase in pro-inflammatory
mediators and loss of astroglia and neurons. These effects were
reduced with RU486 administration (183, 184). The prefrontal
cortex is important in many brain functions and is a target for
neurodegenerative diseases. It has been reported that in this
brain region, TNFα expression and activation of MAPK signal-
ing pathway is upregulated by chronic stress after intracortical
LPS injection in a GR-dependent manner suggesting a syner-
gistic effect between inflammation and stress. This fact could
ultimately explain the relationship described between stress
and some neurodegenerative pathologies (183, 184). GCs ACTIONS IN THE BRAIN For example, GR activation during chronic
stress increases LPS-induced NFκB activation and TNFα, IL1β,
and iNOS expression in the hippocampus and frontal cortex, AUTHOR CONTRIBUTIONS AL: wrote, discussed, and corrected the manuscript. MB:
discussed and corrected the manuscript. CS: discussed and cor-
rected the manuscript, performed the figures. RG: discussed and
corrected the manuscript, performed the figures. AS: corrected
the manuscript. EA: discussed and corrected the manuscript. 4. Refojo D, Liberman AC, Holsboer F, Arzt E. Transcription factor-mediated
molecular mechanisms involved in the functional cross-talk between GCs ACTIONS IN THE BRAIN In order to
investigate if stress-induced GCs is responsible for the response
of brain immune cells to pro-inflammatory stimuli, animals
were acutely stressed and 24 h later hippocampal microglia were
challenged with LPS ex vivo. Treatment in vivo with RU486 and
adrenalectomized inhibited the microglial pro-inflammatory
response, indicating that stress-induced GCs are able to sensitize
the microglial pro-inflammatory function (185, 186). Therefore,
stress may act “priming” central innate immunity to a subse-
quent immune challenge by making the neuroimmune context
more responsive to inflammation, also favoring GC insensitivity
or reducing the HPA response (187). In addition, acute restraint
stress, inescapable tail shock and other stressors induce many
inflammatory mediators, reduce immunoregulatory proteins
and trigger microglia activation and proliferation (188–193). In addition, GCs have been reported to increase the expression
of the purinergic receptor P2Y2R (Figure 3B) which promotes
the secretion of inflammatory mediators in response to ATP
(194). Recent data also indicate that GCs induce the expression
of NLRP3 (NLRP3: nucleotide-binding domain, leucine-rich-
containing family, pyrin domain-containing 3) in macrophages,
which is a critical component of the inflammasome (Figure 3B). The GC-dependent induction of NLRP3 sensitizes the cells to
extracellular ATP and significantly enhances the ATP-mediated
release of pro-inflammatory molecules. This effect was specific
for GCs and dependent on the GR and suggests that GCs sen-
sitize the initial inflammatory response in the context of acute
cellular damage or death (32). In addition, GCs and TNFα were
shown to coregulate immune gene expression when combined
(195). These results suggest that the final outcome of GCs pro- or
anti-inflammatory activity depends on the activation state and
signaling context. GCs are also able to modulate the inflamma-
tory response to LPS in different ways according to the brain
region (180, 182). For example, GR activation during chronic
stress increases LPS-induced NFκB activation and TNFα, IL1β,
d iNOS
i
i
h hi
d f
l but exhibits contrary effects in the hypothalamus (182). It is
important to keep in mind that a pro-inflammatory context
does not necessarily mean that damage will take place. Timing
is a key parameter that will determine the final outcome of the
inflammatory response. While exaggerated inflammation can
favor neuronal dysfunction and cell death, pro-inflammatory
mediators may at first induce the removal of the pathogen, the
recruitment of immune cells and initiate tissue remodeling in
order to appropriately cope with the pathogen and therefore,
restoring homeostasis. GCs ACTIONS IN THE BRAIN injected with LPS in the prefrontal cortex or the hippocampus,
exhibited microglia activation, an increase in pro-inflammatory
mediators and loss of astroglia and neurons. These effects were
reduced with RU486 administration (183, 184). The prefrontal
cortex is important in many brain functions and is a target for
neurodegenerative diseases. It has been reported that in this
brain region, TNFα expression and activation of MAPK signal-
ing pathway is upregulated by chronic stress after intracortical
LPS injection in a GR-dependent manner suggesting a syner-
gistic effect between inflammation and stress. This fact could
ultimately explain the relationship described between stress
and some neurodegenerative pathologies (183, 184). In order to
investigate if stress-induced GCs is responsible for the response
of brain immune cells to pro-inflammatory stimuli, animals
were acutely stressed and 24 h later hippocampal microglia were
challenged with LPS ex vivo. Treatment in vivo with RU486 and
adrenalectomized inhibited the microglial pro-inflammatory
response, indicating that stress-induced GCs are able to sensitize
the microglial pro-inflammatory function (185, 186). Therefore,
stress may act “priming” central innate immunity to a subse-
quent immune challenge by making the neuroimmune context
more responsive to inflammation, also favoring GC insensitivity
or reducing the HPA response (187). In addition, acute restraint
stress, inescapable tail shock and other stressors induce many
inflammatory mediators, reduce immunoregulatory proteins
and trigger microglia activation and proliferation (188–193). In addition, GCs have been reported to increase the expression
of the purinergic receptor P2Y2R (Figure 3B) which promotes
the secretion of inflammatory mediators in response to ATP
(194). Recent data also indicate that GCs induce the expression
of NLRP3 (NLRP3: nucleotide-binding domain, leucine-rich-
containing family, pyrin domain-containing 3) in macrophages,
which is a critical component of the inflammasome (Figure 3B). The GC-dependent induction of NLRP3 sensitizes the cells to
extracellular ATP and significantly enhances the ATP-mediated
release of pro-inflammatory molecules. This effect was specific
for GCs and dependent on the GR and suggests that GCs sen-
sitize the initial inflammatory response in the context of acute
cellular damage or death (32). In addition, GCs and TNFα were
shown to coregulate immune gene expression when combined
(195). These results suggest that the final outcome of GCs pro- or
anti-inflammatory activity depends on the activation state and
signaling context. GCs are also able to modulate the inflamma-
tory response to LPS in different ways according to the brain
region (180, 182). Frontiers in Endocrinology | www.frontiersin.org diseases. Microbiol Mol Biol Rev (2016) 80:495–522. doi:10.1128/MMBR.
00064-15
3. Ratman D, Vanden Berghe W, Dejager L, Libert C, Tavernier J, Beck IM, et al.
How glucocorticoid receptors modulate the activity of other transcription
factors: a scope beyond tethering. Mol Cell Endocrinol (2013) 380:41–54.
doi:10.1016/j.mce.2012.12.014
4. Refojo D, Liberman AC, Holsboer F, Arzt E. Transcription factor-mediated
molecular mechanisms involved in the functional cross-talk between 1. Liberman AC, Druker J, Perone MJ, Arzt E. Glucocorticoids in the regulation
of transcription factors that control cytokine synthesis. Cytokine Growth
Factor Rev (2007) 18:45–56. doi:10.1016/j.cytogfr.2007.01.005 diseases. Microbiol Mol Biol Rev (2016) 80:495–522. doi:10.1128/MMBR.
00064-15 REFERENCES Sousa AR, Lane SJ, Cidlowski JA, Staynov DZ, Lee TH. Glucocorticoid resis-
tance in asthma is associated with elevated in vivo expression of the gluco-
corticoid receptor beta-isoform. J Allergy Clin Immunol (2000) 105:943–50. doi:10.1067/mai.2000.106486 17. Kleiman A, Tuckermann JP. Glucocorticoid receptor action in beneficial and
side effects of steroid therapy: lessons from conditional knockout mice. Mol
Cell Endocrinol (2007) 275:98–108. doi:10.1016/j.mce.2007.05.009 l 38. Chikanza IC. Mechanisms of corticosteroid resistance in rheumatoid arthri-
tis: a putative role for the corticosteroid receptor beta isoform. Ann N Y Acad
Sci (2002) 966:39–48. doi:10.1111/j.1749-6632.2002.tb04200.x 18. Barnes PJ, Adcock IM. Glucocorticoid resistance in inflammatory diseases. Lancet (2009) 373:1905–17. doi:10.1016/S0140-6736(09)60326-3 19. Bertini R, Bianchi M, Ghezzi P. Adrenalectomy sensitizes mice to the
lethal effects of interleukin 1 and tumor necrosis factor. J Exp Med (1988)
167:1708–12. doi:10.1084/jem.167.5.1708 39. Honda M, Orii F, Ayabe T, Imai S, Ashida T, Obara T, et al. Expression of glu-
cocorticoid receptor beta in lymphocytes of patients with glucocorticoid-re-
sistant ulcerative colitis. Gastroenterology (2000) 118:859–66. doi:10.1016/
S0016-5085(00)70172-7 20. Edwards CK III, Yunger LM, Lorence RM, Dantzer R, Kelley KW. The
pituitary gland is required for protection against lethal effects of Salmonella
typhimurium. Proc Natl Acad Sci U S A (1991) 88:2274–7. doi:10.1073/
pnas.88.6.2274 40. Piotrowski P, Burzynski M, Lianeri M, Mostowska M, Wudarski M,
Chwalinska-Sadowska H, et al. Glucocorticoid receptor beta splice variant
expression in patients with high and low activity of systemic lupus erythe-
matosus. Folia Histochem Cytobiol (2007) 45:339–42. 21. MacPhee IA, Antoni FA, Mason DW. Spontaneous recovery of rats from
experimental allergic encephalomyelitis is dependent on regulation of the
immune system by endogenous adrenal corticosteroids. J Exp Med (1989)
169:431–45. doi:10.1084/jem.169.2.431 41. Koga Y, Matsuzaki A, Suminoe A, Hattori H, Kanemitsu S, Hara T. Differential
mRNA expression of glucocorticoid receptor alpha and beta is associated
with glucocorticoid sensitivity of acute lymphoblastic leukemia in children. Pediatr Blood Cancer (2005) 45:121–7. doi:10.1002/pbc.20308 22. Ramachandra RN, Sehon AH, Berczi I. Neuro-hormonal host defence
in endotoxin shock. Brain Behav Immun (1992) 6:157–69. doi:10.1016/
0889-1591(92)90015-G 42. Shahidi H, Vottero A, Stratakis CA, Taymans SE, Karl M, Longui CA, et al. Imbalanced expression of the glucocorticoid receptor isoforms in cultured
lymphocytes from a patient with systemic glucocorticoid resistance and
chronic lymphocytic leukemia. Biochem Biophys Res Commun (1999)
254:559–65. doi:10.1006/bbrc.1998.9980 23. Refojo D, Liberman AC, Giacomini D, Carbia Nagashima A, Graciarena M,
Echenique C, et al. REFERENCES 1. Liberman AC, Druker J, Perone MJ, Arzt E. Glucocorticoids in the regulation
of transcription factors that control cytokine synthesis. Cytokine Growth
Factor Rev (2007) 18:45–56. doi:10.1016/j.cytogfr.2007.01.005 1. Liberman AC, Druker J, Perone MJ, Arzt E. Glucocorticoids in the regulation
of transcription factors that control cytokine synthesis. Cytokine Growth
Factor Rev (2007) 18:45–56. doi:10.1016/j.cytogfr.2007.01.005 2. Petta I, Dejager L, Ballegeer M, Lievens S, Tavernier J, De Bosscher K,
et al. The interactome of the glucocorticoid receptor and its influence on
the actions of glucocorticoids in combatting inflammatory and infectious May 2018 | Volume 9 | Article 235 Frontiers in Endocrinology | www.frontiersin.org 8 Liberman et al. GC-Mediated Immune Regulation cytokines and glucocorticoids. Immunol Cell Biol (2001) 79:385–94. doi:10.1046/j.1440-1711.2001.01023.x 25. Evans RM. The steroid and thyroid hormone receptor superfamily. Science
(1988) 240:889–95. doi:10.1126/science.3283939 26. Kumar R, Thompson EB. Gene regulation by the glucocorticoid receptor:
structure: function relationship. J Steroid Biochem Mol Biol (2005) 94:383–94. doi:10.1016/j.jsbmb.2004.12.046 5. Stahn C, Buttgereit F. Genomic and nongenomic effects of glucocorticoids. Nat Clin Pract Rheumatol (2008) 4:525–33. doi:10.1038/ncprheum0898 6. Busillo JM, Cidlowski JA. The five Rs of glucocorticoid action during inflam-
mation: ready, reinforce, repress, resolve, and restore. Trends Endocrinol
Metab (2013) 24:109–19. doi:10.1016/j.tem.2012.11.005 27. Beck IM, Vanden Berghe W, Vermeulen L, Yamamoto KR, Haegeman G,
De Bosscher K. Crosstalk in inflammation: the interplay of glucocorticoid
receptor-based mechanisms and kinases and phosphatases. Endocr Rev
(2009) 30:830–82. doi:10.1210/er.2009-0013 7. D’Mello C, Le T, Swain MG. Cerebral microglia recruit monocytes into
the brain in response to tumor necrosis factoralpha signaling during
peripheral organ inflammation. J Neurosci (2009) 29:2089–102. doi:10.1523/
JNEUROSCI.3567-08.2009 28. Glass CK, Rose DW, Rosenfeld MG. Nuclear receptor coactivators. Curr Opin
Cell Biol (1997) 9:222–32. doi:10.1016/S0955-0674(97)80066-X 8. Konsman JP, Parnet P, Dantzer R. Cytokine-induced sickness behaviour:
mechanisms and implications. Trends Neurosci (2002) 25:154–9. doi:10.1016/
S0166-2236(00)02088-9 29. Bledsoe RK, Montana VG, Stanley TB, Delves CJ, Apolito CJ, McKee DD,
et al. Crystal structure of the glucocorticoid receptor ligand binding domain
reveals a novel mode of receptor dimerization and coactivator recognition. Cell (2002) 110:93–105. doi:10.1016/S0092-8674(02)00817-6 9. Plotkin SR, Banks WA, Kastin AJ. Comparison of saturable transport
and extracellular pathways in the passage of interleukin-1 alpha across
the blood-brain barrier. J Neuroimmunol (1996) 67:41–7. doi:10.1016/
S0165-5728(96)00036-7 30. Cain DW, Cidlowski JA. Specificity and sensitivity of glucocorticoid signaling
in health and disease. Best Pract Res Clin Endocrinol Metab (2015) 29:545–56. doi:10.1016/j.beem.2015.04.007 10. REFERENCES Rivest S, Lacroix S, Vallières L, Nadeau S, Zhang J, Laflamme N. How the
blood talks to the brain parenchyma and the paraventricular nucleus of the
hypothalamus during systemic inflammatory and infectious stimuli. Proc Soc
Exp Biol Med (2000) 223:22–38. doi:10.1111/j.1525-1373.2000.22304.x 31. Oakley RH, Cidlowski JA. Cellular processing of the glucocorticoid receptor
gene and protein: new mechanisms for generating tissue-specific actions of
glucocorticoids. J Biol Chem (2011) 286:3177–84. doi:10.1074/jbc.R110. 179325 11. Watkins LR, Goehler LE, Relton JK, Tartaglia N, Silbert L, Martin D, et al. Blockade of interleukin-1 induced hyperthermia by subdiaphragmatic
vagotomy: evidence for vagal mediation of immune-brain communication. Neurosci Lett (1995) 183:27–31. doi:10.1016/0304-3940(94)11105-R 32. Busillo JM, Azzam KM, Cidlowski JA. Glucocorticoids sensitize the innate
immune system through regulation of the NLRP3 inflammasome. J Biol
Chem (2011) 286:38703–13. doi:10.1074/jbc.M111.275370 33. Ligr M, Li Y, Logan SK, Taneja S, Melamed J, Lepor H, et al. Mifepristone
inhibits GRbeta coupled prostate cancer cell proliferation. J Urol (2012)
188:981–8. doi:10.1016/j.juro.2012.04.102 fi 12. Capuron L, Miller AH. Immune system to brain signaling: neuropsychophar-
macological implications. Pharmacol Ther (2011) 130:226–38. doi:10.1016/j. pharmthera.2011.01.014 34. Lewis-Tuffin LJ, Jewell CM, Bienstock RJ, Collins JB, Cidlowski JA. Human
glucocorticoid receptor beta binds RU-486 and is transcriptionally active. Mol Cell Biol (2007) 27:2266–82. doi:10.1128/MCB.01439-06 13. Pereda MP, Lohrer P, Kovalovsky D, Perez Castro C, Goldberg V, Losa M,
et al. Interleukin-6 is inhibited by glucocorticoids and stimulates ACTH
secretion and POMC expression in human corticotroph pituitary adenomas. Exp Clin Endocrinol Diabetes (2000) 108:202–7. doi:10.1055/s-2000-7887 35. Charmandari E, Chrousos GP, Ichijo T, Bhattacharyya N, Vottero A,
Souvatzoglou E, et al. The human glucocorticoid receptor (hGR) beta iso-
form suppresses the transcriptional activity of hGRalpha by interfering with
formation of active coactivator complexes. Mol Endocrinol (2005) 19:52–64. doi:10.1210/me.2004-0112 h 14. Turrin NP, Rivest S. Unraveling the molecular details involved in the inti-
mate link between the immune and neuroendocrine systems. Exp Biol Med
(Maywood) (2004) 229:996–1006. doi:10.1177/153537020422901003 15. Cruz-Topete D, Cidlowski JA. One hormone, two actions: anti- and pro-in-
flammatory effects of glucocorticoids. Neuroimmunomodulation (2015)
22:20–32. doi:10.1159/000362724 f 36. Oakley RH, Jewell CM, Yudt MR, Bofetiado DM, Cidlowski JA. The dom-
inant negative activity of the human glucocorticoid receptor beta isoform. Specificity and mechanisms of action. J Biol Chem (1999) 274:27857–66. doi:10.1074/jbc.274.39.27857 16. Howell MP, Muglia LJ. Effects of genetically altered brain glucocorticoid
receptor action on behavior and adrenal axis regulation in mice. Front
Neuroendocrinol (2006) 27:275–84. doi:10.1016/j.yfrne.2006.05.001 37. REFERENCES Integrating systemic information at the molecular level:
cross-talk between steroid receptors and cytokine signaling on different target
cells. Ann N Y Acad Sci (2003) 992:196–204. doi:10.1111/j.1749-6632.2003. tb03150.x 43. Lu NZ, Collins JB, Grissom SF, Cidlowski JA. Selective regulation of bone cell
apoptosis by translational isoforms of the glucocorticoid receptor. Mol Cell
Biol (2007) 27:7143–60. doi:10.1128/MCB.00253-07 24. Ruzek MC, Pearce BD, Miller AH, Biron CA. Endogenous glucocorticoids
protect against cytokine-mediated lethality during viral infection. J Immunol
(1999) 162:3527–33. 44. Cain DW, Cidlowski JA. Immune regulation by glucocorticoids. Nat Rev
Immunol (2017) 17:233–47. doi:10.1038/nri.2017.1 44. Cain DW, Cidlowski JA. Immune regulation by glucocorticoids. Nat Rev
Immunol (2017) 17:233–47. doi:10.1038/nri.2017.1 May 2018 | Volume 9 | Article 235 Frontiers in Endocrinology | www.frontiersin.org 9 Liberman et al. GC-Mediated Immune Regulation hormone receptors. PLoS One (2010) 5:e11717. doi:10.1371/journal. pone.0011717 hormone receptors. PLoS One (2010) 5:e11717. doi:10.1371/journal. pone.0011717 45. Cooper MS, Stewart PM. 11Beta-hydroxysteroid dehydrogenase type 1 and
its role in the hypothalamus-pituitary-adrenal axis, metabolic syndrome,
and inflammation. J Clin Endocrinol Metab (2009) 94:4645–54. doi:10.1210/
jc.2009-1412 65. Holownia A, Mroz RM, Kolodziejczyk A, Chyczewska E, Braszko JJ. Increased
FKBP51 in induced sputum cells of chronic obstructive pulmonary disease
patients after therapy. Eur J Med Res (2009) 14(Suppl 4):108–11. 46. Stegk JP, Ebert B, Martin HJ, Maser E. Expression profiles of human
11beta-hydroxysteroid dehydrogenases type 1 and type 2 in inflammatory
bowel diseases. Mol Cell Endocrinol (2009) 301:104–8. doi:10.1016/j. mce.2008.10.030 t
66. Storer CL, Dickey CA, Galigniana MD, Rein T, Cox MB. FKBP51 and
FKBP52 in signaling and disease. Trends Endocrinol Metab (2011) 22:481–90. doi:10.1016/j.tem.2011.08.001 47. Zbánková S, Bryndová J, Leden P, Kment M, Svec A, Pácha J. 11Beta-
hydroxysteroid dehydrogenase 1 and 2 expression in colon from patients with
ulcerative colitis. J Gastroenterol Hepatol (2007) 22:1019–23. doi:10.1111/
j.1440-1746.2006.04529.x 67. Woodruff PG, Boushey HA, Dolganov GM, Barker CS, Yang YH, Donnelly S,
et al. Genome-wide profiling identifies epithelial cell genes associated with
asthma and with treatment response to corticosteroids. Proc Natl Acad Sci
U S A (2007) 104:15858–63. doi:10.1073/pnas.0707413104 U S A (2007) 104:15858–63. doi:10.1073/pnas.0707413104 48. Liberman AC, Antunica-Noguerol M, Arzt E. Modulation of the glucocor-
ticoid receptor activity by post-translational modifications. Nucl Recept Res
(2014) 1:1–15. doi:10.11131/2014/101086 68. Matsushita R, Hashimoto A, Tomita T, Yoshitawa H, Tanaka S, Endo H, et al. Enhanced expression of mRNA for FK506-binding protein 5 in bone marrow
CD34 positive cells in patients with rheumatoid arthritis. REFERENCES Gene-stress-
epigenetic regulation of FKBP5: clinical and translational implications. Neuropsychopharmacology (2016) 41:261–74. doi:10.1038/npp.2015.235 i 56. Paakinaho V, Kaikkonen S, Makkonen H, Benes V, Palvimo JJ. SUMOylation
regulates the chromatin occupancy and anti-proliferative gene programs of
glucocorticoid receptor. Nucleic Acids Res (2014) 42:1575–92. doi:10.1093/
nar/gkt1033 76. Zimmermann P, Brückl T, Nocon A, Pfister H, Binder EB, Uhr M, et al. Interaction of FKBP5 gene variants and adverse life events in predicting
depression onset: results from a 10-year prospective community study. Am
J Psychiatry (2011) 168:1107–16. doi:10.1176/appi.ajp.2011.10111577 57. Davies L, Karthikeyan N, Lynch JT, Sial EA, Gkourtsa A, Demonacos C,
et al. Cross talk of signaling pathways in the regulation of the glucocorticoid
receptor function. Mol Endocrinol (2008) 22:1331–44. doi:10.1210/me. 2007-0360 77. De Bosscher K, Haegeman G. Minireview: latest perspectives on antiin-
flammatory actions of glucocorticoids. Mol Endocrinol (2009) 23:281–91. doi:10.1210/me.2008-0283 i 58. Antunica-Noguerol M, Budziñski ML, Druker J, Gassen NC, Sokn MC, Senin
S, et al. The activity of the glucocorticoid receptor is regulated by SUMO
conjugation to FKBP51. Cell Death Differ (2016) 23:1579–91. doi:10.1038/
cdd.2016.44 78. Desmet SJ, De Bosscher K. Glucocorticoid receptors: finding the middle
ground. J Clin Invest (2017) 127:1136–45. doi:10.1172/JCI88886 79. Surjit M, Ganti KP, Mukherji A, Ye T, Hua G, Metzger D, et al. Widespread
negative response elements mediate direct repression by agonist-liganded
glucocorticoid receptor. Cell (2011) 145:224–41. doi:10.1016/j.cell.2011. 03.027 59. Kotaja N, Karvonen U, Jänne OA, Palvimo JJ. The nuclear receptor inter-
action domain of GRIP1 is modulated by covalent attachment of SUMO-1. J Biol Chem (2002) 277:30283–8. doi:10.1074/jbc.M204768200 80. Hudson WH, Youn C, Ortlund EA. The structural basis of direct gluco-
corticoid-mediated transrepression. Nat Struct Mol Biol (2013) 20:53–8. doi:10.1038/nsmb.2456 60. Panse VG, Hardeland U, Werner T, Kuster B, Hurt E. A proteome-wide
approach identifies sumoylated substrate proteins in yeast. J Biol Chem
(2004) 279:41346–51. doi:10.1074/jbc.M407950200 81. Löwenberg M, Stahn C, Hommes DW, Buttgereit F. Novel insights into
mechanisms of glucocorticoid action and the development of new glu-
cocorticoid receptor ligands. Steroids (2008) 73:1025–9. doi:10.1016/j. steroids.2007.12.002 61. Pountney DL, Raftery MJ, Chegini F, Blumbergs PC, Gai WP. NSF, Unc-18-1,
dynamin-1 and HSP90 are inclusion body components in neuronal intra-
nuclear inclusion disease identified by anti-SUMO-1-immunocapture. Acta
Neuropathol (2008) 116:603–14. doi:10.1007/s00401-008-0437-4 82. Boldizsar F, Talaber G, Szabo M, Bartis D, Palinkas L, Nemeth P, et al. Emerging pathways of non-genomic glucocorticoid (GC) signalling in
T cells. Immunobiology (2010) 215:521–6. doi:10.1016/j.imbio.2009.10.003 62. REFERENCES Clin Exp Rheumatol
(2010) 28:87–90. 49. Golebiowski F, Matic I, Tatham MH, Cole C, Yin Y, Nakamura A, et al. System-wide changes to SUMO modifications in response to heat shock. Sci
Signal (2009) 2:ra24. doi:10.1126/scisignal.2000282 69. Baker RG, Hayden MS, Ghosh S. NF-kappaB, inflammation, and metabolic
disease. Cell Metab (2011) 13:11–22. doi:10.1016/j.cmet.2010.12.008 50. Holmstrom S, Van Antwerp ME, Iñiguez-Lluhi JA. Direct and distinguish-
able inhibitory roles for SUMO isoforms in the control of transcriptional
synergy. Proc Natl Acad Sci U S A (2003) 100:15758–63. doi:10.1073/pnas. 2136933100 70. Erlejman AG, De Leo SA, Mazaira GI, Molinari AM, Camisay MF,
Fontana V, et al. NF-kappaB transcriptional activity is modulated by
FK506-binding proteins FKBP51 and FKBP52: a role for peptidyl-prolyl
isomerase activity. J Biol Chem (2014) 289:26263–76. doi:10.1074/jbc.M114. 582882 51. Le Drean Y, Mincheneau N, Le Goff P, Michel D. Potentiation of glucocorti-
coid receptor transcriptional activity by sumoylation. Endocrinology (2002)
143:3482–9. doi:10.1210/en.2002-220135 i 71. Hayden MS, Ghosh S. NF-kappaB in immunobiology. Cell Res (2011)
21:223–44. doi:10.1038/cr.2011.13 52. Tian S, Poukka H, Palvimo JJ, Jänne OA. Small ubiquitin-related modifier-1
(SUMO-1) modification of the glucocorticoid receptor. Biochem J (2002)
367:907–11. doi:10.1042/bj20021085 72. Nakamura N, Shimaoka Y, Tougan T, Onda H, Okuzaki D, Zhao H, et al. Isolation and expression profiling of genes upregulated in bone marrow-de-
rived mononuclear cells of rheumatoid arthritis patients. DNA Res (2006)
13:169–83. doi:10.1093/dnares/dsl006 53. Iñiguez-Lluhí JA, Pearce D. A common motif within the negative regulatory
regions of multiple factors inhibits their transcriptional synergy. Mol Cell Biol
(2000) 20:6040–50. doi:10.1128/MCB.20.16.6040-6050.2000 73. Park J, Kim M, Na G, Jeon I, Kwon YK, Kim JH, et al. Glucocorticoids
modulate NF-kappaB-dependent gene expression by up-regulating FKBP51
expression in Newcastle disease virus-infected chickens. Mol Cell Endocrinol
(2007) 278:7–17. doi:10.1016/j.mce.2007.08.002 54. Carbia-Nagashima A, Gerez J, Perez-Castro C, Paez-Pereda M, Silberstein S,
Stalla GK, et al. RSUME, a small RWD-containing protein, enhances
SUMO conjugation and stabilizes HIF-1alpha during hypoxia. Cell (2007)
131:309–23. doi:10.1016/j.cell.2007.07.044 74. Binder EB, Salyakina D, Lichtner P, Wochnik GM, Ising M, Pütz B, et al. Polymorphisms in FKBP5 are associated with increased recurrence of
depressive episodes and rapid response to antidepressant treatment. Nat
Genet (2004) 36:1319–25. doi:10.1038/ng1479 55. Druker J, Liberman AC, Antunica-Noguerol M, Gerez J, Paez-Pereda M,
Rein T, et al. RSUME enhances glucocorticoid receptor SUMOylation
and transcriptional activity. Mol Cell Biol (2013) 33:2116–27. doi:10.1128/
MCB.01470-12 75. Zannas AS, Wiechmann T, Gassen NC, Binder EB. REFERENCES Liberman AC, Refojo D, Druker J, Toscano M, Rein T, Holsboer F, et al. The
activated glucocorticoid receptor inhibits the transcription factor T-bet by
direct protein-protein interaction. FASEB J (2007) 21:1177–88. doi:10.1096/
fj.06-7452com 113. Liu Y, Cousin JM, Hughes J, Van Damme J, Seckl JR, Haslett C, et al. Glucocorticoids promote nonphlogistic phagocytosis of apoptotic leuko-
cytes. J Immunol (1999) 162:3639–46. 114. McColl A, Bournazos S, Franz S, Perretti M, Morgan BP, Haslett C, et al. Glucocorticoids induce protein S-dependent phagocytosis of apoptotic
neutrophils by human macrophages. J Immunol (2009) 183:2167–75. doi:10.4049/jimmunol.0803503 j
95. John S, Sabo PJ, Johnson TA, Sung MH, Biddie SC, Lightman SL, et al. Interaction of the glucocorticoid receptor with the chromatin landscape. Mol
Cell (2008) 29:611–24. doi:10.1016/j.molcel.2008.02.010 96. John S, Sabo PJ, Thurman RE, Sung MH, Biddie SC, Johnson TA, et al. Chromatin accessibility pre-determines glucocorticoid receptor binding
patterns. Nat Genet (2011) 43:264–8. doi:10.1038/ng.759 115. Franchimont D. Overview of the actions of glucocorticoids on the immune
response: a good model to characterize new pathways of immunosuppres-
sion for new treatment strategies. Ann N Y Acad Sci (2004) 1024:124–37. doi:10.1196/annals.1321.009 97. De Bosscher K, Beck IM, Haegeman G. Classic glucocorticoids versus
non-steroidal glucocorticoid receptor modulators: survival of the fittest
regulator of the immune system? Brain Behav Immun (2010) 24:1035–42. doi:10.1016/j.bbi.2010.06.010 116. Sauer J, Castren M, Hopfner U, Holsboer F, Stalla GK, Arzt E. Inhibition
of lipopolysaccharide-induced monocyte interleukin-1 receptor antagonist
synthesis by cortisol: involvement of the mineralocorticoid receptor. J Clin
Endocrinol Metab (1996) 81:73–9. doi:10.1210/jc.81.1.73 j
98. De Bosscher K, Haegeman G, Elewaut D. Targeting inflammation using
selective glucocorticoid receptor modulators. Curr Opin Pharmacol (2010)
10:497–504. doi:10.1016/j.coph.2010.04.007 117. Ehrchen J, Steinmüller L, Barczyk K, Tenbrock K, Nacken W, Eisenacher M,
et al. Glucocorticoids induce differentiation of a specifically activated,
anti-inflammatory subtype of human monocytes. Blood (2007) 109:1265–74. doi:10.1182/blood-2006-02-001115 99. Liberman AC, Castro CN, Antunica Noguerol MA, Barcala Tabarrozzi AE,
Druker J, Perone MJ, et al. Immunomodulation by glucocorticoids: from
basic research to clinical implications. Curr Immunol Rev (2010) 6:371–80. basic research to clinical implications. Curr Immunol Rev (2010 118. Vallelian F, Schaer CA, Kaempfer T, Gehrig P, Duerst E, Schoedon G,
et al. Glucocorticoid treatment skews human monocyte differentiation
into a hemoglobin-clearance phenotype with enhanced heme-iron recy-
cling and antioxidant capacity. Blood (2010) 116:5347–56. doi:10.1182/
blood-2010-04-277319 100. Barnes PJ. Glucocorticosteroids: current and future directions. Br J Pharmacol
(2011) 163:29–43. doi:10.1111/j.1476-5381.2010.01199.x 101. REFERENCES Pascual G, Glass CK. Nuclear receptors versus inflammation: mechanisms
of transrepression. Trends Endocrinol Metab (2006) 17:321–7. doi:10.1016/j. tem.2006.08.005 p
107. Vayssière BM, Dupont S, Choquart A, Petit F, Garcia T, Marchandeau C,
et al. Synthetic glucocorticoids that dissociate transactivation and AP-1
transrepression exhibit antiinflammatory activity in vivo. Mol Endocrinol
(1997) 11:1245–55. doi:10.1210/me.11.9.1245 89. Jonat C, Rahmsdorf HJ, Park KK, Cato AC, Gebel S, Ponta H, et al. Antitumor promotion and antiinflammation: down-modulation of AP-1
(Fos/Jun) activity by glucocorticoid hormone. Cell (1990) 62:1189–204. doi:10.1016/0092-8674(90)90395-U 108. Sugimoto MA, Vago JP, Teixeira MM, Sousa LP. Annexin A1 and the res-
olution of inflammation: modulation of neutrophil recruitment, apoptosis,
and clearance. J Immunol Res (2016) 2016:8239258. doi:10.1155/2016/
8239258 90. Helmberg A, Auphan N, Caelles C, Karin M. Glucocorticoid-induced
apoptosis of human leukemic cells is caused by the repressive function of the
glucocorticoid receptor. EMBO J (1995) 14:452–60. 109. Errasfa M, Russo-Marie F. A purified lipocortin shares the anti-inflamma-
tory effect of glucocorticosteroids in vivo in mice. Br J Pharmacol (1989)
97:1051–8. doi:10.1111/j.1476-5381.1989.tb12561.x 91. Auphan N, DiDonato JA, Rosette C, Helmberg A, Karin M. Immunosuppression by glucocorticoids: inhibition of NF-kappa B activity
through induction of I kappa B synthesis. Science (1995) 270:286–90. doi:10.1126/science.270.5234.286 110. Buckingham JC, John CD, Solito E, Tierney T, Flower RJ, Christian H, et al. Annexin 1, glucocorticoids, and the neuroendocrine-immune interface. Ann
N Y Acad Sci (2006) 1088:396–409. doi:10.1196/annals.1366.002 92. Vacca A, Felli MP, Farina AR, Martinotti S, Maroder M, Screpanti I, et al. Glucocorticoid receptor-mediated suppression of the interleukin 2 gene
expression through impairment of the cooperativity between nuclear
factor of activated T cells and AP-1 enhancer elements. J Exp Med (1992)
175:637–46. doi:10.1084/jem.175.3.637 111. Barczyk K, Ehrchen J, Tenbrock K, Ahlmann M, Kneidl J, Viemann D, et al. Glucocorticoids promote survival of anti-inflammatory macrophages via
stimulation of adenosine receptor A3. Blood (2010) 116:446–55. doi:10.1182/
blood-2009-10-247106 93. De Bosscher K, Schmitz ML, Vanden Berghe W, Plaisance S, Fiers
W, Haegeman G. Glucocorticoid-mediated repression of nuclear fac-
tor-kappaB-dependent transcription involves direct interference with
transactivation. Proc Natl Acad Sci U S A (1997) 94:13504–9. doi:10.1073/
pnas.94.25.13504 112. Fadok VA, Bratton DL, Konowal A, Freed PW, Westcott JY, Henson PM. Macrophages that have ingested apoptotic cells in vitro inhibit proinflamma-
tory cytokine production through autocrine/paracrine mechanisms involv-
ing TGF-beta, PGE2, and PAF. J Clin Invest (1998) 101:890–8. doi:10.1172/
JCI1112 94. REFERENCES Zhou W, Ryan JJ, Zhou H. Global analyses of sumoylated proteins in
Saccharomyces cerevisiae. Induction of protein sumoylation by cellular
stresses. J Biol Chem (2004) 279:32262–8. doi:10.1074/jbc.M404173200 83. Croxtall JD, Choudhury Q, Flower RJ. Glucocorticoids act within minutes to
inhibit recruitment of signalling factors to activated EGF receptors through a
receptor-dependent, transcription-independent mechanism. Br J Pharmacol
(2000) 130:289–98. doi:10.1038/sj.bjp.0703272 i 63. Pratt WB, Toft DO. Regulation of signaling protein function and trafficking
by the hsp90/hsp70-based chaperone machinery. Exp Biol Med (Maywood)
(2003) 228:111–33. doi:10.1177/153537020322800201 64. Schülke JP, Wochnik GM, Lang-Rollin I, Gassen NC, Knapp RT, Berning B,
et al. Differential impact of tetratricopeptide repeat proteins on the steroid 84. Ayroldi E, Cannarile L, Migliorati G, Nocentini G, Delfino DV, Riccardi C. Mechanisms of the anti-inflammatory effects of glucocorticoids: genomic May 2018 | Volume 9 | Article 235 Frontiers in Endocrinology | www.frontiersin.org 10 GC-Mediated Immune Regulation Liberman et al. receptor modulators in stress and immune systems. Eur J Pharmacol (2008)
583:290–302. doi:10.1016/j.ejphar.2007.11.076 receptor modulators in stress and immune systems. Eur J Pharmacol (2008)
583:290–302. doi:10.1016/j.ejphar.2007.11.076 and nongenomic interference with MAPK signaling pathways. FASEB J
(2012) 26:4805–20. doi:10.1096/fj.12-216382 and nongenomic interference with MAPK signaling pathways. FASEB J
(2012) 26:4805–20. doi:10.1096/fj.12-216382 104. Belvisi MG, Wicks SL, Battram CH, Bottoms SE, Redford JE, Woodman P,
et al. Therapeutic benefit of a dissociated glucocorticoid and the relevance of
in vitro separation of transrepression from transactivation activity. J Immunol
(2001) 166:1975–82. doi:10.4049/jimmunol.166.3.1975 85. Scheinman RI, Gualberto A, Jewell CM, Cidlowski JA, Baldwin AS Jr. Characterization of mechanisms involved in transrepression of NF-kappa
B by activated glucocorticoid receptors. Mol Cell Biol (1995) 15:943–53. doi:10.1128/MCB.15.2.943 86. De Bosscher K, Vanden Berghe W, Haegeman G. The interplay between the
glucocorticoid receptor and nuclear factor-kappaB or activator protein-1:
molecular mechanisms for gene repression. Endocr Rev (2003) 24:488–522. doi:10.1210/er.2002-0006 105. De Bosscher K, Beck IM, Dejager L, Bougarne N, Gaigneaux A, Chateauvieux
S, et al. Selective modulation of the glucocorticoid receptor can distinguish
between transrepression of NF-kappaB and AP-1. Cell Mol Life Sci (2014)
71:143–63. doi:10.1007/s00018-013-1367-4 87. De Bosscher K, Vanden Berghe W, Haegeman G. Cross-talk between
nuclear receptors and nuclear factor kappaB. Oncogene (2006) 25:6868–86. doi:10.1038/sj.onc.1209935 106. De Bosscher K, Vanden Berghe W, Beck IM, Van Molle W, Hennuyer N,
Hapgood J, et al. A fully dissociated compound of plant origin for inflam-
matory gene repression. Proc Natl Acad Sci U S A (2005) 102:15827–32. doi:10.1073/pnas.0505554102 88. REFERENCES Vandevyver S, Dejager L, Van Bogaert T, Kleyman A, Liu Y, Tuckermann J,
et al. Glucocorticoid receptor dimerization induces MKP1 to protect against
TNF-induced inflammation. J Clin Invest (2012) 122:2130–40. doi:10.1172/
JCI60006 119. Ayroldi E, Riccardi C. Glucocorticoid-induced leucine zipper (GILZ): a new
important mediator of glucocorticoid action. FASEB J (2009) 23:3649–58. doi:10.1096/fj.09-134684 102. Ronchetti S, Migliorati G, Riccardi C. GILZ as a mediator of the anti-inflam-
matory effects of glucocorticoids. Front Endocrinol (2015) 6:170. doi:10.3389/
fendo.2015.00170 j
120. Cohen N, Mouly E, Hamdi H, Maillot MC, Pallardy M, Godot V, et al. GILZ expression in human dendritic cells redirects their maturation and
prevents antigen-specific T lymphocyte response. Blood (2006) 107:2037–44. doi:10.1182/blood-2005-07-2760 103. De Bosscher K, Van Craenenbroeck K, Meijer OC, Haegeman G. Selective
transrepression versus transactivation mechanisms by glucocorticoid May 2018 | Volume 9 | Article 235 Frontiers in Endocrinology | www.frontiersin.org 11 Liberman et al. GC-Mediated Immune Regulation 121. Elftman MD, Norbury CC, Bonneau RH, Truckenmiller ME. Corticosterone
impairs dendritic cell maturation and function. Immunology (2007)
122:279–90. doi:10.1111/j.1365-2567.2007.02637.x 139. Mosmann TR, Coffman RL. TH1 and TH2 cells: different patterns of lym-
phokine secretion lead to different functional properties. Annu Rev Immunol
(1989) 7:145–73. doi:10.1146/annurev.iy.07.040189.001045 122. Hamdi H, Godot V, Maillot MC, Prejean MV, Cohen N, Krzysiek R, et al. Induction of antigen-specific regulatory T lymphocytes by human dendritic
cells expressing the glucocorticoid-induced leucine zipper. Blood (2007)
110:211–9. doi:10.1182/blood-2006-10-052506 140. Farrar JD, Ouyang W, Löhning M, Assenmacher M, Radbruch A,
Kanagawa O, et al. An instructive component in T helper cell type 2
(Th2) development mediated by GATA-3. J Exp Med (2001) 193:643–50. doi:10.1084/jem.193.5.643 123. Hontelez S, Karthaus N, Looman MW, Ansems M, Adema GJ. DC-SCRIPT
regulates glucocorticoid receptor function and expression of its target
GILZ in dendritic cells. J Immunol (2013) 190:3172–9. doi:10.4049/
jimmunol.1201776 141. Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Lafaille JJ,
et al. The orphan nuclear receptor RORgammat directs the differentiation
program of proinflammatory IL-17+ T helper cells. Cell (2006) 126:1121–33. doi:10.1016/j.cell.2006.07.035 124. Luther C, Adamopoulou E, Stoeckle C, Brucklacher-Waldert V, Rosenkranz D,
Stoltze L, et al. Prednisolone treatment induces tolerogenic dendritic cells
and a regulatory milieu in myasthenia gravis patients. J Immunol (2009)
183:841–8. doi:10.4049/jimmunol.0802046 142. Yang XO, Pappu BP, Nurieva R, Akimzhanov A, Kang HS, Chung Y, et al. T helper 17 lineage differentiation is programmed by orphan nuclear recep-
tors ROR alpha and ROR gamma. Immunity (2008) 28:29–39. doi:10.1016/j. REFERENCES immuni.2007.11.016 125. Rutella S, Lemoli RM. Regulatory T cells and tolerogenic dendritic cells:
from basic biology to clinical applications. Immunol Lett (2004) 94:11–26. doi:10.1016/j.imlet.2004.04.015 143. Campbell DJ, Ziegler SF. FOXP3 modifies the phenotypic and functional
properties of regulatory T cells. Nat Rev Immunol (2007) 7:305–10. doi:10.1038/nri2061 126. Cao Y, Bender IK, Konstantinidis AK, Shin SC, Jewell CM, Cidlowski JA,
et al. Glucocorticoid receptor translational isoforms underlie maturational
stage-specific glucocorticoid sensitivities of dendritic cells in mice and
humans. Blood (2013) 121:1553–62. doi:10.1182/blood-2012-05-432336 144. Ramírez F, Fowell DJ, Puklavec M, Simmonds S, Mason D. Glucocorticoids
promote a TH2 cytokine response by CD4+ T cells in vitro. J Immunol (1996)
156:2406–12. 145. Franchimont D, Galon J, Gadina M, Visconti R, Zhou Y, Aringer M, et al. Inhibition of Th1 immune response by glucocorticoids: dexamethasone
selectively inhibits IL-12-induced Stat4 phosphorylation in T lymphocytes. J Immunol (2000) 164:1768–74. doi:10.4049/jimmunol.164.4.1768 h 127. Cronstein BN, Kimmel SC, Levin RI, Martiniuk F, Weissmann G. A mechanism for the antiinflammatory effects of corticosteroids: the glu-
cocorticoid receptor regulates leukocyte adhesion to endothelial cells and
expression of endothelial-leukocyte adhesion molecule 1 and intercellular
adhesion molecule 1. Proc Natl Acad Sci U S A (1992) 89:9991–5. doi:10.1073/
pnas.89.21.9991 146. Barnes PJ. Th2 cytokines and asthma: an introduction. Respir Res (2001)
2:64–5. doi:10.1186/rr39 147. Liberman AC, Druker J, Refojo D, Holsboer F, Arzt E. Glucocorticoids inhibit
GATA-3 phosphorylation and activity in T cells. FASEB J (2009) 23:1558–71. doi:10.1096/fj.08-121236 128. Pitzalis C, Pipitone N, Bajocchi G, Hall M, Goulding N, Lee A, et al. Corticosteroids inhibit lymphocyte binding to endothelium and intercellular
adhesion: an additional mechanism for their anti-inflammatory and immu-
nosuppressive effect. J Immunol (1997) 158:5007–16. 148. Maneechotesuwan K, Yao X, Ito K, Jazrawi E, Usmani OS, Adcock IM,
et al. Suppression of GATA-3 nuclear import and phosphorylation: a novel
mechanism of corticosteroid action in allergic disease. PLoS Med (2009)
6:e1000076. doi:10.1371/journal.pmed.1000076 f
129. Pitzalis C, Pipitone N, Perretti M. Regulation of leukocyte-endothelial
interactions by glucocorticoids. Ann N Y Acad Sci (2002) 966:108–18. doi:10.1111/j.1749-6632.2002.tb04208.x 149. Biola A, Andréau K, David M, Sturm M, Haake M, Bertoglio J, et al. The glucocorticoid receptor and STAT6 physically and functionally
interact in T-lymphocytes. FEBS Lett (2000) 487:229–33. doi:10.1016/
S0014-5793(00)02297-3 h 130. Weber PS, Toelboell T, Chang LC, Tirrell JD, Saama PM, Smith GW, et al. REFERENCES Mechanisms of glucocorticoid-induced down-regulation of neutrophil
L-selectin in cattle: evidence for effects at the gene-expression level and
primarily on blood neutrophils. J Leukoc Biol (2004) 75:815–27. doi:10.1189/
jlb.1003505 150. Banuelos J, Cao Y, Shin SC, Lu NZ. Immunopathology alters Th17 cell
glucocorticoid sensitivity. Allergy (2017) 72:331–41. doi:10.1111/all.13051 j
131. Besedovsky L, Born J, Lange T. Endogenous glucocorticoid receptor sig-
naling drives rhythmic changes in human T-cell subset numbers and the
expression of the chemokine receptor CXCR4. FASEB J (2014) 28:67–75. doi:10.1096/fj.13-237958 151. Ziolkowska M, Koc A, Luszczykiewicz G, Ksiezopolska-Pietrzak K, Klimczak
E, Chwalinska-Sadowska H, et al. High levels of IL-17 in rheumatoid
arthritis patients: IL-15 triggers in vitro IL-17 production via cyclosporin
A-sensitive mechanism. J Immunol (2000) 164:2832–8. doi:10.4049/
jimmunol.164.5.2832 j
132. Fischer HJ, Schweingruber N, Lühder F, Reichardt HM. The potential role of
T cell migration and chemotaxis as targets of glucocorticoids in multiple scle-
rosis and experimental autoimmune encephalomyelitis. Mol Cell Endocrinol
(2013) 380:99–107. doi:10.1016/j.mce.2013.04.001 152. Momcilović M, Miljković Z, Popadić D, Marković M, Savić E, Ramić Z,
et al. Methylprednisolone inhibits interleukin-17 and interferon-gamma
expression by both naive and primed T cells. BMC Immunol (2008) 9:47. doi:10.1186/1471-2172-9-47 133. Reichardt HM, Kaestner KH, Tuckermann J, Kretz O, Wessely O, Bock R,
et al. DNA binding of the glucocorticoid receptor is not essential for survival. Cell (1998) 93:531–41. doi:10.1016/S0092-8674(00)81183-6 153. McKinley L, Alcorn JF, Peterson A, Dupont RB, Kapadia S, Logar A, et al. TH17 cells mediate steroid-resistant airway inflammation and airway
hyperresponsiveness in mice. J Immunol (2008) 181:4089–97. doi:10.4049/
jimmunol.181.6.4089 134. Bouillet P, Metcalf D, Huang DC, Tarlinton DM, Kay TW, Köntgen F, et al. Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses,
leukocyte homeostasis, and to preclude autoimmunity. Science (1999)
286:1735–8. doi:10.1126/science.286.5445.1735 154. Ramesh R, Kozhaya L, McKevitt K, Djuretic IM, Carlson TJ, Quintero MA,
et al. Pro-inflammatory human Th17 cells selectively express P-glycoprotein
and are refractory to glucocorticoids. J Exp Med (2014) 211:89–104. doi:10.1084/jem.20130301 135. Knudson CM, Tung KS, Tourtellotte WG, Brown GA, Korsmeyer SJ. Bax-
deficient mice with lymphoid hyperplasia and male germ cell death. Science
(1995) 270:96–9. doi:10.1126/science.270.5233.96 155. Schewitz-Bowers LP, Lait PJ, Copland DA, Chen P, Wu W, Dhanda AD, et al. Glucocorticoid-resistant Th17 cells are selectively attenuated by cyclosporine
A. Proc Natl Acad Sci U S A (2015) 112:4080–5. doi:10.1073/pnas.1418316112 136. Villunger A, Michalak EM, Coultas L, Müllauer F, Böck G, Ausserlechner MJ,
et al. REFERENCES p53- and drug-induced apoptotic responses mediated by BH3-
only proteins puma and noxa. Science (2003) 302:1036–8. doi:10.1126/
science.1090072 156. Banuelos J, Shin S, Cao Y, Bochner BS, Morales-Nebreda L, Budinger GR,
et al. BCL-2 protects human and mouse Th17 cells from glucocorticoid-in-
duced apoptosis. Allergy (2016) 71:640–50. doi:10.1111/all.12840 hl 137. Yin XM, Wang K, Gross A, Zhao Y, Zinkel S, Klocke B, et al. Bid-deficient
mice are resistant to Fas-induced hepatocellular apoptosis. Nature (1999)
400:886–91. doi:10.1038/23730 157. Zhao J, Lloyd CM, Noble A. Th17 responses in chronic allergic airway inflam-
mation abrogate regulatory T-cell-mediated tolerance and contribute to air-
way remodeling. Mucosal Immunol (2013) 6:335–46. doi:10.1038/mi.2012.76 f 138. Purton JF, Monk JA, Liddicoat DR, Kyparissoudis K, Sakkal S, Richardson SJ,
et al. Expression of the glucocorticoid receptor from the 1A promoter cor-
relates with T lymphocyte sensitivity to glucocorticoid-induced cell death. J Immunol (2004) 173:3816–24. doi:10.4049/jimmunol.173.6.3816 158. Chen X, Murakami T, Oppenheim JJ, Howard OMZ. Differential response of
murine CD4+CD25+ and CD4+CD25- T cells to dexamethasone-induced
cell death. Eur J Immunol (2004) 34:859–69. doi:10.1002/eji.200324506 May 2018 | Volume 9 | Article 235 Frontiers in Endocrinology | www.frontiersin.org 12 Liberman et al. GC-Mediated Immune Regulation 159. Chen X, Oppenheim JJ, Winkler-Pickett RT, Ortaldo JR, Howard OM. Glucocorticoid amplifies IL-2-dependent expansion of functional FoxP3(+)
CD4(+)CD25(+) T regulatory cells in vivo and enhances their capacity
to suppress EAE. Eur J Immunol (2006) 36:2139–49. doi:10.1002/eji. 200635873 178. Frank MG, Miguel ZD, Watkins LR, Maier SF. Prior exposure to gluco-
corticoids sensitizes the neuroinflammatory and peripheral inflammatory
responses to E. coli lipopolysaccharide. Brain Behav Immun (2010) 24:19–30. doi:10.1016/j.bbi.2009.07.008 179. Johnson JD, O’Connor KA, Deak T, Stark M, Watkins LR, Maier SF. Prior
stressor exposure sensitizes LPS-induced cytokine production. Brain Behav
Immun (2002) 16:461–76. doi:10.1006/brbi.2001.0638 160. Braitch M, Harikrishnan S, Robins RA, Nichols C, Fahey AJ, Showe L, et al. Glucocorticoids increase CD4CD25 cell percentage and Foxp3 expression
in patients with multiple sclerosis. Acta Neurol Scand (2009) 119:239–45. doi:10.1111/j.1600-0404.2008.01090.x 180. Johnson JD, O’Connor KA, Hansen MK, Watkins LR, Maier SF. Effects of
prior stress on LPS-induced cytokine and sickness responses. Am J Physiol
Regul Integr Comp Physiol (2003) 284:R422–32. doi:10.1152/ajpregu.00230. 2002 161. Engler JB, Kursawe N, Solano ME, Patas K, Wehrmann S, Heckmann N, et al. Glucocorticoid receptor in T cells mediates protection from autoimmunity
in pregnancy. Proc Natl Acad Sci U S A (2017) 114:E181–90. doi:10.1073/
pnas.1617115114 181. REFERENCES Stress-induced glucocorticoids as a
neuroendocrine alarm signal of danger. Brain Behav Immun (2013) 33:1–6. doi:10.1016/j.bbi.2013.02.004 168. Masferrer JL, Seibert K, Zweifel B, Needleman P. Endogenous glucocorticoids
regulate an inducible cyclooxygenase enzyme. Proc Natl Acad Sci U S A
(1992) 89:3917–21. doi:10.1073/pnas.89.9.3917 188. Blandino P Jr, Barnum CJ, Solomon LG, Larish Y, Lankow BS, Deak T. Gene
expression changes in the hypothalamus provide evidence for regionally-
selective changes in IL-1 and microglial markers after acute stress. Brain
Behav Immun (2009) 23:958–68. doi:10.1016/j.bbi.2009.04.013 169. Drew PD, Chavis JA. Inhibition of microglial cell activation by cortisol. Brain
Res Bull (2000) 52:391–6. doi:10.1016/S0361-9230(00)00275-6 170. Tanaka J, Fujita H, Matsuda S, Toku K, Sakanaka M, Maeda N. Glucocorticoid-
and mineralocorticoid receptors in microglial cells: the two receptors medi-
ate differential effects of corticosteroids. Glia (1997) 20:23–37. doi:10.1002/
(SICI)1098-1136(199705)20:1<23::AID-GLIA3>3.0.CO;2-6 189. Frank MG, Baratta MV, Sprunger DB, Watkins LR, Maier SF. Microglia
serve as a neuroimmune substrate for stress-induced potentiation of CNS
pro-inflammatory cytokine responses. Brain Behav Immun (2007) 21:47–59. doi:10.1016/j.bbi.2006.03.005 171. Glezer I, Munhoz CD, Kawamoto EM, Marcourakis T, Avellar MC,
Scavone C. MK-801 and 7-Ni attenuate the activation of brain NF-kappa
B induced by LPS. Neuropharmacology (2003) 45:1120–9. doi:10.1016/
S0028-3908(03)00279-X 190. García-Bueno B, Madrigal JL, Pérez-Nievas BG, Leza JC. Stress mediators
regulate brain prostaglandin synthesis and peroxisome proliferator-acti-
vated receptor-gamma activation after stress in rats. Endocrinology (2008)
149:1969–78. doi:10.1210/en.2007-0482 172. Blais V, Turrin NP, Rivest S. Cyclooxygenase 2 (COX-2) inhibition increases
the inflammatory response in the brain during systemic immune stimuli. J Neurochem (2005) 95:1563–74. doi:10.1111/j.1471-4159.2005.03480.x 191. Madrigal JL, Hurtado O, Moro MA, Lizasoain I, Lorenzo P, Castrillo A, et al. The increase in TNF-alpha levels is implicated in NF-kappaB activation
and inducible nitric oxide synthase expression in brain cortex after immo-
bilization stress. Neuropsychopharmacology (2002) 26:155–63. doi:10.1016/
S0893-133X(01)00292-5 173. Blais V, Zhang J, Rivest S. In altering the release of glucocorticoids, ketoro-
lac exacerbates the effects of systemic immune stimuli on expression of
proinflammatory genes in the brain. Endocrinology (2002) 143:4820–7. doi:10.1210/en.2002-220598 192. Madrigal JL, Moro MA, Lizasoain I, Lorenzo P, Fernández AP, Rodrigo J,
et al. Induction of cyclooxygenase-2 accounts for restraint stress-induced
oxidative status in rat brain. Neuropsychopharmacology (2003) 28:1579–88. doi:10.1038/sj.npp.1300187 174. Nadeau S, Rivest S. Endotoxemia prevents the cerebral inflammatory
wave induced by intraparenchymal lipopolysaccharide injection: role of
glucocorticoids and CD14. J Immunol (2002) 169:3370–81. doi:10.4049/
jimmunol.169.6.3370 193. REFERENCES Hermoso MA, Matsuguchi T, Smoak K, Cidlowski JA. Glucocorticoids
and tumor necrosis factor alpha cooperatively regulate toll-like recep-
tor 2 gene expression. Mol Cell Biol (2004) 24:4743–56. doi:10.1128/
MCB.24.11.4743-4756.2004 162. Wüst S, van den Brandt J, Tischner D, Kleiman A, Tuckermann JP, Gold R,
et al. Peripheral T cells are the therapeutic targets of glucocorticoids in exper-
imental autoimmune encephalomyelitis. J Immunol (2008) 180:8434–43. doi:10.4049/jimmunol.180.12.8434 182. Munhoz CD, Lepsch LB, Kawamoto EM, Malta MB, Lima Lde S, Avellar MC,
et al. Chronic unpredictable stress exacerbates lipopolysaccharide-induced
activation of nuclear factor-kappaB in the frontal cortex and hippocampus
via glucocorticoid secretion. J Neurosci (2006) 26:3813–20. doi:10.1523/
JNEUROSCI.4398-05.2006 163. Olsen PC, Kitoko JZ, Ferreira TP, de-Azevedo CT, Arantes AC, Martins
MA. Glucocorticoids decrease Treg cell numbers in lungs of allergic mice. Eur J Pharmacol (2015) 747:52–8. doi:10.1016/j.ejphar.2014.11.034 h 164. Sorrells SF, Caso JR, Munhoz CD, Sapolsky RM. The stressed CNS: when glu-
cocorticoids aggravate inflammation. Neuron (2009) 64:33–9. doi:10.1016/j. neuron.2009.09.032 183. de Pablos RM, Villarán RF, Argüelles S, Herrera AJ, Venero JL, Ayala A,
et al. Stress increases vulnerability to inflammation in the rat prefrontal cor-
tex. J Neurosci (2006) 26:5709–19. doi:10.1523/JNEUROSCI.0802-06.2006 165. Butler LD, Layman NK, Riedl PE, Cain RL, Shellhaas J, Evans GF, et al. Neuroendocrine regulation of in vivo cytokine production and effects: I. In vivo regulatory networks involving the neuroendocrine system, interleu-
kin-1 and tumor necrosis factor-alpha. J Neuroimmunol (1989) 24:143–53. doi:10.1016/0165-5728(89)90108-2 184. Espinosa-Oliva AM, de Pablos RM, Villarán RF, Argüelles S, Venero JL,
Machado A, et al. Stress is critical for LPS-induced activation of microglia
and damage in the rat hippocampus. Neurobiol Aging (2011) 32:85–102. doi:10.1016/j.neurobiolaging.2009.01.012 h 185. Frank MG, Thompson BM, Watkins LR, Maier SF. Glucocorticoids mediate
stress-induced priming of microglial pro-inflammatory responses. Brain
Behav Immun (2012) 26:337–45. doi:10.1016/j.bbi.2011.10.005 166. Goujon E, Parnet P, Cremona S, Dantzer R. Endogenous glucocor-
ticoids down regulate central effects of interleukin-1 beta on body
temperature and behaviour in mice. Brain Res (1995) 702:173–80. doi:10.1016/0006-8993(95)01041-9 186. Frank MG, Watkins LR, Maier SF. Stress- and glucocorticoid-induced
priming of neuroinflammatory responses: potential mechanisms of stress-in-
duced vulnerability to drugs of abuse. Brain Behav Immun (2011) 25(Suppl
1):S21–8. doi:10.1016/j.bbi.2011.01.005 167. Goujon E, Parnet P, Layé S, Combe C, Dantzer R. Adrenalectomy enhances
pro-inflammatory cytokines gene expression, in the spleen, pituitary and
brain of mice in response to lipopolysaccharide. Brain Res Mol Brain Res
(1996) 36:53–62. doi:10.1016/0169-328X(95)00242-K 187. Frank MG, Watkins LR, Maier SF. 196. De Bosscher K. Selective glucocorticoid receptor modulators. J Steroid
Biochem Mol Biol (2010) 120:96–104. doi:10.1016/j.jsbmb.2010.02.027 Conflict of Interest Statement: The authors declare that there is no conflict of
interest that could be perceived as prejudicing the impartiality of the research
reported. The reviewer LD and handling Editor declared their shared affiliation. Copyright © 2018 Liberman, Budziñski, Sokn, Gobbini, Steininger and Arzt. This
is an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). The use, distribution or reproduction in other forums
is permitted, provided the original author(s) and the copyright owner are credited
and that the original publication in this journal is cited, in accordance with accepted
academic practice. No use, distribution or reproduction is permitted which does not
comply with these terms. GC-Mediated Immune Regulation REFERENCES O’Connor KA, Johnson JD, Hammack SE, Brooks LM, Spencer RL,
Watkins LR, et al. Inescapable shock induces resistance to the effects of
dexamethasone. Psychoneuroendocrinology (2003) 28:481–500. doi:10.1016/
S0306-4530(02)00035-5 j
175. Nadeau S, Rivest S. Glucocorticoids play a fundamental role in protecting
the brain during innate immune response. J Neurosci (2003) 23:5536–44. doi:10.1523/JNEUROSCI.23-13-05536.2003 194. Ding Y, Gao ZG, Jacobson KA, Suffredini AF. Dexamethasone enhances
ATP-induced inflammatory responses in endothelial cells. J Pharmacol Exp
Ther (2010) 335:693–702. doi:10.1124/jpet.110.171975 l 176. Soulet D, Rivest S. Bone-marrow-derived microglia: myth or reality? Curr
Opin Pharmacol (2008) 8:508–18. doi:10.1016/j.coph.2008.04.002 177. Carrillo-de Sauvage MÁ, Maatouk L, Arnoux I, Pasco M, Sanz Diez A,
Delahaye M, et al. Potent and multiple regulatory actions of microglial
glucocorticoid receptors during CNS inflammation. Cell Death Differ (2013)
20:1546–57. doi:10.1038/cdd.2013.108 195. Lannan EA, Galliher-Beckley AJ, Scoltock AB, Cidlowski JA. Proinflammatory
actions of glucocorticoids: glucocorticoids and TNFalpha coregulate
gene expression in vitro and in vivo. Endocrinology (2012) 153:3701–12. doi:10.1210/en.2012-1020 May 2018 | Volume 9 | Article 235 Frontiers in Endocrinology | www.frontiersin.org 13 GC-Mediated Immune Regulation Liberman et al. Copyright © 2018 Liberman, Budziñski, Sokn, Gobbini, Steininger and Arzt. This
is an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). The use, distribution or reproduction in other forums
is permitted, provided the original author(s) and the copyright owner are credited
and that the original publication in this journal is cited, in accordance with accepted
academic practice. No use, distribution or reproduction is permitted which does not
comply with these terms. May 2018 | Volume 9 | Article 235 Frontiers in Endocrinology | www.frontiersin.org 14
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Parametric Decay Instabilities during Electron Cyclotron Resonance Heating of Fusion Plasmas, Problems and Possibilities
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1 Introduction fusion research, decays to two daughter waves once the
pump wave amplitude exceeds a threshold determined by
the nonlinear interaction strength between the three waves. Energy conservation in the three-wave process requires the
frequency, f, and wave vector, k, of the pump wave to
equal the sum of those of the daughter waves, i.e., Parametric decay instabilities (PDIs) of millimeter wave
radiation from gyrotron sources used for electron cy-
clotron resonance heating (ECRH) of magnetically con-
fined fusion plasmas have been a topic of significant inter-
est in recent years. The main reason for this is that PDIs
can generate strong microwave signals capable of dam-
aging microwave diagnostics [1], as well as transfering a
significant fraction of the ECRH power from electromag-
netic waves to quasi-electrostatic plasma waves, altering
the heating and current drive characteristics from those
expected based on linear ECRH theory [2, 3]. However,
the microwave signals caused by PDIs can also serve as
plasma diagnostics, e.g., allowing the occurrence of O-
X-B heating to be confirmed [4, 5] and the properties of
edge-localized modes to be explored [6]. Additionally,
the low-frequency waves produced by some PDIs also al-
low generation of a moderate number of fast ions in pure
ECRH plasmas [7–11]. In the present Paper, we provide
an overview of the ECRH PDIs studied in recent years and
assess their potential impact on the O-mode ECRH scenar-
ios planned for ITER [12]. f0 = f1 + f2,
k0 = k1 + k2,
(1) (1) where subscript 0 refers to the pump wave, while sub-
scripts 1 and 2 refer to the daughter waves. Equation (1)
requires the daughter waves excited by PDIs to be shifted
in frequency relative to the pump wave, meaning that their
frequencies will often be outside the protective diagnostic
filters around the ECRH frequency in the case of ECRH
PDIs. This is the root cause of the microwave diagnos-
tics damage associated with such PDIs [1]. Traditionally,
magnetic confinement fusion ECRH PDIs were only ex-
pected in connection with extremely high power ECRH
provided by pulsed free-electron maser sources [13] or
in scenarios with lower ECRH power where a significant
fraction of the ECRH power would reach the upper hy-
brid resonance (UHR) with X-mode polarization [4, 5, 9–
11, 14–30]. PDIs are expected in the above cases due
to the occurrence of electric fields with large amplitudes. Parametric Decay Instabilities during Electron Cyclotron Resonance Heating
of Fusion Plasmas, Problems and Possibilities S.K. Hansen1,∗, S.K. Nielsen2, J. Stober3, J. Rasmussen2, M. Salewski2, M. Willensdorfer3, M. Hoelzl3,
the ASDEX Upgrade Team4 S.K. Nielsen2, J. Stober3, J. Rasmussen2, M. Salewski2, M. Willensdorfer3, M. Hoelzl3, M. Stejner2, and
4 1Plasma Science and Fusion Center, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
2Department of Physics, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark
3Max-Planck-Institut für Plasmaphysik, D-85748 Garching b. München, Germany
4See Author List in H. Meyer for the ASDEX Upgrade Team, Nucl. Fusion 59, 112014 (2019) 1Plasma Science and Fusion Center, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
2Department of Physics, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark
3Max-Planck-Institut für Plasmaphysik, D-85748 Garching b. München, Germany
4See Author List in H. Meyer for the ASDEX Upgrade Team, Nucl. Fusion 59, 112014 (2019) Abstract. We review parametric decay instabilities (PDIs) expected in connection with electron cyclotron
resonance heating (ECRH) of magnetically confined fusion plasmas, with a specific focus on conditions relevant
for the ITER tokamak. PDIs involving upper hybrid (UH) waves are likely to occur in O-mode ECRH scenarios
at ITER if electron density profiles allowing trapping of UH waves near the ECRH frequency are present. Such
PDIs may occur near the plasma center in ITER full-field scenarios heated by 170 GHz O-mode ECRH and on
the high-field side of half-field ITER plasmas heated by 110 GHz or 104 GHz O-mode ECRH. Additionally,
110 GHz O-mode ECRH of half-field ITER scenarios may have low ECRH absorption, due to the electron
cyclotron resonance being located on the high-field side of the main plasma. This potentially allows PDIs
driven by a significant amount of ECRH radiation reaching the UH resonance in X-mode to occur, as X-mode
radiation can be generated by reflection of unabsorbed O-mode radiation from the high-field side wall. The
occurrence of PDIs during ECRH may damage microwave diagnostics, such as the electron cyclotron emission
and low-field side reflectometer systems at ITER, as well as complicate the calculation of heating and current
drive characteristics. However, if PDIs are induced in a controlled manner, they may provide novel diagnostic
tools and allow the generation of a moderate fast ion population in plasmas heated only by ECRH. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
(http://creativecommons.org/licenses/by/4.0/). EPJ Web of Conferences 277, 01002 (2023)
EC21 EPJ Web of Conferences 277, 01002 (2023)
EC21 EPJ Web of Conferences 277, 01002 (2023) https://doi.org/10.1051/epjconf/202327701002 ∗e-mail: soerenkh@psfc.mit.edu 2 Theoretical Background ITER is a large tokamak with minor radius a = 2 m and
major radius R0 = 6.2 m [12]. The magnetic field strength,
B, is approximately proportional to 1/R, with R being the
distance from the symmetry axis. Thus, B ≈B0
R0
R ,
(2) (2) where B0 is the field at the magnetic axis; in full-field sce-
narios, B0 = 5.3 T, while B0 = 2.65 T in half-field scenar-
ios [12]. For quasi-perpendicular ECRH injection in the
non-relativistic limit, absorption occurs when f0 is equal
to multiples of the electron cyclotron frequency [61], [ , ,
,
],
p [
,
]
In spite of previous expectations, strong PDI-like mi-
crowave signals were observed in connection with ECRH
of plasma scenarios with tearing modes at the TEXTOR
tokamak that did not fulfill any of the above criteria
[37, 38]. This was explained in terms of ECRH PDIs in-
volving trapped daughter waves [39], for which the con-
vective losses normally limiting the occurrence of PDIs in
inhomogeneous plasmas [31–33] are essentially removed. Specifically, the TEXTOR results at higher densities were
explained in terms of decay of X-mode ECRH waves into
two trapped UH waves near the O-point of the magnetic is-
land [40–47]. PDIs of this kind have also been found at the
ASDEX Upgrade tokamak [1, 6, 34], in low-temperature
plasma filament experiments [2], and in particle-in-cell
simulations [48–50]. The PDIs leading to microwave di-
agnostics damage at ASDEX Upgrade [1] and significant
anomalous X-mode ECRH absorption in low-temperature
plasma filaments [2] were both of the above type. More
recently, an explanation of strong microwave signals ob-
served at the Wendelstein 7-X stellarator in terms of decay
of an X-mode ECRH wave into a trapped UH wave and a
propagating X-mode wave around the O-point of a divertor
island [51, 52], has also provided a potential explanation
of the strong microwave signals observed at lower densi-
ties in TEXTOR [37, 38]. Apart from the PDIs driven by
X-mode ECRH, PDIs driven by O-mode ECRH, relevant
for the full-field ITER scenarios [12], have also been in-
vestigated. Particularly, decay of an O-mode ECRH wave
to a trapped UH wave and a low-frequency lower hybrid
wave has been investigated theoretically [53–56], in a low-
temperature plasma filament [3], and at the FTU tokamak
[57–59]. 1 Introduction This is due to the fact that X-mode
waves cannot reach the UHR directly from the usual low-
field side ECRH launcher position in tokamaks, owing to
the R-cutoffalways occurring on the low-field side of the
UHR. The analysis of PDIs at the UHR is distinct for
strongly overdense plasmas [18, 19, 22, 29], commonly
found in spherical tokamaks, and underdense/weakly over-
dense plasmas [17, 24–28, 30], commonly found in con-
ventional tokamaks and during plasma start-up, owing to
the different dispersion properties of the electron Bernstein
waves (EBWs) involved in the PDI in these cases. Specific
scenarios in which PDIs at the UHR are of interest in con-
nection with magnetic confinement fusion include funda-
mental X-mode [9–11, 17] and O-mode [9, 11, 21, 24–28]
ECRH with optically thin/gray resonances, O-X-B heating
[4, 5, 18–20, 29], and EBW start-up [35, 36]. pedestals could lead to PDIs near the plasma edge during
O-mode ECRH [60]. In this Paper, we review the basic
conditions under which the above PDIs may occur and as-
sess their relevance for the O-mode ECRH scenarios pro-
posed for ITER [12], in a manner similar to the investiga-
tions in Section 4 of [1], but covering different scenarios. The remainder of the Paper is organized as follows. Section 2 discusses the theoretical background of the em-
ployed criteria. In Section 3, the criteria are applied to the
O-mode ECRH scenarios planned at ITER. Finally, Sec-
tion 4 presents our conclusions. 1 Introduction These large amplitude electric fields allow the convective
losses normally suppressing PDIs in inhomogeneous plas- In a PDI, a strong quasi-coherent pump wave, e.g., the
millimeter waves used for ECRH in magnetic confinement https://doi.org/10.1051/epjconf/202327701002 EPJ Web of Conferences 277, 01002 (2023)
EC21 EPJ Web of Conferences 277, 01002 (2023) mas [31–33] to be overcome. In the free-electron maser
case, the electric field amplitude is large enough to over-
come the losses in the bulk plasma [13], while the UHR
case relies on enhancement of the electric field ampli-
tude associated with X-mode waves near the UHR, where
their group velocity becomes very small [34]. Although
PDIs involving X-mode waves reaching the UHR can oc-
cur at relatively low ECRH power levels without strict re-
quirements on the plasma conditions [25, 26], they have
generally only been considered to play a role in certain
ECRH scenarios. This is due to the fact that X-mode
waves cannot reach the UHR directly from the usual low-
field side ECRH launcher position in tokamaks, owing to
the R-cutoffalways occurring on the low-field side of the
UHR. The analysis of PDIs at the UHR is distinct for
strongly overdense plasmas [18, 19, 22, 29], commonly
found in spherical tokamaks, and underdense/weakly over-
dense plasmas [17, 24–28, 30], commonly found in con-
ventional tokamaks and during plasma start-up, owing to
the different dispersion properties of the electron Bernstein
waves (EBWs) involved in the PDI in these cases. Specific
scenarios in which PDIs at the UHR are of interest in con-
nection with magnetic confinement fusion include funda-
mental X-mode [9–11, 17] and O-mode [9, 11, 21, 24–28]
ECRH with optically thin/gray resonances, O-X-B heating
[4, 5, 18–20, 29], and EBW start-up [35, 36]. mas [31–33] to be overcome. In the free-electron maser
case, the electric field amplitude is large enough to over-
come the losses in the bulk plasma [13], while the UHR
case relies on enhancement of the electric field ampli-
tude associated with X-mode waves near the UHR, where
their group velocity becomes very small [34]. Although
PDIs involving X-mode waves reaching the UHR can oc-
cur at relatively low ECRH power levels without strict re-
quirements on the plasma conditions [25, 26], they have
generally only been considered to play a role in certain
ECRH scenarios. 2 Theoretical Background Additionally, it has recently been suggested that
trapped low-frequency waves existing as a consequence
of the strong gradients in the ITER and ASDEX Upgrade | fce| =
eB
2πme
≈eB0
2πme
R0
R ,
(3) (3) where e is the elementary charge and me is the electron
mass. Additionally in the cold plasma limit, O-mode radi-
ation has a cutoffat the electron plasma frequency, fpe =
s
e2ne
4π2ϵ0me
,
(4) (4) where ne is the electron density and ϵ0 is the permittivity
of vacuum. The UHR occurs for X-mode radiation at the
UH frequency, fUH =
q
f 2pe + f 2ce,
(5) (5) in cold plasmas, although we note that its properties are
substantially influenced by warm plasma effects at elec-
tron temperatures Te ≳3 keV [1, 62]. ECRH radia-
tion with a frequency of f0 will encounter the cold UHR
( f0 = fUH) at an ne value of nUH
e
≈ϵ0me
e2
4π2 f 2
0 −e2B2
0
m2e
R2
0
R2
. (6) (6) We investigate nUH
e
for O-mode ECRH radiation at ITER,
as such radiation may drive a PDI involving a trapped UH
wave and a low-frequency lower hybrid wave in the pres-
ence of an inhomogeneous ne profile with a local maxi-
mum slightly above nUH
e
[53–56]. Additionally, in connec-
tion with O-mode ECRH at ITER, we investigate whether
scenarios are likely to occur in which a significant amount
of X-mode radiation reaches the UHR upon reflection
offthe high-field side wall, as PDIs have been observed 2 EPJ Web of Conferences 277, 01002 (2023) EPJ Web of Conferences 277, 01002 (2023)
EC21 https://doi.org/10.1051/epjconf/202327701002 Figure 1. nUH
e
, nG
e , ¯nmin
e , f0 = |fce|, and f0 = 2|fce| of the O-
mode ECRH scenarios planned for ITER [12]. The top panel
shows the situation for the full-field scenario (B0 = 5.3 T, Ip =
15 MA) heated by the main ECRH system, f0 = 170 GHz. The
middle and bottom panels show the situations for O-mode ECRH
of the half-field scenario (B0 = 2.65 T, Ip = 7.5 MA) with f0 =
110 GHz and f0 = 104 GHz, respectively. in such scenarios at ASDEX Upgrade, even without UH
wave trapping [23–26]. We note that the trapping-ne of
the UH wave is slightly different from the value in Eq. 3 Potential O-Mode ECRH PDI Scenarios
at ITER Figure 1. nUH
e
, nG
e , ¯nmin
e , f0 = |fce|, and f0 = 2|fce| of the O-
mode ECRH scenarios planned for ITER [12]. The top panel
shows the situation for the full-field scenario (B0 = 5.3 T, Ip =
15 MA) heated by the main ECRH system, f0 = 170 GHz. The
middle and bottom panels show the situations for O-mode ECRH
of the half-field scenario (B0 = 2.65 T, Ip = 7.5 MA) with f0 =
110 GHz and f0 = 104 GHz, respectively. We plot nUH
e
, nG
e , ¯nmin
e
, along with the (cold) fundamen-
tal (f0 = | fce|) and second-harmonic (f0 = 2| fce|) electron
cyclotron resonances (ECRs) for the O-mode ECRH sce-
narios planned at ITER [12] in Fig. 1. The plots cover
R ∈[4.11 m, 8.48 m], corresponding to the R-range of the
ITER vacuum vessel in the midplane, while the R-ticks at
R0 −a = 4.2 m, R0 = 6.2 m, and R0 + a = 8.2 m represent
the extent of the main plasma in the midplane [12]. The top panel of Fig. 1 shows the situation for fun-
damental O-mode ECRH of the full-field ITER scenario
(B0 = 5.3 T, Ip = 15 MA) using the main ITER ECRH
system operating at f0 = 170 GHz [12]. In this case,
nUH
e
attains values from 0 to nG
e in the central part of the
plasma. Since the ECRH waves are injected from the
low-field side (large R) [12] and thus encounter the re-
gion with nUH
e
∈]0, nG
e [ before they are absorbed around
the fundamental ECR, PDIs involving decay of an O-mode
ECRH wave to a trapped UH wave and a low-frequency
lower hybrid wave are likely occur if a region allowing
UH wave trapping is present near the plasma center in
full-field ITER scenarios. It is also possible to operate
plasmas with core ne-values approaching 2nG
e when the
plasma is fueled by pellets [64], in which case trapped UH
wave PDIs may be possible over a wider range of the full-
field ITER plasmas; the requirement that ne < nG
e at the plasma edge means that there will always be some point
with R < R0 + a at which f0 = fUH in stable plasmas. 2 Theoretical Background (6),
since it is down-shifted relative to f0 by the frequency of
the low-frequency lower hybrid wave in accordance with
Eq. (1). However, this shift can be ignored, as Eq. (6) al-
ready ignores finite-Te effects, which will modify the UHR
condition in the core of ITER plasmas [1, 12, 62]. Equa-
tion (6) should thus be considered a rough estimate of the
ne-value at which UH wave trapping and field enhance-
ment of X-mode ECRH waves may occur. In addition to nUH
e
, we consider the ne-limits and typi-
cal ne-values at ITER. To avoid disruptions, ne should not
exceed the Greenwald density, nG
e [1020 m−3] = Ip [MA]
π(a [m])2 ,
(7) (7) (7) at the plasma edge [63, 64]; Ip is the plasma current, which
is 15 MA in standard full-field ITER scenarios and 7.5 MA
in half-field ITER scenarios [12]. We note that the core-
ne can exceed nG
e in the case of peaked plasma profiles
obtained by pellet fueling [64] and that the empirical nG
e
given by Eq. (7) may underestimate the ne-limit in ITER
[65], meaning that nG
e does not represent a hard limit on ne
in ITER, but still serves as a useful upper estimate of the
edge-ne. Further, the line-averaged ne values leading to the
minimum L−H threshold, ¯nmin
e
, which are representative of
the lower bound of ne in the core of typical H-mode plas-
mas, are given by 5×1019 m−3 in full-field ITER scenarios
and 2.5 × 1019 m−3 in half-field ITER scenarios according
to [12, 66]. 4 Conclusion We have investigated the potential occurrence of PDIs in
O-mode ECRH scenarios at ITER [12], using an approach
similar to that employed for the ITER X-mode ECRH sce-
narios in Section 4 of [1]. We found that PDIs are likely
to occur near the UHR in connection with phenomena
leading to non-monotonic ne-profiles allowing UH wave
trapping in a wide variety of O-mode ECRH scenarios at
ITER, as well as in situations with X-mode radiation re-
flected offthe high-field side wall reaching the UHR with-
out restrictions on the ne-profiles in scenarios with limited
ECRH absorption. Specifically, trapped UH wave PDIs
may occur near the plasma center during 170 GHz ECRH
in full-field scenarios (B0 = 5.3 T, Ip = 15 MA), while
PDIs requiring limited ECRH absorption are unlikely in
these scenarios. Trapped UH wave PDIs may also occur
on the high-field side during 110 GHz or 104 GHz ECRH
of half-field scenarios (B0 = 2.65 T, Ip = 7.5 MA). Such
scenarios may further allow PDIs requiring limited ECRH
absorption at sufficiently low B0. For 110 GHz ECRH,
a B0 > 2.66 T was found to make such PDIs unlikely for
ECRH beams launched from the outboard midplane, while
a less strict requirement of B0 > 2.52 T was found to apply
for 104 GHz ECRH. It is possible to mitigate the poten-
tial damage to microwave diagnostics due to ECRH PDIs
by installing additional filters around the frequencies ex-
pected to be affected and by minimizing the abilitiy of the
radiometers to pick up strong signals generated inside the
ECRH beams [1]. While the f0 = 110 GHz and f0 = 104 GHz cases are
similar in terms of their behavior with respect to PDIs in-
volving a trapped UH wave, the conclusions regarding the
possibility of having a significant amount of X-mode radi-
ation reaching the UHR due to reflections from the high-
field side wall and driving PDIs differ. For f0 = 110 GHz,
the middle panel of Fig. 1 shows that the fundamental
ECR is located on the far high-field side, slightly outside
the main plasma, meaning that its absorption is highly de-
pendent on the precise value of B0 and the angle at which
the O-mode ECRH waves are launched [24–26, 61]. 3 Potential O-Mode ECRH PDI Scenarios
at ITER Opera-
tion with pellet fueling above nG
e would allow the trapped
UH wave PDIs to occur near the plasma center and on the
low-field side in addition to the high-field side; once again,
the requirement of ne < nG
e at the plasma edge means that
there will always be some point with R < R0 + a at which
f0 = fUH in stable plasmas. of PDIs relying on a significant amount of X-mode ECRH
power reaching the UHR. In the case of f0 = 104 GHz,
shown in the bottom panel of Fig. 1, the fundamental ECR
occurs well inside main plasma around the midplane for
B0 = 2.65 T, so no PDIs relying on X-mode ECRH waves
reaching the UHR are expected in this case. Additionally,
Bmin
0
≈2.52 T for f0 = 104 GHz, meaning that operation at
B0 values somewhat below 2.65 T is also possible without
the occurrence of PDIs relying on X-mode ECRH power
around the UHR. Finally, we note that 104 GHz radiation
might also be preferable for avoiding strong trapped UH
wave PDIs involving X-mode ECRH pump waves [62]. 3 Potential O-Mode ECRH PDI Scenarios
at ITER Trapped UH wave PDIs in the ITER full-field scenarios
may create strong microwave signals with frequency shifts
∼10 GHz from f0 = 170 GHz [1]. This makes them a po-
tential risk to the ITER electron cyclotron emission (ECE)
and low-field side reflectometer systems, operating in the
frequency ranges 70−1000 GHz [67, 68] and 30−165 GHz
[69], respectively. PDIs involving a significant amount of
X-mode ECRH power reflected from the high-field side
wall reaching the UHR are unlikely to occur in the full-
field ITER scenario, as the fundamental ECR is located
close to the plasma center and therefore expected to be op-
tically thick for the O-mode ECRH waves [61]. The middle and bottom panels of Fig. 1 show the sit-
uation for fundamental O-mode ECRH of the half-field 3 https://doi.org/10.1051/epjconf/202327701002 EPJ Web of Conferences 277, 01002 (2023) ITER scenario (B0 = 2.65 T, Ip = 7.5 MA) using the two
lower-frequency ECRH options considered for ITER to
enable ECRH start-up in the one-third-field scenarios [12],
f0 = 110 GHz (middle panel) and f0 = 104 GHz (bottom
panel). In these cases, nUH
e
covers the range from 0 to
nG
e on the high-field side of the plasma. Since the ECRH
waves are still injected from the low-field side, they will
encounter the region with nUH
e
∈]0, nG
e [ before potentially
being absorbed at the fundamental ECR [61]; the second-
harmonic ECR for f0 = 110 GHz which occurs far on the
low-field side, outside the main plasma, is optically thin
due to the low ne and Te in this region [61]. This means
that 110 GHz and 104 GHz ECRH waves are also likely to
drive PDIs involving decay of the O-mode ECRH waves
into trapped UH waves and low-frequency lower hybrid
waves in regions allowing UH wave trapping on the high-
field side in ITER half-field scenarios. Such trapped UH
wave PDIs would again pose a risk to the ECE [67, 68] and
low-field side reflectometer [69] systems at ITER. References [1] S.K. Hansen, A.S. Jacobsen, M. Willensdorfer, S.K. Nielsen, J. Stober, K. Höfler, M. Maraschek, R. Fis-
cher, M. Dunne, the EUROfusion MST1 team et al.,
Plasma Phys. Control. Fusion 63, 095002 (2021) [21] S. Kubo, M. Nishiura, K. Tanaka, D. Moseev,
S. Ogasawara,
T. Shimozuma,
Y. Yoshimura,
H. Igami, H. Takahashi, T.I. Tsujimura et al., J. In-
strum. 11, C06005 (2016) [2] A.B. Altukhov, V.I. Arkhipenko, A.D. Gurchenko,
E.Z. Gusakov, A.Yu. Popov, L.V. Simonchik, M.S. Usachonak, EPL 126, 15002 (2019) [22] A.V. Arefiev, I.Y. Dodin, A. Köhn, E.J. Du Toit,
E. Holzhauer, V.F. Shevchenko, R.G.L. Vann, Nucl. Fusion 57, 116024 (2017) [3] L. Simonchik,
A. Altukhov,
V. Arkhipenko,
A. Gurchenko, E. Gusakov, A. Popov, M. Usa-
chonak, Europhys. Conf. Abstracts 43C, P2.4012
(2019) [23] S.K. Nielsen, P.K. Michelsen, S.K. Hansen, S.B. Korsholm, F. Leipold, J. Rasmussen, M. Salewski,
M. Schubert, M. Stejner, J. Stober et al., Phys. Scr. 92, 024001 (2017) [4] H.P. Laqua, V. Erckmann, H.J. Hartfuß, H. Laqua,
W7-AS Team, ECRH Group, Phys. Rev. Lett. 78,
3467 (1997) [24] S.K. Hansen, S.K. Nielsen, M. Salewski, M. Stejner,
J. Stober, the ASDEX Upgrade team, Plasma Phys. Control. Fusion 59, 105006 (2017) [5] H.P. Laqua, Plasma Phys. Control. Fusion 49, R1
(2007) [25] S.K. Hansen, S.K. Nielsen, J. Stober, J. Rasmussen,
M. Stejner, the ASDEX Upgrade team, EPJ Web
Conf. 203, 02007 (2019) [6] S.K. Hansen, S.K. Nielsen, J. Stober, J. Rasmussen,
M. Stejner, M. Hoelzl, T. Jensen, the ASDEX Up-
grade team, Nucl. Fusion 60, 106008 (2020) [26] S.K. Hansen, S.K. Nielsen, J. Stober, J. Rasmussen,
M. Salewski, M. Stejner, ASDEX Upgrade Team,
Phys. Plasmas 26, 062102 (2019) [7] M. Gryaznevich, A. Nicolai, V. Chuyanov, Tokamak
Energy Ltd. Team, Probl. At. Sci. Technol. Ser. Ther-
monucl. Fusion 44, 107 (2021) [27] M.G. Senstius, S.K. Nielsen, R.G.L. Vann, EPJ Web
Conf. 203, 01010 (2019) [8] S.K. Nielsen, M.P. Gryaznevich, A.S. Jacobsen,
T. Jensen, M. Jessen, S.B. Korsholm, J. Rasmussen,
M. Salewski, M.G. Senstius, V. Naulin et al., Fusion
Eng. Des. 166, 112288 (2021) [28] M.G. Senstius, S.K. Nielsen, R.G. Vann, S.K. Hansen, Plasma Phys. Control. Fusion 62, 025010
(2020) [29] M.G. Senstius, S.J. Freethy, J. Allen, S.K. Nielsen,
Rev. Sci. Instrum. 93, 103522 (2022) [9] R. Wilhelm, V. Erckmann, G. Janzen, W. Kasparek,
G. Müller, E. Räuchle, P.G. Schüller, K. Schwörer,
M. Thumm, the W VII-A team, Plasma Phys. Con-
trol. Fusion 26, 1433 (1984) [30] J. Han, Z. Acknowledgements [14] S. Hiroe, H. Ikegami, Phys. Rev. Lett. 19, 1414
(1967) S.K. Hansen acknowledges support by an Internationali-
sation Fellowship (CF19-0738) from the Carlsberg Foun-
dation. This work was supported by a research grant
(15483) from VILLUM FONDEN. This work has been
carried out within the framework of the EUROfusion Con-
sortium, funded by the European Union via the Euratom
Research and Training Programme (Grant Agreement No
101052200 — EUROfusion). Views and opinions ex-
pressed are however those of the author(s) only and do not
necessarily reflect those of the European Union or the Eu-
ropean Commission. Neither the European Union nor the
European Commission can be held responsible for them. [15] B. Grek, M. Porkolab, Phys. Rev. Lett. 30, 836
(1973) [16] M. Okabayashi, K. Chen, M. Porkolab, Phys. Rev. Lett. 31, 1113 (1973) [17] F.S. McDermott, G. Bekefi, K.E. Hackett, J.S. Levine, M. Porkolab, Phys. Fluids 25, 1488 (1982) [18] V. Shevchenko, G. Cunningham, A. Gurchenko,
E. Gusakov, B. Lloyd, M. O’Brien, A. Saveliev,
A. Surkov, F. Volpe, M. Walsh, Fusion Sci. Technol. 52, 202 (2007) [19] E.Z. Gusakov, A.V. Surkov, Plasma Phys. Control. Fusion 49, 631 (2007) [20] A. Köhn, G. Birkenmeier, A. Chusov, P. Diez,
A. Feuer, U. Höfel, H. Höhnle, E. Holzhauer,
W. Kasparek, S. Merli et al., Plasma Phys. Control. Fusion 55, 014010 (2013) 4 Conclusion If
virtually no particles inside the last closed flux surface are
able to fulfill the ECR resonance condition [61], a signifi-
cant amount of the injected O-mode radiation may reflect
offthe high-field side wall and (for a specific range of
angles, which can be computed using [24]) re-enter the
plasma as X-mode radiation that can drive PDIs at the
UHR [17, 24–26]. This mechanism has been demonstrated
experimentally in ASDEX Upgrade [24–26] and can be as-
sessed for given ITER plasma conditions and ECRH beam
geometries using the theory of [24, 30]. We note that by
slightly increasing B0, such that the fundamental ECR oc-
curs inside the last closed flux surface at the midplane, the
absorption of 110 GHz ECRH radiation from the midplane
launchers can be made more certain, which should also
limit the occurrence of PDIs relying on X-mode radiation
reaching the UHR. The minimum value of B0 at which
ECR absorption around the midplane is guaranteed, Bmin
0 ,
is given by the condition that f0 = | fce| at R = R0 −a, from
which Eq. (3) yields While trapped UH wave PDIs have been found to
lead to diagnostics damage [1] and significant anomalous
ECRH absorption [2, 3], which would generally be detri-
mental to the operation of the ITER ECRH system, we
note that PDIs may also allow novel diagnostics and heat-
ing schemes to be implemented. For instance, PDIs in-
volving X-mode radiation reaching the UHR are being
considered for generating fast ions with ECRH in spherical
tokamaks [7, 8], although we note that the efficacy of this
scheme has yet to be demonstrated, and trapped UH wave
PDIs occurring near the plasma edge have already been
used to investigate edge-localized mode and L-mode blob
characteristics at ASDEX Upgrade [6]. Since PDIs involv-
ing low-frequency trapped waves in the density pedestal
are expected in connection with O-mode ECRH at ITER
[60], it would be of particular interest to assess their value
as an edge diagnostic. Bmin
0
≈2π f0
me
e
R0 −a
R0
. (8) (8) For f0 = 110 GHz, we find Bmin
0
≈2.66 T, so operating
at B0 slightly above 2.65 T should prevent the occurrence 4 https://doi.org/10.1051/epjconf/202327701002 EPJ Web of Conferences 277, 01002 (2023) References Tartari,
E. Alessi, O. D’Arcangelo, J. Instrum. 15, C01046
(2020) [40] A.Yu. Popov, E.Z. Gusakov, Plasma Phys. Control. Fusion 57, 025022 (2015) [60] E.Z. Gusakov, A.Yu. Popov, Phys. Rev. Lett. 128,
065001 (2022) [41] E.Z. Gusakov, A.Yu. Popov, Phys. Plasmas 23,
082503 (2016) [61] M. Bornatici, R. Cano, O. De Barbieri, F. Engel-
mann, Nucl. Fusion 23, 1153 (1983) [42] E.Z. Gusakov, A.Yu. Popov, Nucl. Fusion 59,
104003 (2019) [62] S.K. Hansen, S.K. Nielsen, J. Stober, the EUROfu-
sion MST1 team, the ASDEX Upgrade team, Rel-
ativistic analysis of upper hybrid wave propagation
and trapping (2023), submitted to Phys. Plasmas [43] E.Z. Gusakov, A.Yu. Popov, P.V. Tretinnikov, Nucl. Fusion 59, 106040 (2019) [44] E.Z. Gusakov, A.Yu. Popov, Plasma Phys. Control. Fusion 62, 025028 (2020) [63] M. Greenwald, Plasma Phys. Control. Fusion 44,
R27 (2002) [45] E.Z. Gusakov, A.Yu. Popov, Phys. Plasmas 27,
082502 (2020) [64] P.T. Lang, C. Angioni, M. Bernert, A. Bock,
T.M.J. Engelhardt, R. Fischer, O.J.W.F. Kardaun,
B. Ploeckl, M. Prechtl, W. Suttrop et al., Nucl. Fu-
sion 60, 092003 (2020) [46] E.Z. Gusakov, A.Yu. Popov, Plasma Phys. Control. Fusion 63, 125017 (2021) [47] E.Z. Gusakov, A.Yu. Popov, Plasma Phys. Rep. 48,
327 (2022) [65] M. Giacomin, A. Pau, P. Ricci, O. Sauter, T. Eich, the
ASDEX Upgrade team, JET Contributors, the TCV
team, Phys. Rev. Lett. 128, 185003 (2022) [48] M.G. Senstius, S.K. Nielsen, R.G.L. Vann, Phys. Plasmas 27, 062102 (2020) [49] M.G. Senstius, S.K. Nielsen, R.G.L. Vann, Plasma
Phys. Control. Fusion 63, 065018 (2021) [66] Y.R. Martin, T. Takizuka, ITPA CDBM H-mode
Threshold Database Working Group, J. Phys.: Conf. Ser. 123, 012033 (2008) [50] M.G. Senstius, E.Z. Gusakov, A.Yu. Popov, S.K. Nielsen, Plasma Phys. Control. Fusion 64, 115001
(2022) [67] V.S. Udintsev, S. Danani, G. Taylor, T. Giacomin,
J. Guirao, S. Pak, S. Hughes, L. Worth, G. Vayakis,
M.J. Walsh et al., EPJ Web Conf. 203, 03003 (2019) [51] A. Tancetti, S.K. Nielsen, J. Rasmussen, E.Z. Gusakov, A.Yu. Popov, D. Moseev, T. Stange, M.G. Senstius, C. Killer, M. Vecséi et al., Nucl. Fusion 62,
074003 (2022) [68] Y. Liu, V.S. Udintsev, S. Danani, G. Paraiso,
G. Taylor, M.E. Austin, A. Basile, J.H. Beno,
B. Bunkowski, R. Feder et al., J. Instrum. 17, C04019
(2022) [52] A. Tancetti, S.K. Nielsen, J. Rasmussen, D. Moseev,
T. Stange, S. Marsen, M. Vecséi, C. Killer, G.A. Wur-
den, T. Jensen et al., Plasma Phys. Control. References Gao, S.K. Hansen, Kinetic theory of para-
metric decay instabilities near the upper hybrid reso-
nance in plasmas (2023), accepted by Phys. Plasmas [10] D.G. Bulyginskii, V.V. D’yachenko, M.A. Irzak,
M.M. Larionov, L.S. Levin, G.A. Serebrenyi, N.V. Shustova, Sov. J. Plasma Phys. 12, 77 (1986) [31] A.D. Piliya, Decay Instability in Weakly Inhomoge-
neous Plasma, in Proc. 10th Int. Conf. Phenomena
in Ionized Gases (Oxford), edited by R.N. Franklin
(Donald Parsons & Co. Ltd., Oxford, 1971), p. 320 [11] Z.A. Pietrzyk, A. Pochelon, R. Behn, A. Bondeson,
M. Dutch, T.P. Goodman, M.Q. Tran, D.R. Whaley,
Nucl. Fusion 33, 197 (1993) [32] M.N. Rosenbluth, Phys. Rev. Lett. 29, 565 (1972) [33] A.D. Piliya, Sov. Phys.−JETP 37, 629 (1973) [12] ITER Organization, Tech. Rep. ITR-18-003, ITER
Organization, St. Paul-lez-Durance (2018), https:
//www.iter.org/technical-reports [34] S.K. Hansen,
Ph.D. thesis,
Technical
Uni-
versity
of
Denmark,
Kgs. Lyngby
(2019),
https://pure.mpg.de/rest/items/item_
3182239/component/file_3182252/content [13] M. Porkolab, B.I. Cohen, Nucl. Fusion 28, 239
(1988) 5 https://doi.org/10.1051/epjconf/202327701002 EPJ Web of Conferences 277, 01002 (2023) [35] V. Shevchenko, M.R. O’Brien, D. Taylor, A.N. Saveliev, MAST Team, Nucl. Fusion 50, 022004
(2010) [53] E.Z. Gusakov, A.Yu. Popov, A.N. Saveliev, E.V. Sysoeva, Plasma Phys. Control. Fusion 59, 075002
(2017) [54] E.Z. Gusakov, A.Yu. Popov, Phys. Plasmas 25,
012101 (2018) [36] V.F. Shevchenko, Y.F. Baranov, T. Bigelow, J.B. Caughman, S. Diem, C. Dukes, P. Finburg, J. Hawes,
C. Gurl, J. Griffiths et al., EPJ Web Conf. 87, 02007
(2015) [55] E.Z. Gusakov, A.Yu. Popov, A.N. Saveliev, Plasma
Phys. Control. Fusion 61, 025006 (2019) [37] E. Westerhof,
S.K. Nielsen,
J.W. Oosterbeek,
M. Salewski,
M.R. De Baar,
W.A. Bongers,
A. Bürger, B.A. Hennen, S.B. Korsholm, F. Leipold
et al., Phys. Rev. Lett. 103, 125001 (2009) [56] E.Z. Gusakov, A.Yu. Popov, Plasma Phys. Control. Fusion 63, 015016 (2021) [57] A. Bruschi, E. Alessi, W. Bin, O. D’Arcangelo,
B. Baiocchi, F. Belli, G. Calabrò, I. Casiraghi,
V. Cocilovo, L. Figini et al., Nucl. Fusion 57, 076004
(2017) [38] S.K. Nielsen,
M. Salewski,
E. Westerhof,
W. Bongers,
S.B. Korsholm,
F. Leipold,
J.W. Oosterbeek, D. Moseev, M. Stejner, the TEXTOR
team, Plasma Phys. Control. Fusion 55, 115003
(2013) [58] A. Bruschi,
E. Alessi,
B. Baiocchi,
W. Bin,
O. D’Arcangelo, F. Fanale, L. Figini, C. Galperti,
S. Garavaglia, G. Gittini et al., EPJ Web Conf. 203,
02005 (2019) [39] E.Z. Gusakov, V.I. Fedorov, Sov. J. Plasma Phys. 5,
463 (1979) [59] B. Baiocchi, W. Bin, A. Bruschi, L. Figini, U. References Fusion
65, 015001 (2023) [69] C.M. Muscatello,
C. Anderson,
J. Anderson,
A. Basile, R.L. Boivin, M. Duco, D.K. Finkenthal,
A. Gattuso, J. Klabacha, G.J. Kramer et al., Nucl. Fusion 60, 066005 (2020) 6 6
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Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
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Aline Cássia Figueira1* , Valdecir Luccas1 1Instituto de Tecnologia de Alimentos, Cereal Chocotec, Campinas/SP - Brasil. 1Instituto de Tecnologia de Alimentos, Cereal Chocotec, Campinas/SP - Brasil. *Corresponding Author: Aline Cássia Figueira, Instituto de tecnologia de Alimentos, Cereal Chocotec, Avenida
Brasil, 2880, Jardim Brasil, CEP: 13070-178, Campinas/SP – Brasil, e-mail: aline.cassia.figueira@gmail.com Cite as: Figueira, A. C., & Luccas, V. (2022). Physicochemical characterization of national and commercial cocoa
butter used in Brazil to make chocolate. Brazilian Journal of Food Technology, 25, e2022033. https://doi.org/10.1590/1981-6723.03322 ORIGINAL ARTICLE ORIGINAL ARTICLE Abstract Cocoa butter is one of the most important ingredients in chocolate production as it is responsible for important
characteristics of chocolates such as hardness, snap, mold shrinkage, fat bloom stability and melting. It appears that
factors such as the geographic area and climate where cocoa is grown directly affect the chemical composition of
cocoa butter, so fruits grown in Africa and Asia have greater thermal stability. In this sense, two samples of cocoa
butter, a Brazilian one from the south of Bahia and a commercial one (consisting of a mixture of butters from
different origins) were studied and compared. The results obtained in this study showed that Brazilian cocoa butter
had a higher content of Saturated Fatty Acids (SFA) and a higher content of unsaturated triacylglycerols compared
to commercial butter. It also showed a faster polymorphic transition and a higher maximum solids content
compared to commercial cocoa butter, indicated by isothermal crystallization analysis. Based on the analyzes carried
out, it was found that this cocoa butter studied, from the Forasteiro amelonado species and coming from several
farms in the south of Bahia, presented greater thermal stability in relation to commercial cocoa butter, i.e., different
from that presented in other studies in the literature. Keywords: Thermal behavior; Fatty acids; Triacylglycerols; Isothermal crystallization; Cooling curve; Solid curve. Cite as: Figueira, A. C., & Luccas, V. (2022). Physicochemical characterization of national and commercial cocoa
butter used in Brazil to make chocolate. Brazilian Journal of Food Technology, 25, e2022033.
https://doi.org/10.1590/1981-6723.03322 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited. Caracterização físico-química de manteigas de cacau nacional
e comercial utilizadas no Brasil para fabricação de chocolate Caracterização físico-química de manteigas de cacau nacional
e comercial utilizadas no Brasil para fabricação de chocolate Aline Cássia Figueira1* , Valdecir Luccas1 Aline Cássia Figueira1* , Valdecir Luccas1 Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 1 Introduction Cocoa butter is one of the most important raw materials used in chocolate production, being one of
the ingredients with the highest value-added product, corresponding to 25 and 36%, or more, of the
value of the final product. It is also one of the most important ingredients, where it represents the
continuous phase of chocolate and acts as a dispersing matrix for cocoa, milk, and sugar solids. The
importance of this ingredient is also related to the quality present in the final product, since it is
responsible for the characteristics of hardness, snap (breakdown at room temperature), complete
melting in the mouth, contraction during demolding and chocolate shine, in addition to the product
stability to fat bloom (Suri & Basu, 2022). Among the most predominant fatty acids in the structure of cocoa butter, oleic acid (C18:1), stearic
acid (C18:0) and palmitic acid (C16:0) stood out (Jin et al., 2021). The literature has shown that
factors such as geographic area and climatic conditions where cocoa is grown directly influence the
composition of triacylglycerols (TAGs) and the fatty acid profile of cocoa butter, thus modifying its
melting point and solids profile. According to Beckett (2008), the closer to the equator the cocoa was
grown, the harder the butter would be. In addition, cocoa butter from fruits grown in South America
contains a higher content of oleic acid than those from fruits grown in Africa and Asia (Beckett, 2008). Studies have been shown that Brazilian cocoa butter had a lower content of monounsaturated
triacylglycerols and a higher content of di and polyunsaturated triacylglycerols (Ribeiro et al., 2012). In this way, it has a softer texture, which interferes with the thermal stability of chocolates produced
with this raw material and, consequently, contributes to a reduction in its commercial value. According to Beckett (2008), while the cocoa butter from Malaysia had a Solid Fat Content (SFC) of
81%, the Brazilian one had only approximately 65% of this content. For this reason, most of the cocoa
butter used by Brazilian industries in chocolate making is imported, as they are more resistant to heat,
but more expensive. As an alternative to this issue, many cocoa processors in Brazil sell a blend of
cocoa butters from different origins, but this blend has a percentage of only 20 to 30% of national
butter in the mixture, which is considered a low value. HIGHLIGHTS: •
Oleic, stearic and palmitic acids are the most predominant fatty acids of cocoa butter •
Oleic, stearic and palmitic acids are the most predominant fatty acids of cocoa butter •
The Brazilian sample presented characteristics close to the commercial sample •
The Brazilian sample presented characteristics close to the commercial sample p
p
p
•
It is not recommended to use only the solids curve for selecting cocoa butters for application in
chocolates •
It is not recommended to use only the solids curve for selecting cocoa butters for application in
chocolates Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 Resumo A manteiga de cacau é um dos ingredientes mais importantes na fabricação de chocolate, pois é responsável por
características importantes desse produto como dureza, snap, contração na desmoldagem, estabilidade ao fat
bloom e derretimento. Fatores como a área geográfica e o clima onde o cacaueiro é cultivado afetam diretamente
a composição química da manteiga de cacau, de modo que frutos cultivados na África e Ásia apresentam maior
estabilidade térmica. Nesse sentido, duas amostras de manteiga de cacau, uma brasileira proveniente do sul da
Bahia e uma comercial (constituída por uma mistura de manteigas de diversas origens) foram estudadas e
comparadas. Os resultados obtidos neste estudo mostraram que a manteiga de cacau brasileira apresentou maior
teor de ácidos graxos saturados e maior teor de triacilgliceróis disaturados (S2U), em relação à manteiga comercial. This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distributio
and reproduction in any medium, provided the original work is properly cited. Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 1/9 Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
Figueira, A. C., & Luccas, V. Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
Figueira, A. C., & Luccas, V. Também apresentou uma transição polimórfica mais rápida e um teor máximo de sólidos maior em relação à
manteiga de cacau comercial, indicados pela análise de isoterma de cristalização. Com base nas análises realizadas,
constatou-se que esta manteiga de cacau estudada, da espécie de cacaueiro forasteiro amelonado, apresentou uma
maior estabilidade térmica em relação à manteiga de cacau comercial, resultado diferente dos apresentados em
outros estudos da literatura. Palavras-chave: Comportamento térmico; Ácidos graxos; Triacilgliceróis; Isoterma de cristalização; Curva de
resfriamento; Curva de sólidos. 1 Introduction In this way, this work aimed to evaluate the
physicochemical characteristics of Brazilian cocoa butter sample, from the south of Bahia and
commercial one (blend of cocoa butter from different origins). Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 2/9 Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
Figueira, A. C., & Luccas, V. Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
Figueira, A. C., & Luccas, V. 2.2.2 Triacylglycerol composition The triacylglycerol composition was determined according to the AOCS Ce 5-86 methodology (American
Oil Chemists’ Society, 2009). A CGC model Agilent 6850 Series Gc System and capillary column DB-17
HT Agilent Catalog: 122-1811 (50% phenyl – ethylpolysiloxane) with dimensions of 10 m, internal diameter
of 0.25 mm and 0. 15 μm film were used. The chromatograph operating conditions were: column flow = 1.0
mL/min.; linear velocity = 40 cm/sec; detector temperature: 375 °C; injector temperature: 360 °C; Oven
temperature: 250 – 350 °C (5 °C/min), 350 °C for 20 minutes; carrier gas: Helium; injected volume: 1.0 μL,
split 1:100; and sample concentration: 10 mg/mL tetrahydrofuran (THF). TAGs were identified through the
comparison of retention times, according to the procedures of Antoniosi Filho et al. (1995). The quantity was
determined by peak area normalization. The analysis was carried out in triplicate. 2.1 Material The Brazilian cocoa butter used in this study was granted by the Cocoa Innovation Center (CIC), being
extracted from cocoa beans of the Forasteiro amelonado species, from several farms located in the south of
Bahia. Commercial cocoa butter was obtained from the company Olam Cocoa, Brazil, that it was a
commercial cocoa butter, made up of a mixture of butters from different origins, the proportion of which was
not known. 2.2.1 Fatty acids composition The fatty acid composition was determined according to the AOCS Ce 2-66 method (American Oil
Chemists’ Society, 2009). A Capillary Gas Chromatography (CGC) model Agilent 6850 Series GC System
and an Agilent DB-23 capillary column (50% cyanopropyl) - methylpolysiloxane, with dimensions of 60 m,
internal diameter of 0.25 mm and 0.25 μm thickness of movie were used. The identification of fatty acids
was performed by comparing the retention time obtained with the respective standards. The analysis was
performed in triplicate. The conditions used for chromatograph operation were: column flow = 1.0 mL/min.;
linear velocity = 24 cm/sec; detector temperature: 280°C; injector temperature: 250 °C; oven temperature:
110 °C for 5 min, 110 – 215 °C (5 °C/min), 215 °C for 24 min.; carrier gas: Helium; and injected volume:
1.0 μL. The identification of the fatty acids present in the sample was carried out by comparing the retention times
of the peaks with those of the respective fatty acid standards. The quantification of fatty acids was determined
through the normalization of the peak areas obtained by the chromatogram, expressed in mass percentage. The analysis was carried out in triplicate. 2.3 Statistical analysis The results obtained were submitted to the t test for two samples to determine significant differences in
the means at a significance level of 5% (p ≤ 0.05), using the Minitab 19 software. 2.2.5 Isothermal crystallization The sample was melted and kept at 70 °C for 1 h in a TCON 2000 dry bath, with a temperature
range of 0 to 70 °C (Duratech, USA), to erase its crystalline history. After this period, the sample was
kept in a thermal bath at 17.5 °C ± 0.5 °C and readings were taken every 1 min in a Bruker pc120
Minispec NMR Spectroscopy (Rheinstetten, Germany), until reaching equilibrium (250 min). The
crystallization kinetics was characterized according to the induction period data, which corresponds
to the time required for the beginning of the formation of a stable nucleus and maximum SFC
(SFCmax). The methodology used for the isothermal crystallization analysis was proposed by
Ribeiro et al. (2009). The analysis was carried out in duplicate. 2.2.6 Cooling curve The cooling curves of the cocoa butter samples were obtained on a Multitherm TC temperature meter
(Buhler, Switzerland). The sample was heated to 70 °C and packed in aluminum capsules, specific to the
equipment. The equipment's cooling compartment was kept at a temperature of 17.6 °C for the analysis. The
result was evaluated using the Buhler Crystallization Index (BCI) (Zeng et al., 2021). 2.2.4 Melting point The melting point of the samples was obtained through the solids curve, corresponding to the temperature
at which the sample presented 4% of solids. 2.2.3 Solid Fat Content (%SFC) The SFC of cocoa butters was determined according to the AOCS Cd 16b-93 method, using a Bruker
pc120 Minispec Nuclear Magnetic Resonance Spectroscopy (NMR Spectroscopy) (Silberstreifen,
Rheinstetten, Germany) (American Oil Chemists’ Society, 2009). First, the sample was tempered using
Duratech Tcon 2000 dry baths (Carmel, USA), where it was subjected to 0 °C for 90 min, 26 °C for 40 h and,
again, 0 °C for 90 min. After this preparation, the sample was kept for 1 h at the temperatures analyzed (10,
20, 25, 30 and 35 °C) before taking the reading. The analysis was carried out in triplicate. Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 3/9 Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
Figueira, A. C., & Luccas, V. Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
Figueira, A. C., & Luccas, V. Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 3.1 Fatty acids composition The results presented in Table 1 indicate that the fatty acids present in greater amounts in cocoa butter are
palmitic (C16:0), stearic (C18:0) and oleic (C18:1) acids, which represent more than 90% of the fatty acids
in the sample. The levels obtained for these fatty acids are close to the values found in the literature, which
vary from 24 to 27% for palmitic acid, 33 to 36% for stearic acid and 32 to 35% for oleic acid (Ribeiro et al.,
2012). According to Beckett (2008), the fact that cocoa butter is relatively simple in relation to the main fatty
acids makes this fat have very interesting physical properties, such as the fact that it melts in a small
temperature range, and melts quickly in the mouth, besides being responsible for the glow and snap of
chocolates. It was also verified that the national cocoa butter had a higher content of Saturated Fatty Acids (SFA) and
a lower content of Unsaturated Fatty Acids (UFAs) in relation to commercial cocoa butter. The literature has
shown that factors such as geographic area and climatic conditions where cocoa is grown directly influence
the composition of TAGs and the fatty acid profile of cocoa butter, modifying its melting point and solid
profile. According to Beckett (2008), the closer to the equator the cocoa was grown, the harder the butter
would be. In addition, cocoa butter from fruits grown in South America contains a higher content of oleic
acid than those from fruits grown in Africa and Asia (Beckett, 2008). Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 4/9 Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
Figueira, A. C., & Luccas, V. Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
Figueira, A. C., & Luccas, V. Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
Figueira, A. C., & Luccas, V. Table 1. Fatty acids composition (%) present in commercial and national cocoa butter (CB). Table 1. Fatty acids composition (%) present in commercial and national cocoa butter (CB). 3.1 Fatty acids composition Fatty acids
National CB
Commercial CB
C14:0 – myristic
0.1 ± 0.00
0.1 ± 0.02
C16:0 – palmitic
26.7 ± 0.02
25.1 ± 0.01
C16:1 – palmitoleic
0.3 ± 0.03
0.3 ± 0.01
C17:0 – margaric
0.3 ± 0.00
0.3 ± 0.00
C18:0 – stearic
33.6 ± 0.03
33.3 ± 0.24
C18:1 – oleic
34.3 ± 0.03
35.1 ± 0.10
C18:2 – linoleic
2.9 ± 0.02
4.1 ± 0.26
C18:3 – linolenic
0.2 ± 0.00
0.3 ± 0.02
C20:0 – arachidic
1.2 ± 0.00
1.1 ± 0.01
C20:1 – gadoleic
0.1 ± 0.01
0.1 ± 0.00
C22:0 – behenic
0.2 ± 0.00
0.2 ± 0.00
C24:0 – lignoceric
0.1 ± 0.00
0.0 ± 0.02
∑ Saturated
62.2 ± 0.05
60.2 ± 0.21
∑ Unsaturated
37.8 ± 0.09
39.8 ± 0.14 3.2 TAG composition The TAGs are the major components of oils and fats, and they are responsible for the properties of these
lipids, such as spreadability, melting in the mouth, crystallization profile and type of polymorph. Table 2
present the TAG composition of South of Bahia and commercial cocoa butter. For both butters, it was
possible to obtain 14 types of TAGs. The TAGs of the types Palmitic-Oleic-Palmitic (POP), Palmitic-Oleic-
Stearic (POSt) and Stearic-Oleic-Stearic (StOSt) were the predominant ones, where they presented a total of
84.5% in national butter and 78.5% in commercial butter. These values agree with the range of these
triglycerides reported in the literature, which varies from 72 to 93% (Jin et al., 2021). However, contrary to what was expected, commercial cocoa butter had a higher content (9.8%) of TAGs of the
types Palmitic-Oleic-Oleic (POO) and Stearic-Oleic-Oleic (StOO) compared to national butter (6.6%). In a study
carried out by Ribeiro et al. (2012), the authors found a higher percentage of POO and StOO in Bahia (8.5%) and
Espírito Santo (9.3%) butters in relation to the commercial butters evaluated (around 5%). Furthermore, national
cocoa butter presented lower content of TAGs trisaturated (0.9%) compared to commercial cocoa butter (1.3%). These TAGs have a higher melting point and can accelerate the fat crystallization. Table 2. Triacylglycerol composition present in commercial and national cocoa butter (CB). TAG (%)
National CB
Commercial CB
PPP
0.2 ± 0.0
0.2 ± 0.1
PPSt
0.4 ± 0.2
0.5 ± 0.1
POP
20.6 ± 0.2
17.6 ± 0.2
PLP
1.9 ± 0.2
2.1 ± 0.0
POSt
40.6 ± 0.8
37.4 ± 0.9
POO
3.3 ± 0.3
4.5 ± 0.0
PLSt
2.7 ± 0.2
3.5 ± 0.4
PLO
0.4 ± 0.1
0.7 ± 0.4
PStSt
0.3 ± 0.5
0.6 ± 0.3
StOSt
23.3 ± 0.2
23.5 ± 0.4
StOO
3.3 ± 0.4
5.3 ± 0.1
StLO
1.5 ± 0.0
2.0 ± 0.0
OOO
0.2 ± 0.3
0.9 ± 0.2
StOA
1.3 ± 0.0
1.3 ± 0.4
ΣS3
0.9 ± 0.7
1.3 ± 0.5
ΣS2U
90.4 ± 1.5
85.3 ± 2.3
ΣU2S
8.5 ± 0.8
12.5 ± 0.4
P P l iti (
t
t d) St St
i (
t
t d) O Ol i (
t
t d) A A
hidi (
t
t d) L Li
l i (di
t
t d) Table 2. Triacylglycerol composition present in commercial and national cocoa butter (CB). Braz. J. P: Palmitic (saturated); St: Stearic (saturated); O: Oleic (monounsaturated); A: Arachidic (saturated); L: Linoleic (diunsaturated
Triacylglycerols: S3 = trisaturated, S2U = disaturated–monounsaturated e U2S = diunsaturated–monosaturated. urated); St: Stearic (saturated); O: Oleic (monounsaturated); A: Arachidic (saturated); L: Linoleic (diunsaturated). 3.3 Solid Fat Content (%SFC) One of the indications of the fat melting profile is through the solids curve, which relates the solid fraction
of fat in relation to its total mass (SFC) when the sample is subjected to different temperatures. The solids
curves obtained for the national CB and commercial CB are shown in Figure 1. At all temperatures evaluated,
Bahia cocoa butter presented a lower solid fat content in relation to commercial cocoa butter, presenting a
similar behavior to that obtained by Ribeiro et al. (2012). The authors evaluated the SFC of different
commercial cocoa butters, where, at 10 °C, this content ranged from 83.9 to 85.4%, while for national butter,
the value obtained by the authors was 77.4%. In the present work, at the same temperature, it was possible
to obtain a value of 80% for national butter and 86.4% for commercial butter. In a study carried out by Shukla (2006), the authors obtained a variation in SFC from 74.8 to 83.7%, at a
temperature of 25 °C, for butters from Ghana, India, Nigeria, Malaysia, and Sri Lanka. In the present work,
at 25 °C, a value of 57.5% was obtained for national butter and 65.4% for commercial butter, lower values
compared to imported butters. Ribeiro et al. (2012) also obtained values like those obtained in this study for
Bahia and commercial butter. The authors stated that national cocoa butters, especially those obtained in
Espírito Santo (Brazil), have greater softness in relation to imported cocoa butters and, therefore, to be used
in tropical regions, they need to be mixed with butters from foreign origins to obtain higher levels of SFC at
various temperatures. Importantly, other analyses, such as isothermal crystallization and thermal behavior,
must also be considered before determining whether one cocoa butter is harder than the other. Figure 1. Solid Fat Content (SFC) of national cocoa butter and commercial cocoa butter (CB). Figure 1. Solid Fat Content (SFC) of national cocoa butter and commercial cocoa butter (CB). Through the solids curve, it was possible to quantify several physical parameters of fats. A very important
parameter that can be extracted from the solids curve is the variation of the solid fat content from 25 to 35 °C,
called ∆S. This parameter is widely used in the food industry to assess the quality of cocoa butter (Luccas, 2001). 3.2 TAG composition Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 5/9 Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
Figueira, A. C., & Luccas, V. Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
Figueira, A. C., & Luccas, V. Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 3.4 Melting point From the solids curve, it was also possible to obtain the value of the melting temperature of the samples,
which corresponded to the temperature at which the solid fat content of the sample was equivalent to 4%. It
is considered that at this value the fat is completely in the liquid state, with no residual fraction of solids
(Ribeiro et al., 2012). The melting temperature obtained for each of the butters evaluated is shown in Table
4. According to Shukla (2006), the melting temperature of cocoa butter could vary in a range of 32 to 35 °C. This variation in temperature is not relevant because cocoa butter has a relatively uniform chemical
composition, since the predominant TAGs in cocoa butter (POP, POS and SOS) have a uniform chemical
structure, where oleic acid occupies the second position (Suri & Basu, 2022). The values determined for the
national CB and commercial CB are within this temperature range, and both values were very close. Table 4. Melting point of national and commercial cocoa butter (CB). Samples
Melting point (°C)
National CB
34.40 ± 0.04
Commercial CB
34.66 ± 0.02 Table 4. Melting point of national and commercial cocoa butter (CB). Table 4. Melting point of national and commercial cocoa butter (CB). 3.3 Solid Fat Content (%SFC) It is desirable that fats used for chocolate production have high values of ∆S, thus indicating that the fat has good
melting properties in the mouth, associated with the release of flavor and sensation of freshness. The solid fat
content at 25 °C represents the hardness of the fat. Fats with higher values of S25 °C and lower values of ∆S25 °C – 35
°C are characterized by being harder and with greater resistance to heat, respectively. The presence of solid fat at
temperatures above 35 °C is undesirable, since it causes a sensory perception of waxy fat, and is easily perceptible
during tasting (Luccas, 2001). For cocoa butter to be used in the manufacture of chocolates, it must have a solid
fat content above 50% at 25 °C and must not have high solids content at 35 °C (Luccas, 2001). The values obtained for S25 °C, ∆S25 °C – 35 °C, and S35 °C are shown in Table 3. The cocoa butters showed significant
differences (p < 0.05) concerning the fat content solid at 25 °C and 35 °C, where commercial butter showed higher
SFC values than Bahia cocoa butter. Regarding the ΔS values, it was found that commercial cocoa butter presented
a higher value compared to Bahia cocoa butter, with a significant difference between the samples at a significance
level of 5%. Both butters had low solids content at 35 °C, as expected for cocoa butter samples. Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 6/9 Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
Figueira, A. C., & Luccas, V. Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
Figueira, A. C., & Luccas, V. Table 3. Comparison between the physical characteristics of national cocoa butter (Bahia) and commercial cocoa
butter (CB). Samples
S25 °C
∆S25 °C - 35 °C
S35 °C
National CB (Bahia)
57.51 ± 0.04b
57.30 ± 0.16b
0.21 ± 0.17b
Commercial CB
65.38 ± 0.19a
63.96 ± 0.34a
1.42 ± 0.15a
Values with different letters in the same column are significantly different (p ≤ 0.05). Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 3.5 Isothermal crystallization The graph of Figure 2 shows the curves obtained from the isothermal crystallization of the analyzed cocoa
butters. The isothermal crystallization analysis was performed at 17.5 °C, according to Marangoni &
McGauley (2003). It is possible to verify that, in both cocoa butters, it was possible to obtain a crystallization
with an induction period called “𝜏𝜏1” and in a second step, a second induction period called “𝜏𝜏2” was obtained. In addition, the crystallization curve showed a sigmoidal shape. Ribeiro et al. (2012) also observed this
behavior of cocoa butter. It follows that the first induction time (𝜏𝜏1) is related to the time required for the
formation of a crystal nucleus. The presence of this plateau in the cocoa butter isothermal crystallization is
related to its polymorphic transition, where the transition from a less stable to a more stable form occurs. Figure 2. Isothermal crystallization at 17.5 °C of national and commercial cocoa butters (CB). Figure 2. Isothermal crystallization at 17.5 °C of national and commercial cocoa butters (CB). Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 7/9 Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
Figueira, A. C., & Luccas, V. Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
Figueira, A. C., & Luccas, V. Table 5 presents the results of the two induction periods (𝜏𝜏1 and 𝜏𝜏2) and the maximum solids content
obtained for both butters. It is found that national cocoa butter had a higher 𝜏𝜏1 value than commercial one. This behavior may be related to the TAG composition of the fat. According to Suri & Basu (2022), different
types of cocoa butter submitted to the same crystallization condition may have different crystallization rates. This fact can be attributed to the minority components present in cocoa butter, such as glycolipids,
phospholipids and tri-saturated TAGs, which can act as “seeds” or crystallization nuclei and accelerate the
crystallization of fat (Suri & Basu, 2022). Based on the data of the TAG composition of the cocoa butters in
this study, it was possible to note that commercial cocoa butter had a TAG content (S3) of approximately
40% more than national cocoa butter (Bahia). Such TAGs had a higher melting point, thus being the first to
crystallize, therefore the commercial butter started to crystallize first. 3.5 Isothermal crystallization 𝜏 Regarding the values of 𝜏𝜏2, it appears that the national cocoa butter showed a faster polymorphic transition
than the commercial one. This fact can be attributed to the TAG composition of cocoa butters, since national
cocoa butter had a higher content of symmetrical TAGs (90.4%) in relation to commercial cocoa butter
(85.3%), and these TAGs are responsible for accelerating the polymorphic transition of these fats. Regarding
the maximum solids content, it appears that, although the national cocoa butter has presented itself as softer
by the solids curve analysis, when evaluated at a temperature of 17.5 °C, it could reach equilibrium with a
higher solids content when compared to commercial cocoa butter. Table 5. Induction periods and SFCmax obtained for national and commercial cocoa butters (CB). Samples
τ1 (min)
τ2 (min)
SFCmax (%)
National CB
22.00 ± 0.71a
55.00 ± 1.41a
73.77 ± 0.42a
Commercial CB
14.75 ± 0.35b
62.00 ± 5.60a
59.00 ± 0.71b
Values with different letters in the same column are significantly different (p ≤ 0.05). Table 5. Induction periods and SFCmax obtained for national and commercial cocoa butters (CB). Table 6. Results obtained by analyzing the cooling curve of national and commercial cocoa butters (CB).
Samples
Tn (°C)
tn (min)
Q (°C/min)
BCI
National CB
18.8
16.7
0.22
5.4
Commercial CB
20.2
9.2
0.12
3.5
Tn corresponds to the temperature at the beginning of crystallization; tn corresponds to the time of start of crystallization; Q corresponds to
the crystallization rate. 3.6 Cooling curve The cooling curve provides information on the crystallization of cocoa butter, making it possible to assume
whether it has the necessary or desired hardness for making chocolates. The analysis is based on a
calorimetric principle, where heat is released from cocoa butter during cooling. The analysis is performed at
a temperature of 17.6 °C to maximize the expansion of the cocoa butter exothermic reaction. The BCI index
was created by Buhler (manufacturer of the equipment used) and evaluates the cooling curve of cocoa butter
as a whole, considering the nucleation and crystals growth. Through this value it is possible to know the
crystallization of the raw material used is suitable for chocolate production. According to the manufacturer,
fats with indexes above 4.0 are recommended for chocolate production, as they have high hardness and rapid
crystallization. However, some companies use cocoa butter to make chocolates with BCI values up to 3.5. Table 6 presents the values determined for the cocoa butters in this study. It is verified that national cocoa
butter (Bahia) presented a BCI value of 5.4, which is characterized as a very hard fat, with rapid
crystallization. The commercial cocoa butter evaluated had an index of 3.5, thus characterizing a butter with
moderate crystallization capacity (Zeng et al., 2021). Furthermore, the data obtained for crystallization start
temperatures also showed that domestic cocoa butter started to crystallize before commercial cocoa butter,
suggesting it is a harder cocoa butter. Table 6. Results obtained by analyzing the cooling curve of national and commercial cocoa butters (CB). Samples
Tn (°C)
tn (min)
Q (°C/min)
BCI
National CB
18.8
16.7
0.22
5.4
Commercial CB
20.2
9.2
0.12
3.5
Tn corresponds to the temperature at the beginning of crystallization; tn corresponds to the time of start of crystallization; Q corresponds to
the crystallization rate. Table 6. Results obtained by analyzing the cooling curve of national and commercial cocoa butters (CB). Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 8/9 Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
Figueira, A. C., & Luccas, V. Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
Figueira, A. C., & Luccas, V. 4 Conclusion The results obtained in this study showed that Brazilian cocoa butter had a higher content of SFA and a
higher content of unsaturated TAGs compared to commercial cocoa butter. In addition, national cocoa butter
also showed a faster polymorphic transition and a higher SFCmax compared to commercial cocoa butter. Although Brazilian cocoa butters are characterized by being softer when compared to butters originated from
countries such as Ghana and Malaysia, it could be noted in the sample studied, Forasteiro amelonado species,
which came from several farms located in the South of Bahia, that this species presented physicochemical
characteristics close to the commercial sample, used by national chocolate industries, unlike the results
presented in other studies. This indicated that, probably, the South region of Bahia has some species
consisting of significant thermal resistance. References American Oil Chemists’ Society – AOCS. (2009). Official methods and recommended practices of the American Oil Chemists’
Society (6th ed). Champaign: AOCS. Beckett, S. T. (2008). The science of chocolate. Cambridge: RSC Paperbacks. Jin, J., Jin, Q., Akoh, C., & Wang, X. (2021). StOSt-rich fats in the manufacture of heat-stable chocolates and their potential
impacts on fat bloom behaviors. Trends in Food Science & Technology, 118, 418-430. http://dx.doi.org/10.1016/j.tifs.2021.10.005 Jin, J., Jin, Q., Akoh, C., & Wang, X. (2021). StOSt-rich fats in the manufacture of heat-stable chocolates and their potential
impacts on fat bloom behaviors. Trends in Food Science & Technology, 118, 418-430. http://dx.doi.org/10.1016/j.tifs.2021.10.005 Luccas, V. (2001). Fracionamento térmico e obtenção de gorduras de cupuaçu alternativas à manteiga de cacau para uso na
fabricação de chocolate (Tese de doutorado). Universidade Estadual de Campinas, Faculdade de Engenharia Química,
Campinas. Luccas, V. (2001). Fracionamento térmico e obtenção de gorduras de cupuaçu alternativas à manteiga de cacau para uso na
fabricação de chocolate (Tese de doutorado). Universidade Estadual de Campinas, Faculdade de Engenharia Química,
Campinas. Marangoni, A. G., & McGauley, S. E. (2003). Relationship between crystallization behavior and structure in cocoa butte
Growth & Design, 3(1), 95-108. http://dx.doi.org/10.1021/cg025580l Marangoni, A. G., & McGauley, S. E. (2003). Relationship between crystallization behavior and structure in cocoa butter. Crystal
Growth & Design, 3(1), 95-108. http://dx.doi.org/10.1021/cg025580l Ribeiro, A. P. B., Grimaldi, R., Gioielli, L. A., Santos, A. O., Cardoso, L. P., & Gonçalves, L. A. G. (2009). Thermal behavior,
microstructure, polymorphism, and crystallization properties of zero trans fats from soybean oil and fully hydrogenated soybean
oil. Food Biophysics, 4(2), 106-118. http://dx.doi.org/10.1007/s11483-009-9106-y Ribeiro, A. P. B., Silva, R. C., Gioielli, L. A., Almeida Gonçalves, M. I., Grimaldi, R., Gonçalves, L. A. G., & Guenter Kieckbusch,
T. (2012). Physico-chemical properties of Brazilian cocoa butter and industrial blends. Part I - Chemical composition, solid fat
content and consistency. Grasas y Aceites, 63(1), 79-88. http://dx.doi.org/10.3989/gya.069011 Shukla, V. (2006). Cocoa butter, cocoa butter equivalents, and cocoa butter substitutes. In C. Akoh (Ed.), Handbook of
functional lipids (pp. 279 -307). Boca Raton: CRC Press. Suri, T., & Basu, S. (2022). Heat resistant chocolate development for subtropica and tropical climates: A review. Food Science &
Nutrition, 62(20), 5603-5622. PMid:33635177. Suri, T., & Basu, S. (2022). Heat resistant chocolate development for subtropica and tropical climates: A review. Food Science &
Nutrition, 62(20), 5603-5622. PMid:33635177. Zeng, Y., Braun, P., Keller, J. Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 References P., Paggios, K., Jacob, J., & Pirhalla, S. (2021). Exothermal analysis of cocoa butter, cocoa liquor,
and chocolate mass with MultiTherm TC™. The Green Machine, 32(2), 24-29. http://dx.doi.org/10.21748/inform.02.2021.24 Zeng, Y., Braun, P., Keller, J. P., Paggios, K., Jacob, J., & Pirhalla, S. (2021). Exothermal analysis of cocoa butter, cocoa liquor,
and chocolate mass with MultiTherm TC™. The Green Machine, 32(2), 24-29. http://dx.doi.org/10.21748/inform.02.2021.24 Funding: Acknowledgement to FAPESP (The São Paulo Research
Foundation) for the financial support: projects numbers
2019/09376-0 and 2020/07015-7. Received: Apr. 04, 2022; Accepted: July 16, 2022 Received: Apr. 04, 2022; Accepted: July 16, 2022 Received: Apr. 04, 2022; Accepted: July 16, 2022
Associate Editor: Maria Gabriela Bello Koblitz Associate Editor: Maria Gabriela Bello Koblitz Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 9/9
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Spread of Amphibian Chytrid Fungus across Lowland Populations of Túngara Frogs in Panamá
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PloS one
| 2,016
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cc-by
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Spread of Amphibian Chytrid Fungus across
Lowland Populations of Túngara Frogs in
Panamá Sofía Rodríguez-Brenes1*, David Rodriguez2, Roberto Ibáñez3,4, Michael J. Ryan1,3 Sofía Rodríguez-Brenes1*, David Rodriguez2, Roberto Ibáñez3,4, Michael J. Ryan1,3
1 Department of Integrative Biology, University of Texas, Austin, Texas, United States of America,
2 Department of Biology, Texas State University, San Marcos, Texas, United States of America,
3 Smithsonian Tropical Research Institute, Balboa, Panamá, 4 Departamento de Zoología, Universidad de
Panamá, Panamá, Panamá Sofía Rodríguez-Brenes1*, David Rodriguez2, Roberto Ibáñez3,4, Michael J. Ryan1,3
1 Department of Integrative Biology, University of Texas, Austin, Texas, United States of America,
2 Department of Biology, Texas State University, San Marcos, Texas, United States of America,
3 Smithsonian Tropical Research Institute, Balboa, Panamá, 4 Departamento de Zoología, Universidad de
Panamá, Panamá, Panamá 1 Department of Integrative Biology, University of Texas, Austin, Texas, United States of America,
2 Department of Biology, Texas State University, San Marcos, Texas, United States of America,
3 Smithsonian Tropical Research Institute, Balboa, Panamá, 4 Departamento de Zoología, Universidad d
Panamá, Panamá, Panamá a1111 * sofiarb@utexas.edu * sofiarb@utexas.edu * sofiarb@utexas.edu Abstract Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is
an emergent infectious disease partially responsible for worldwide amphibian population
declines. The spread of Bd along highland habitats (> 500 meters above sea level, m a.s.l.)
of Costa Rica and Panamá is well documented and has been linked to amphibian popula-
tion collapses. In contrast, data are scarce on the prevalence and dispersal of Bd in lowland
habitats where amphibians may be infected but asymptomatic. Here we describe the
spread (2009 to 2014) of Bd across lowland habitats east of the Panamá Canal (< 500 m
a.s.l.) with a focus on the Túngara frog (Physalaemus [Engystomops] pustulosus), one of
the most common and abundant frog species in this region. Highland populations in western
Panamá were already infected with Bd at the start of the study, which was consistent with
previous studies indicating that Bd is enzootic in this region. In central Panamá, we col-
lected the first positive samples in 2010, and by 2014, we detected Bd from remote sites in
eastern Panamá (Darién National Park). We discuss the importance of studying Bd in low-
land species, which may serve as potential reservoirs and agents of dispersal of Bd to high-
land species that are more susceptible to chytridiomycosis. OPEN ACCESS Citation: Rodríguez-Brenes S, Rodriguez D, Ibáñez
R, Ryan MJ (2016) Spread of Amphibian Chytrid
Fungus across Lowland Populations of Túngara
Frogs in Panamá. PLoS ONE 11(5): e0155745. doi:10.1371/journal.pone.0155745 Editor: Louise A. Rollins-Smith, Vanderbilt University
School of Medicine, UNITED STATES
Received: September 29, 2015
Accepted: May 3, 2016
Published: May 13, 2016 Editor: Louise A. Rollins-Smith, Vanderbilt University
School of Medicine, UNITED STATES
Received: September 29, 2015
Accepted: May 3, 2016
Published: May 13, 2016
Copyright: © 2016 Rodríguez-Brenes 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. Editor: Louise A. Rollins-Smith, Vanderbilt University
School of Medicine, UNITED STATES
Received: September 29, 2015
Accepted: May 3, 2016
Published: May 13, 2016 Editor: Louise A. Rollins-Smith, Vanderbilt University
School of Medicine, UNITED STATES
Received: September 29, 2015
Accepted: May 3, 2016
Published: May 13, 2016
Copyright: © 2016 Rodríguez-Brenes 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. Published: May 13, 2016 Copyright: © 2016 Rodríguez-Brenes 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. RESEARCH ARTICLE Data Availability Statement: All data are available
from the DRYAD Digital Repository (doi:10.5061/
dryad.6bp92). Data Availability Statement: All data are available
from the DRYAD Digital Repository (doi:10.5061/
dryad.6bp92). Wildlife extinctions are not typically attributed to infectious diseases, yet there are a few exam-
ples showing pathogens as the direct cause of local extinctions [1–3]. Some of them have been
linked to chytridiomycosis, an emerging infectious disease [4,5]. In amphibians, chytridiomy-
cosis results from a skin infection caused by the chytrid fungus Batrachochytrium dendrobati-
dis (Bd). In the tropics, the most severe declines have been documented in comparatively
cooler and humid areas above 500 m [6], most likely because lower temperatures (17–25°C)
are optimal for Bd growth and water facilitates the propagation and dispersal of the aquatic,
flagellated Bd zoospores [7]. Although the rapid spread of Bd into apparently Bd-free areas Funding: This work was funded by National Science
Foundation (http://www.nsf.gov/), IBN 0517328 to
MJR. The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript. Competing Interests: The authors have declared
that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0155745
May 13, 2016 1 / 8 Amphibian Chytrid Spreads across Tropical Lowlands throughout the world is well-documented [8–12], the process by which Bd spreads is still not
clearly understood [13]. Bd has rapidly spread throughout the highlands of Central America. In the late 1980s, the dis-
appearance of golden toads (Incilius periglenes) and declines of other anuran populations in the
protected cloud forest of Monteverde, Costa Rica (Fig 1), were the first alerts to what later became
a predictable pattern of declines spreading towards Panamá [14,15]. Shortly thereafter, between
1993 and 1997 additional cases of amphibian population declines or extinctions were reported in
the highlands close to the border with Panamá at Las Tablas, Costa Rica, and Fortuna, Panamá
(Fig 1)[16,17]. The sites experiencing population declines were located at altitudes above 500 m,
and the fastest declining species were stream dwellers with aquatic tadpoles [6]. Bd was spreading
west to east in a wave like pattern [8]. High elevation sites in western and central Panamá were
then intensively studied prior to and during the arrival of Bd, these studies documented amphib-
ian population declines after the arrival of Bd in this region (Fig 1)[16–19]. Only two studies have investigated Bd infection spread in eastern Panamá. Rebollar et al. [20] sampled populations along the lowlands of east Panamá, and Woodhams et al. Methods We sampled Túngara frog populations in Panamá from 2009 to 2014, each year during the
rainy season (June to November), which falls within their reproductive season. In most cases,
populations were sampled during two reproductive seasons. Lowland sites in east and central
Panamá ranged from 12 to 67 m in altitude (Fig 1). We also sampled highland populations at
El Valle (elevation 600 m; Fig 1) in central Panamá, and at Chiriquí near the town of Cuesta de
Piedra (elevation 460–967 m; Fig 1) in western Panamá. This altitude is the highest known
Túngara population in Panamá. Chiriquí was also our western most site and was located 30 km
east of sites where other amphibian species experienced declines in 1993 [17]. In central Panamá, we sampled two lowland sites east of the Panamá Canal, Gamboa and
Summit, ranging from 46 to 98 m in elevation (Fig 1). These two lowland sites are located on
the eastern side of the Panamá Canal but are separated by the Chagres River. Gamboa is a
small town north of the river that is surrounded by rainforest. Here we included Pipeline Road,
which intersects a protected rainforest in the Soberanía National Park. Summit is located south
of the river and includes portions of the Soberanía National Park. We sampled along the main
road, on trails and dirt roads within the National Park. In east Panamá, we sampled four sites. Metetí and Yaviza are located along and at the very
end of the Inter-American Highway, respectively (Fig 1). Here, we sampled disturbed, defor-
ested habitats, in puddles on dirt roads and cattle ranches. Further east, along the Tuira River,
we sampled at El Real (Fig 1), a small town surrounded by less disturbed habitat and in Darién
National Park. In Darién National Park, we sampled around the Rancho Frío field station (Fig
1) at an average of 50 m of elevation. This site is predominantly primary rainforest with a tall
canopy, thick lianas and an understory dominated by palms. Data Availability Statement: All data are available
from the DRYAD Digital Repository (doi:10.5061/
dryad.6bp92). [19] sam-
pled sites in central Panamá. These studies did not detect a clear pattern of wave-like spread
from west to east as observed in the highlands of Panamá. It is also possible that another wave
of Bd from South America crossed into Panamá [8]. Currently, the dynamics of Bd spread in
the lowlands of Panamá, where environmental conditions are not ideal for Bd, are still unclear
and require additional investigation. Evaluating the spread of a pathogen in a single, abundant, and well-characterized host can
help to predict spread dynamics while controlling for host phylogenetic diversity. The Túngara
frog (Physalaemus [Engystomops] pustulosus) is a common species occupying lowland habitats
ranging from Mexico to Colombia, Venezuela, and a small portion of the Guyana Shield. Its Fig 1. Sites where Bd has been detected in the past (filled circles), and sites where we sampled Túngara frog (Physalaemus [Engystomops]
pustulosus) populations (open triangles). Year in parenthesis corresponds to year of decline or the year that Bd was first detected. doi:10.1371/journal.pone.0155745.g001 Fig 1. Sites where Bd has been detected in the past (filled circles), and sites where we sampled Túngara frog (Physalaemus [Engystomops]
pustulosus) populations (open triangles). Year in parenthesis corresponds to year of decline or the year that Bd was first detected. Fig 1. Sites where Bd has been detected in the past (filled circles), and sites where we sampled Túngara frog (Physalaemus [Engystomops]
pustulosus) populations (open triangles). Year in parenthesis corresponds to year of decline or the year that Bd was first detected. doi:10.1371/journal.pone.0155745.g001 doi:10.1371/journal.pone.0155745.g001 PLOS ONE | DOI:10.1371/journal.pone.0155745
May 13, 2016 2 / 8 Amphibian Chytrid Spreads across Tropical Lowlands range parallels many highland regions where Bd and amphibian declines have been extensively
documented [8–13]. The adults and tadpoles of this species use both permanent and ephemeral
bodies of water in habitats ranging from urban areas to pristine forests. Thus, the Túngara frog
is an ideal species in which to characterize the spread of Bd through populations of a lowland
host. Here, we (i) report the Bd infection of túngara frogs at two sites west of the Panamá Canal
where Bd is enzootic, and (ii) present data documenting the spread of Bd in the lowlands of
central and east Panamá from 2009 to 2014. Time scale of surveys We first sampled El Valle in 2009 and then Chiriquí in 2010, where Bd is thought to have
arrived in the mid-1990s according to previous estimations [13]. In 2010, we also sampled
Gamboa and Summit in central Panamá. Woodhams et al. [19] reported Bd in Summit in
2007. We then sampled Metetí and Yaviza in eastern Panamá during 2011, where we expected
the front of the Bd wave to be just arriving. In 2013, we sampled all the populations previously
surveyed. In 2014 we sampled in Gamboa, Yaviza and finally Darién National Park, which was
the eastern most site and where we expected P. pustulosus populations to be Bd naive. Results In total, we sampled 1695 P. pustulosus adults across Panamá from 2009 to 2014 (Fig 1). Sam-
ple size, prevalence, and 95% binomial confidence intervals are shown in Table 1. We consid-
ered sites with sample sizes greater than 60 individuals and no positive samples as Bd-negative
[25]. The site Chiriquí, in western Panamá, was positive for Bd in 2010 and 2013. In central
Panamá, El Valle was positive for Bd in 2009. In 2010, Gamboa samples were negative for Bd,
but Summit, which is only 8 km to the south, was positive. Bd reached Gamboa in 2011, and in
following years we detected Bd positive samples in both Summit and Gamboa. By 2014, preva-
lence had reached 26% in Gamboa (Table 1). Populations in Metetí were Bd naive in 2011 but
positive in 2013. Farther east, Yaviza was naive for Bd in 2013, but by 2014 prevalence reached
approximately 6%. By 2014, Bd was also present in P. pustulosus populations from El Real and
Rancho Frío, Darién National Park (Table 1). Bd sampling We toe-clipped individuals to avoid recapture. To avoid cross contamination, we captured
adults by hand using a new pair of nitrile gloves for each individual and kept them isolated in a
plastic bag until processing. We swabbed the ventral area using a sterile cotton tip dry swab
(Medical Wire & Equipment, model MW113 and MW110) following established procedures
[21]. Swabs were stored in 90% ethanol or they were kept frozen until extraction, thus we
expect no effects of sample storage on estimates of prevalence or infection intensity [22]. To 3 / 8 PLOS ONE | DOI:10.1371/journal.pone.0155745
May 13, 2016 Amphibian Chytrid Spreads across Tropical Lowlands avoid potential cross-contamination between sites, we bleached our rubber boots and vehicle
tires and then rinsed them with tap water before leaving the collection site. Real time quantitative PCR To determine the prevalence of Bd in each population and the intensity of infection for individ-
ual frogs, we processed the swabs using quantitative PCR (qPCR) following the protocol devel-
oped by Boyle et al. [23] and modified by Kriger et al. [24]. For samples collected from 2009 to
2012, we used a Roche LightCycler 480 system with a high confidence setting to detect positive
samples. For samples collected during 2009–2010, we did not quantify the number of zoo-
spores due to lack of Bd standards. For samples collected during 2013 and 2014, we used Taq-
Man1 Fast Advanced Master Mix (Applied Biosystems) and a StepOnePlus™system. We used
a dilution series of genomic DNA from strain JEL423 as our standard reference for the estima-
tions of infection intensity in samples collected from 2011 to 2014. We calculated prevalence
by using the ratio of positive samples to total samples per population and calculated 95% bino-
mial confidence intervals. For each population, we calculated average infection intensity using
the average number of zoospore equivalents (z.e.) inferred from qPCR among positive
individuals. To calculate the rate of Bd spread across sites east of the Canal, we used the distance between
sites and the year we first detected infected frogs. We sampled frogs from June to November. If
Bd arrived after sampling was completed for a given year, then we would have detected it the
following year. We could not calculate the rate of spread in western Panamá because Bd was
already present at the beginning of our sampling period. Thus, we approximated when Tún-
gara frogs were first infected based on historical reports for sites near Chiriquí [8,13,19]. Ethical approval All applicable institutional and/or national guidelines for the care and use of animals were fol-
lowed. The protocol was approved by the Institutional Animal Care and Use Committee of the
Smithsonian Tropical Research Institute (protocol number: 2011-0825-2014-02), and by the
Autoridad Nacional del Ambiente (permit numbers: SE/A-81-09, SE/A-73-10, SE/A-48-10,
SC/A-28-11, SE/A-83-11, SE/A-42-11, SE/A-30-12, SE/A-47-13, SC/A-9-14) Amphibian Chytrid Spreads across Tropical Lowlands Table 1. Bd prevalence and infection intensity per site and year in Túngara frog (Physalaemus [Engystomops] pustulosus) populations sampled in
this study (n = number of individuals sampled; Positive = number of individuals detected positive for Bd; 95% CI = 95% binomial distribution confi-
dence intervals; Average intensity = average of number of zoospore equivalents in infected frogs per population). Sites are arranged west to east by
l
it d Table 1. Bd prevalence and infection intensity per site and year in Túngara frog (Physalaemus [Engystomops] pustulosus) populations sampled in
this study (n = number of individuals sampled; Positive = number of individuals detected positive for Bd; 95% CI = 95% binomial distribution confi-
dence intervals; Average intensity = average of number of zoospore equivalents in infected frogs per population). Sites are arranged west to east by
l
it d Table 1. Bd prevalence and infection intensity per site and year in Túngara frog (Physalaemus [Engystomops] pustulosus) populations sampled in
this study (n = number of individuals sampled; Positive = number of individuals detected positive for Bd; 95% CI = 95% binomial distribution confi-
dence intervals; Average intensity = average of number of zoospore equivalents in infected frogs per population). Sites are arranged west to east by
longit de Table 1. Bd prevalence and infection intensity per site and year in Túngara frog (Physalaemus [Engystomops] pustulosus) populations sampled in
this study (n = number of individuals sampled; Positive = number of individuals detected positive for Bd; 95% CI = 95% binomial distribution confi-
dence intervals; Average intensity = average of number of zoospore equivalents in infected frogs per population). Sites are arranged west to east by
longitude Site
Year
N
Positive
Prevalence % (95% CI)
Average Intensity (± St.Dev)
Chiriquí
2010
41
11
27 (14–43)
data not available
2013
38
16
42 (26–60)
3570 (± 12454)
El Valle
2009
5
3
60 (15–95)
data not available
Gamboa
2010
321
0
0 (0–0.5)
0
2011
111
7
6 (3–13)
86 (± 117)
2012
205
26
13 (8–18)
1617 (± 5485)
2013
166
35
21 (15–29)
536 (± 2061)
2014
84
22
26 (17–37)
209 (± 833)
Summit
2010
12
2
17 (2–48)
data not available
2011
108
2
2 (0–6)
10 (± 7)
2013
120
17
14 (9–22)
89 (± 193)
Metetí
2011
91
0
0 (0–4)
0
2013
94
2
2 (0–8)
7 (± 3)
Yaviza
2013
68
0
0 (0–0.5)
0
2014
41
3
7 (2–20)
6 (± 1)
El Real
2014
40
2
5 (1–17)
6 (± 1)
Rancho Frío
2014
150
11
7 (4–13)
48 (± 101)
doi:10.1371/journal.pone.0155745.t001 The rates of spread, however, vary substantially. Specifically, our data suggest that it took
approximately one year for Bd to move the 8 km distance between Summit and Gamboa,
which are separated by the Chagres River. In contrast, the rate of Bd spread from Summit to
Metetí was 65 km/year, and 42 km/year from Metetí to Yaviza. All data are available from the DRYAD Digital Repository (doi:10.5061/dryad.6bp92). The rates of spread, however, vary substantially. Specifically, our data suggest that it took
approximately one year for Bd to move the 8 km distance between Summit and Gamboa,
which are separated by the Chagres River. In contrast, the rate of Bd spread from Summit to
Metetí was 65 km/year, and 42 km/year from Metetí to Yaviza. All data are available from the DRYAD Digital Repository (doi:10.5061/dryad.6bp92). All data are available from the DRYAD Digital Repository (doi:10.5061/dryad.6bp92). Rates of spread of Bd in Túngara frogs If Bd spread eastward from Summit in central Panamá to Darién in eastern Panamá, then the
front moved at an average rate of 54 km/year among these lowland Túngara frog populations. PLOS ONE | DOI:10.1371/journal.pone.0155745
May 13, 2016 4 / 8 PLOS ONE | DOI:10.1371/journal.pone.0155745
May 13, 2016 Amphibian Chytrid Spreads across Tropical Lowlands We cannot discern whether the source of infection of the Gamboa populations was from
Summit Túngara frogs or from other species of frogs in Gamboa. Regardless, there is a rela-
tively large time lag between when Bd was detected in Túngara frogs from Gamboa compared
to when Bd was first detected in Túngara frogs from Summit. The mechanism by which Bd
spreads is unknown, but it has been suggested that Bd could survive and could be carried in
mud, and thus easily dispersed by humans [26]. In Túngara frogs, Bd did not spread as fast as
expected from Summit to Gamboa, which are only 8 km apart and connected by a well-traveled
road and bridge over the Chagres River. Certain geographic features, like rivers, could impede the spread of Bd. The Chagres
River, which is about 100 m wide and has water all year long, separates Gamboa and Summit. Genetic studies demonstrate that the Chagres River is a geographical barrier for gene flow
between Túngara frog populations [27], thus it is possible that limited migration between
these populations could slow the spread of Bd whether Túngara frogs contracted Bd from
conspecifics or heterospecifics. Bd does seem to have spread rapidly in the lowlands towards
eastern Panamá. An alternative explanation for the spread of Bd from east to west through-
out all of Panamá is that populations in Darién, and elsewhere in far eastern Panamá, were
infected by a wave coming from the south [8]. These two scenarios should be tested through
a phylogenetic analysis of Bd throughout the Túngara frog's range in Central and South
America. As such data are currently unavailable, it is difficult to determine the recent origin
of Bd in eastern Panamá; therefore, our estimates of rate of spread should be viewed with this
potential caveat in mind. Woodhams et al. [19] conservatively estimated that Bd spread east and would have
reached Tortí in September of 2012, but [28] reported two positives out of 93 samples of
other species of frogs at this site in 2010. There are no other published reports on the pres-
ence of Bd among amphibian species from this specific area. In 2011, we sampled in Metetí,
approximately 70 km southeast of Tortí, and found this site to be Bd naive. Rebollar et al. PLOS ONE | DOI:10.1371/journal.pone.0155745
May 13, 2016 Discussion Data for the spread of Bd in the lowlands of Middle America are scarce. Here we estimated the
rate of spread of Bd in Túngara frogs based on the first detection of Bd, and we assumed that
Bd spread in a wave-like fashion [8]. In the lowlands of Panamá, Bd spread at a similar rate
among Túngara frogs (54 km/year) when compared to other amphibian species tested in this
area (30–174 km/year) [19]. There was one exception. The rate of spread from Summit across
the Chagres River to Gamboa was slower than the average (8 km/year). In western Panamá, we sampled in Chiriquí, at elevations where amphibian population
declines have been severe in the past [16]. Since Bd is now enzootic in this area [19] and at El
Valle in central Panamá, it is not surprising that the Túngara frog populations from Chiriquí
and El Valle were positive in 2010, and 2009, respectively. We also found the highest average
loads of Bd in Chiriquí (3570 z.e., Table 1). We could not estimate the rate of Bd dispersal
among Túngara frog populations between Chiriquí and El Valle as they were already infected
at the time we sampled. In 2007, Woodhams et al. [19] reported Bd positive individuals (30%
prevalence) among three species at Soberanía National Park, including the Summit area where
we sampled in 2010, and they suggested that Bd was already enzootic during their study. In the
same year, Gamboa, was still naive and we most likely sampled before Bd arrived. In 2011, we
detected Bd at Gamboa for the first time, one year earlier than previously reported for this area
[20]. 5 / 8 PLOS ONE | DOI:10.1371/journal.pone.0155745
May 13, 2016 References 1. MacPhee RDE, Greenwood AD. Infectious disease, endangerment, and extinction. Int J Evol Biol. 2013 Jan 16; 2013:e571939. 1. MacPhee RDE, Greenwood AD. Infectious disease, endangerment, and extinction. Int J Evol Biol. 2013 Jan 16; 2013:e571939. 2. McCallum H. Disease and the dynamics of extinction. Philos Trans R Soc B Biol Sci. 2012 Oct 19; 367
(1604):2828–39. 3. Smith KF, Sax DF, Lafferty KD. Evidence for the role of infectious disease in species extinction and
endangerment. Conserv Biol. 2006 Oct 1; 20(5):1349–57. PMID: 17002752 3. Smith KF, Sax DF, Lafferty KD. Evidence for the role of infectious disease in species extinction and
endangerment. Conserv Biol. 2006 Oct 1; 20(5):1349–57. PMID: 17002752 4. Collins JP, Crump ML. Extinction in Our Times: Global Amphibian Decline. Oxford University Press;
2009. 477 p. 4. Collins JP, Crump ML. Extinction in Our Times: Global Amphibian Decline. Oxford University Press;
2009. 477 p. 5. Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, McCraw SL, et al. Emerging fungal threats
to animal, plant and ecosystem health. Nature. 2012 Apr 12; 484(7393):186–94. doi: 10.1038/
nature10947 PMID: 22498624 5. Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, McCraw SL, et al. Emerging fungal threats
to animal, plant and ecosystem health. Nature. 2012 Apr 12; 484(7393):186–94. doi: 10.1038/
nature10947 PMID: 22498624 6. Brem FMR, Lips KR. Batrachochytrium dendrobatidis infection patterns among Panamanian amphib-
ian species, habitats and elevations during epizootic and enzootic stages. Dis Aquat Organ. 2008 Sep
24; 81(3):189–202. doi: 10.3354/dao01960 PMID: 18998584 6. Brem FMR, Lips KR. Batrachochytrium dendrobatidis infection patterns among Panamanian amphib-
ian species, habitats and elevations during epizootic and enzootic stages. Dis Aquat Organ. 2008 Sep
24; 81(3):189–202. doi: 10.3354/dao01960 PMID: 18998584 7. Piotrowski JS, Annis SL, Longcore JE. Physiology of Batrachochytrium dendrobatidis, a chytrid patho-
gen of amphibians. Mycologia. 2004 Jan 1; 96(1):9–15. PMID: 21148822 8. Lips KR, Diffendorfer J, Mendelson JR, Sears MW. Riding the wave: reconciling the roles of disease
and climate change in amphibian declines. PLoS Biol. 2008 Mar; 6(3):e72. doi: 10.1371/journal.pbio. 0060072 PMID: 18366257 9. Farrer RA, Weinert LA, Bielby J, Garner TWJ, Balloux F, Clare F, et al. Multiple emergences of geneti-
cally diverse amphibian-infecting chytrids include a globalized hypervirulent recombinant lineage. Proc
Natl Acad Sci. 2011 Nov 15; 108(46):18732–6. doi: 10.1073/pnas.1111915108 PMID: 22065772 10. Fisher MC, Garner TWJ, Walker SF. Global emergence of Batrachochytrium dendrobatidis and
amphibian chytridiomycosis in space, time, and host. [20] recorded Bd positive samples from Nuevo Vigía in 2012, just 26 km east of Metetí,
where we detected low Bd prevalence in 2013, thus supporting a wave-like Bd spread from
west to east in Túngara frogs. Species that carry Bd asymptomatically and share the habitat with more vulnerable species
can potentially function as Bd spreaders. Túngara frog population declines have not been
reported and were not evident during our study. If the prevalence and dispersal of Bd is den-
sity dependent, then the spread of Bd along the lowlands might be enhanced by abundant
and apparently resistant species serving as reservoirs. Túngara frogs are known to disperse
between breeding sites at distances up to 200 m [29]. They also share the habitat with a wide
variety of species; thus, they could contribute to the rapid dispersal of Bd if they are effective
carriers. Moreover, if most species in the lowlands are less susceptible to Bd or Bd is less viru-
lent [7], then the high diversity and abundance of hosts could further facilitate the dispersal
of Bd. Chytridiomycosis is linked to some of the most severe population declines and extinctions
of wildlife yet recorded. While substantial efforts have been aimed at understanding the spread
and pathogenicity in the more vulnerable highland frog species, we know relatively little about
the dynamics of Bd in tropical lowland regions of the world. Lowland species, however, could
be reservoirs and dispersal agents between areas where amphibian species are more vulnerable
to Bd infection. As highlighted by our results, even though lowland regions are typically char-
acterized by less favorable climatic conditions for Bd [7], by harboring asymptomatic Bd infec-
tions, lowland amphibian populations could potentially play an important role in the spread of
Bd across tropical regions. 6 / 8 PLOS ONE | DOI:10.1371/journal.pone.0155745
May 13, 2016 Amphibian Chytrid Spreads across Tropical Lowlands Author Contributions Conceived and designed the experiments: SRB MJR. Performed the experiments: SRB MJR RI
DR. Analyzed the data: SRB DR RI. Contributed reagents/materials/analysis tools: SRB MJR
DR RI. Wrote the paper: SRB MJR DR RI. Acknowledgments For help in the field we thank Emma and Lucy Ryan, Tony Alexander, Alex Jordan, Meghan
Still, Ty Hoskin, Mahudy Díaz, Samuel Sucre, and for logistical support we thank SENA-
FRONT, ANAM, and STRI. We thank Teofil Nakov and Robert Puschendorf for comments on
the manuscript. We also thank Michael R.J. Forstner at Texas State University for instrumenta-
tion support, and Mahudy Díaz at STRI for laboratory assistance. PLOS ONE | DOI:10.1371/journal.pone.0155745
May 13, 2016 References Annu Rev Microbiol. 2009; 63(1):291–310. 11. James TY, Litvintseva AP, Vilgalys R, Morgan JAT, Taylor JW, Fisher MC, et al. Rapid global expan-
sion of the fungal disease chytridiomycosis into declining and healthy amphibian populations. PLoS
Pathog. 2009 May 29; 5(5):e1000458. doi: 10.1371/journal.ppat.1000458 PMID: 19478871 12. Velo-Antón G, Rodríguez D, Savage AE, Parra-Olea G, Lips KR, Zamudio KR. Amphibian-killing fun-
gus loses genetic diversity as it spreads across the New World. Biol Conserv. 2012 Feb; 146(1):213–8. 13. Phillips BL, Puschendorf R. Do pathogens become more virulent as they spread? Evidence from the
amphibian declines in Central America. Proc R Soc B Biol Sci [Internet]. 2013 Sep 7 [cited 2013 Jul 29];
280(1766). Available: http://rspb.royalsocietypublishing.org/content/280/1766/20131290 14. Pounds JA, Crump ML. Amphibian declines and climate disturbance: the case of the Golden Toad and
the Harlequin Frog. Conserv Biol. 1994 Mar 1; 8(1):72–85. 15. Pounds JA, Fogden MPL, Savage JM, Gorman GC. Tests of null models for amphibian declines on a
tropical mountain. Conserv Biol. 1997; 11(6):1307–22. 16. Lips KR. Mass mortality and population declines of anurans at an upland site in western Panama. Con-
serv Biol. 1999; 13(1):117–25. 17. Lips KR. Decline of a tropical montane amphibian fauna. Conserv Biol. 1998; 12(1):106–17. 7 / 8 PLOS ONE | DOI:10.1371/journal.pone.0155745
May 13, 2016 Amphibian Chytrid Spreads across Tropical Lowlands 18. Lips KR, Brem F, Brenes R, Reeve JD, Alford RA, Voyles J, et al. Emerging infectious disease and the
loss of biodiversity in a neotropical amphibian community. Proc Natl Acad Sci U S A. 2006 Feb 28; 103
(9):3165–70. PMID: 16481617 19. Woodhams DC, Kilburn VL, Reinert LK, Voyles J, Medina D, Ibáñez R, et al. Chytridiomycosis and
amphibian population declines continue to spread eastward in Panama. EcoHealth. 2008 Sep 1; 5
(3):268–74. doi: 10.1007/s10393-008-0190-0 PMID: 18807089 20. Rebollar EA, Hughey MC, Harris RN, Domangue RJ, Medina D, Ibáñez R, et al. The lethal fungus
Batrachochytrium dendrobatidis is present in lowland tropical forests of far eastern Panamá. PLoS
ONE. 2014 Apr 16; 9(4):e95484. doi: 10.1371/journal.pone.0095484 PMID: 24740162 21. Hyatt A, Boyle D, Olsen V, Boyle D, Berger L, Obendorf D, et al. Diagnostic assays and sampling proto-
cols for the detection of Batrachochytrium dendrobatidis. Dis Aquat Organ. 2007; 73:175–92. PMID:
17330737 22. Sluys MV, Kriger KM, Phillott AD, Campbell R, Skerratt LF, Hero JM. PLOS ONE | DOI:10.1371/journal.pone.0155745
May 13, 2016 References Storage of samples at high tem-
peratures reduces the amount of amphibian chytrid fungus Batrachochytrium dendrobatidis DNA
detectable by PCR assay. Dis Aquat Organ. 2008 Aug 27; 81(2):93–7. doi: 10.3354/dao01953 PMID:
18924373 23. Boyle D, Boyle D, Olsen V, Morgan J, Hyatt A. Rapid quantitative detection of chytridiomycosis (Batra-
chochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay. Dis Aquat
Organ. 2004; 60:141–8. PMID: 15460858 24. Kriger KM, Hero J, Ashton KJ. Cost efficiency in the detection of chytridiomycosis using PCR assay. Dis Aquat Organ. 2006 Jul 25; 71(2):149–54. PMID: 16956062 25. Skerratt LF, Berger L, Hines HB, McDonald KR, Mendez D, Speare R. Survey protocol for detecting
chytridiomycosis in all Australian frog populations. Dis Aquat Organ. 2008 Jul 7; 80(2):85–94. doi: 10. 3354/dao01923 PMID: 18717061 26. Johnson ML, Speare R. Possible modes of dissemination of the amphibian chytrid Batrachochytrium
dendrobatidis in the environment. Dis Aquat Organ. 2005 Jul 18; 65(3):181–6. PMID: 16119886 27. Lampert KP, Rand AS, Mueller UG, Ryan MJ. Fine-scale genetic pattern and evidence for sex-biased
dispersal in the túngara frog, Physalaemus pustulosus. Mol Ecol. 2003 Dec 1; 12(12):3325–34. PMID:
14629349 28. Küng D, Bigler L, Davis LR, Gratwicke B, Griffith E, Woodhams DC. Stability of microbiota facilitated by
host immune regulation: informing probiotic strategies to manage amphibian disease. PLoS ONE. 2014
Jan 29; 9(1):e87101. doi: 10.1371/journal.pone.0087101 PMID: 24489847 29. Marsh DM, Fegraus EH, Harrison S. Effects of breeding pond isolation on the spatial and temporal
dynamics of pond use by the Tungara frog, Physalaemus pustulosus. J Anim Ecol. 1999 Jul 1; 68
(4):804–14. 8 / 8
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https://openalex.org/W4200093992
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https://zenodo.org/records/5825210/files/Improvement%20of%20test%20methods%20and%20criteria%20for%20evaluation%20of%20resistance%20to%20flame%20propagation%20of%20long%20elements%20of%20the%20wiring%20system.pdf
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Improvement of test methods and criteria for evaluation of resistance to flame propagation of long elements of the wiring system
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Eastern-European journal of enterprise technologies
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cc-by
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IMPROVEMENT OF TEST
METHODS AND CRITERIA
FOR EVALUATION OF
RESISTANCE TO FLAME
PROPAGATION OF LONG
ELEMENTS OF THE
WIRING SYSTEM Using a substrate of a wooden board covered
with a layer of tissue paper with an areal density of
(21±9) g/m2, adopted for testing other elements of the
electrical wiring system, a cable was identified that is
not resistant to flame propagation. It is proposed to
use this substrate for testing the flame propagation of
cables instead of a substrate made of a double layer of
filter paper with a surface density of (80±15) g/m2. In one of three experiments, a cable that was not
resistant to flame propagation was found based on the
criterion of the presence of ignition of the substrate
located under it. To reduce the risk of making an
incorrect decision on compliance, it is proposed that
the assessment of long elements of the wiring system be
carried out according to the rules established for cable
ducts, trays and ladders in EN 50085-1 and IEC 61537. R o s t y s l a v K r a v c h e n k o
Head of Department
Department of Technical Regulation
Center of Research in Fire Protection**
P a v l o I l l i u c h e n k o
Corresponding author
Head of Department*
E-mail: illuchenko@ukr.net
A n d r i i O n y s h c h u k
Junior Researcher*
O l e k s a n d r Z a z y m k o
Researcher*
*Department of Electrical Products
Research and Testing Center**
**Institute of Public Administration
and Research in Civil Protection
Vyshhorodska str., 21, Kyiv, Ukraine, 04074 For the AVVG cable with an outer diameter of 10 mm
to 60 mm, when it touches the blue flame cone of 1 kW,
the correlation coefficient of the dependence of the
length of the charred part on the diameter was 0.969. For
a distance of 100 mm between the sample and the burner
along its axis, a correlation coefficient of 0.985 was
obtained. It is proposed to test cables under the second
condition recommended in IEC 60695-11-2. For two conduits, flame propagation was revealed
when exposed to a 1 kW flame for 120 s and 240 s. However, for these conduits, flame propagation did
not occur under standard conditions of exposure to
such a flame for 20 s and 25 s. IMPROVEMENT OF TEST
METHODS AND CRITERIA
FOR EVALUATION OF
RESISTANCE TO FLAME
PROPAGATION OF LONG
ELEMENTS OF THE
WIRING SYSTEM To identify long elements
of the wiring system that are not resistant to flame
propagation, it is proposed to test them at a duration
of exposure to a flame of 1 kW, established for cables
in IEC 60332-1-2 Keywords: electrical product, electrical and
optical cable, conduit, fire safety, flame propagation,
wiring system Received date 26.10.2021
Accepted date 01.12.2021
Published date 29.12.2021 How to Cite: Kravchenko, R., Illiuchenko, P., Onyshchuk, A., Zazymko, O. (2021). Improvement of test methods and criteria for
evaluation of resistance to flame spread of long elements of the wiring system. Eastern-European Journal of Enterprise Technolo
gies, 6 (10 (114)), 57–68. doi: https://doi.org/10.15587/1729-4061.2021.249105 How to Cite: Kravchenko, R., Illiuchenko, P., Onyshchuk, A., Zazymko, O. (2021). Improvement of test methods and criteria for
evaluation of resistance to flame spread of long elements of the wiring system. Eastern-European Journal of Enterprise Technolo
gies, 6 (10 (114)), 57–68. doi: https://doi.org/10.15587/1729-4061.2021.249105 Ecology Ecology UDC 614.841.41 The test methods for flame propagation of long
elements of the electrical wiring system, in particular,
cables, cable conduits and ducts, are analyzed, and
differences in them are found in the test conditions
and criteria for evaluating the resistance to flame
propagation. DOI: 10.15587/1729-4061.2021.249105 2. Literature review and problem statement In [3], a new model was developed for the rate of heat re
lease during combustion of a horizontally located cable tray
located at the side wall. The resulting model makes it possi
ble to reduce the error in determining the peak rate of heat
release during the combustion of the cable tray. However, it
is advisable to identify discrepancies in the results obtained
by the developed model and standardized test methods, for
example, using the Steiner tunnel. The European Committee for Electrotechnical Stan
dardization (CENELEC) for the method [13] has defined
an additional criterion for assessing the length of the carbon
part of the cable [17]. However, with the introduction of an
additional classification criterion [8], other important cri
teria for assessing the length of the carbon part of the cable
were excluded. Also CENELEC for method [13] has improved the re
quirements for test equipment to ensure constant exposure
to a flame source for a specified duration of its applica
tion [18]. At the same time, such an improvement has not
been proposed for the method [14]. An extended model for estimating the speed of flame
propagation during combustion of a vertically located ca
ble tray, depending on the distance between the cables, is
proposed in [4]. However, it is desirable to investigate the
change in flame propagation speed for cables placed on a
cable ladder. Testing of cables on cable ladders is accepted by
international and European standards. The research results presented in [19] established a sig
nificant variance in the test results according to the meth
od [13] of samples of identical cables obtained in different
laboratories. The author notes that if the obtained length of
the charred part of the sample is more than 152 mm, the test
ing laboratory runs the risk of making an incorrect decision
on the conformity of the cables. However, the factors that
influenced the reproducibility of the comparative test results
have not been identified. The work [5] presents the results of studies of the effect
of the distance between cables and their layers on the height
and width of the flame when burning cables laid in the tun
nel. The paper also presents data on the speed of horizontal
flame propagation along the cables. At the same time, it is ad
visable to determine the dependence of the horizontal flame
propagation on the transverse dimensions of the cables. 2. Literature review and problem statement When using a 1 kW flame source, the International Elec
trotechnical Commission has developed methods for testing
the flame propagation of other long elements of the wiring
system [20–24]. At the same time, these methods differ
in terms of test conditions and criteria for evaluating the
results, which can influence the decision on the conformity
of such elements. This applies to standardized methods for
testing cables with a 1 kW flame. The results of studies of the influence of the angle of
inclination of the cable (0–20°) laid in the tunnel and the
speed of longitudinal air flows (0.35–0.65 m/s) on the heat
ing temperature of the tunnel ceiling from a burning cable
are presented in [6]. But the work does not provide import
ant data on the influence of these parameters on the speed of
propagation of the flame of the cables. In order to reduce the risk of fire in buildings associated
with cables, methods for testing cables laid in bundles for
flame propagation have been improved [7]. These methods
make it possible to additionally determine the indicators of
heat release and smoke production. On the basis of these and
other methods, a new classification of cables for reaction to
fire has been developed. This classification is implemented
in EN 13501-6 [8] to fulfill the requirements of Regula
tion (EU) No 305/2011 [9]. At the same time, the disadvan
tage of this classification is not taking into account all the
criteria for assessing the resistance to flame propagation of
single-laid cables. The lack of an estimate for the duration of
self-combustion and the flammable ability of particles sepa
rated from single laid cables can affect their classification. It should be noted that in practice, standardized test meth
ods for products compared to non-standardized ones are those,
based on the results of which, a decision is made on its compli
ance and use at facilities. The perfection of these methods is an
important influential factor in ensuring the fire safety of both
the product itself and the facilities on which it is used. At the same time, for economic reasons, improvements
in such methods, especially in terms of significant changes
in the design of test equipment, are slowly being introduced
into practice. The complex of factors influencing the assessment of
the fire hazard of electrical products is defined in [2]. 2. Literature review and problem statement Their
influence on the assessment of resistance to flame propaga
tion of long elements of the wiring system has not yet been
adequately studied. To reduce the cost of testing cables of the same brand
with different transverse dimensions, a procedure for ex
tended application of the results for fiber-optic cables has
been developed [10]. Such a procedure has been proposed
for the classification of electrical cables according to their
reaction to fire. All this leads to research on the resistance to flame
propagation of long elements of the electrical wiring system
under conditions of exposure to a 1 kW flame source. At the same time, work [11] did not find a clear relation
ship between fire hazard indicators and the main parameter
χ of the extended application procedure for electrical cables. Therefore, further research is advisable to improve the pro
cedure for the extended application of cable test results. 1. Introduction At the same time, the distance between
the torch and the sample was not given in accordance with
the distance recommended by the basic IEC standard. 1. Introduction ance of these systems with fire safety requirements ensures
compliance with such requirements of each of their elements. Electrical wiring systems are elements of power supply,
power supply and control systems for various equipment and
communication systems in buildings and vehicles. Fires as
sociated with electrical wiring systems can spread the flame
over a considerable distance from the source of the fire. In
the process of their combustion, smoke, toxic and corrosive
volatile combustion products can be released in a dangerous
amount for people and property. Under certain conditions,
the development of a fire from electrical wiring systems led
to catastrophic consequences. In electrical wiring systems, potentially fire hazardous
elements are electrical and optical cables and wires (hereinaf
ter referred to as cables), conduits, trunkings, ductings, trays,
ladders, liquid tight sheathings and powertracks. For long elements of the electrical wiring system, one of
the measures to reduce the emission of hazardous volatile
combustion products is to limit the spread of the flame. Considering that fire hazard tests of long elements of
the wiring system are performed separately, their test con
ditions, criteria for evaluating the results, as well as the
rules for making a decision on compliance should not differ
significantly. The main preventive measure to reduce the risk of fire
from electrical products is the fire hazard test [1]. The prior
ity is the testing of finished products [2]. At the same time, in standard methods, the specified
characteristics, on which the results of assessing the resis
tance to flame propagation of long elements of the wiring In practice, fire hazard testing of all combinations of
wiring systems is problematic. In this regard, the compli 57 s is an open access article under the Creative Commons CC BY license 6/10 ( 114 ) 2021 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 system depend, are different, therefore, studies to determine
the characteristics that significantly affect such an assess
ment are relevant. part of samples [13] and the incendiary ability of particles
separated from them [14]. For these methods, the procedure
for measuring the outer diameter of the samples was deter
mined and the requirements for the duration of the applica
tion of a flame source of 1 kW to samples of flat cables were
improved [15, 16]. 3. The aim and objectives of research The aim of research is to ensure the fire safety of facilities
by improving test methods and criteria for assessing the re
sistance to flame propagation of long elements of the wiring
system. This will help to reduce the number of people killed
and injured and the amount of damage from fires. To meet the requirements of technical regulations [9, 12],
The International Electrotechnical Commission (IEC) has
developed methods for determining the length of the charred 58 Ecology To achieve the aim, the following objectives were set: flame propagation of the cable, a test method was applied ac
cording to IEC 60332-1-3 [14, 16]. During the experiments,
let’s additionally use a base made of a white pinewood board
0
25
700−
mm long,
0
25
300−
mm wide and 10 mm thick, covered
with tissue paper with an areal density of (21±9) g/m2. – to analyze the standardized test methods for flame
propagation of long elements of the wiring system; – to determine the influence of the characteristics of the
bases, placed under the sample of long elements of the wiring
system, on their flammability; The distance between the base and the point of applica
tion of the 1 kW flame was 200 mm. – to determine the influence of the characteristics of the
base, placed under the sample of long elements of the wiring
system, on the assessment of resistance to the propagation of
flame of the cable; Samples for each experiment were six lengths of 4×240
AVVG cable, 54.6 mm in diameter each. The duration of the application of the test flame to the
cable samples was 240 s. – to determine the influence of the duration of appli
cation of the flame source to the sample and the distance
between the burner and the sample on the assessment of the
resistance to flame propagation of cables; In the course of the experiments, the length of the
charred part and the duration of self-combustion of the sam
ples were additionally determined. – to determine the effect of the duration of application of
the flame source to the sample on the assessment of resistance
to flame propagation of cable conduits trunkings and ductings. 4. 4. Methodology for studying the influence of flame
application conditions on the assessment of resistance to
flame propagation of cables To study the influence of the duration of application of
the flame source to the sample and the distance between
the burner and the sample on the assessment of resistance
to flame propagation of cables, test methods and criteria
for evaluating the results were used according to stan
dards [13–17]. 4. 1. Experimental conditions
h
h d
f Research methods of testing and criteria for assessing
the resistance to flame propagation of long elements of the
wiring system [8, 13–17, 20–24] were carried out using ana
lytical and experimental methods. Additional experiments were carried out on the distance
between the burner and the sample, which is measured along
the axis of the burner, equal to 100 mm. To eliminate the influence on the results of experimental
studies of the conditions of air conditioning and the environ
ment and the size of the samples: In all experiments, the distance between the base and the
point of application of the 1 kW flame was 200 mm. The samples for the experiments were six cable sections
AVVG 4×2.5, AVVG 4×16, AVVG 4×35, AVVG 4×120 and
AVVG 4×240 with diameters of 10 mm, 19 mm, 26 mm,
42 mm, and 61 mm. 1) samples and bases for assessing the inflammatory
capacity of particles separated from them were kept for at
least 48 hours at a temperature of (23±2) °С and a relative
humidity of (50±10) %; In the course of the experiments, the duration of self-com
bustion of the samples was additionally determined. 2) the experiments were carried out at a temperature
of (23±2) °С, a relative air humidity of no more than 75 %
and an air flow rate of no more than 0.1 m/s; 2) the experiments were carried out at a temperature
of (23±2) °С, a relative air humidity of no more than 75 %
and an air flow rate of no more than 0.1 m/s; 3) samples of length
25
0
600+
mm were subjected to tests. 4. 5. Methods for studying the effect of the duration
of the application of the flame on the assessment of
resistance to flame propagation of cable conduitstrunk
ings and ductings 3) samples of length
25
0
600+
mm were subjected to tests. 4. 2. Methodology for studying the effect of the char
acteristics of bases on their flammability To study the influence of the characteristics of the bases
placed under the sample of the long elements of the electrical
wiring system on their flammability, two bases were used. To study the effect of the duration of the application of
the flame source to the sample on the assessment of resis
tance to flame propagation of cable conduits trunkings and
ductings, test methods and criteria for evaluating the results
were used in accordance with the standards [21, 23]. The first base consisted of a white pinewood board
0
25
700−
mm long,
0
25
300−
mm wide and 10 mm thick, covered
with tissue paper with an areal density of (21±9) g/m2. Additionally, experiments were carried out on the du
ration of the application of the flame source, established for
cables [13]. The second base was a double layer of filter paper with an
areal density of (80±15) g/m and a square shape with a side
of (300±10) mm. The samples for the experiments were: The samples for the experiments were: The source of the formation of burning drops was a tele
phone wire with polyethylene insulation TRP 2×0.5. a) six segments: a) six segments: 1) corrugated cable conduit made of polyamide with an
outer diameter of 16 mm and a wall thickness of 0.5 mm; The bases were placed under a horizontally located wire
at distances of 150 mm, 200 mm and 600 mm. 2) corrugated polypropylene cable conduit with an outer
diameter of 50 mm and a wall thickness of 0.8 mm; Five experiments were performed at each distance. h
h
b
f d
h h 3) rigid PVC cable conduit with an outer diameter of
52 mm and a wall thickness of 4.1 mm; During the experiments, the number of drops at which the
base ignited was recorded, and the time from the beginning of
the fall of the first burning drop until the base ignited. 4) corrugated cable conduits made of polyethylene with
an outer diameter of 62.5 mm and a wall thickness of 1.4 mm;
b) three segments: 5. Results of experimental studies on resistance to flame
propagation of long elements of the wiring system ditioning of samples are specified compared to other long
elements of the electrical wiring system. The temperature
and relative humidity of the air of such elements is consis
tent with the conditions for the conditioning of electrical
products [25, 26]. 5. 1. The results of the analysis of standardized test
methods for flame propagation of long elements of the
wiring system The duration of conditioning samples of cable conduits,
liquid tight sheathings and cable trunkings, ductings, trays,
ladders (Table 1), respectively, is 5 and 3.5 times longer than
the duration of conditioning samples of electrical products
established in the fundamental standards [25, 26]. For ca
bles, this duration is 2 times less. Tables 1, 2 give the characteristics of test methods and
criteria for assessing the resistance to flame propagation of
long elements of the wiring system. 5. Results of experimental studies on resistance to flame
propagation of long elements of the wiring system According to the Table 1, for cable conduits and liquid
tight sheathings, more stringent conditions for the con Table 1
Characteristics of test methods and criteria for evaluating the resistance to flame propagation of
long elements of the wiring system
Characteristics
Value or description
Cables [13–17]
Powertracks
[20]
Cable trunk
ings, ductings,
trays, ladders
[21, 22]
Cable
conduits
[23]
liquid
tight
sheath
ings [24]
Sample length (E*), mm
600±25
675±10
Conditioning conditions for samples
(23±5) °С;
(50±20) %;
≥16 h
not defined
≥168 h after
manufactur
ing
(23±2) °С;
(50±10) %;
≥240 h
Conditioning conditions of the base to determine
the flammability of particles separated from the
sample
(23±2) °С;
(50±10) %;
≥4 h
not defined
Temperature during testing, °С
23±10
20±5
Chamber volume for protection against air flows, m3
≥1
not used
Dimensions of the metal fence, mm×mm×mm
(1200±25)×
×(300±25)×(450±25)
(
)
+25
+25
0
0
1300
25
700
450
×
×
±
×
(
)
+25
+25
0
0
1300
25
300
450
±
×
×
×
Clamp width (support) (A*), mm
not defined (≈25 mm accord�
ing to IEC 332-1:1979)
25
Distance between clamps (resistors) (D*), mm
550±5
550±10
Distance between the lower clamp and the point of
application of the flame source to the sample (C*), mm
75±10
100±5
Distance between the upper edge of the lower
clamp and the bottom of the guard (F*), mm
≈75
500±10;
550±10 (for trays and ladders)
Width of white pine board covered
with tissue paper, mm
not used
0
25
700
0
25
300
Distance between burner and sample (B*), mm
62±16
(blue cone height)
100±10
Duration of application of the flame source
to the sample, s
t±2 (Table 2)
60±2
+1
0t
(Table 2)
Distance between the point of application of the
flame source to the sample and the upper limit
of the rounded zone of the sample (L1), mm
<425±5 1)
<400±15
Distance between the point of application of the
flame source to the sample and the lower boundary
of the carbon zone of the sample (L2), mm
≤65±5 1)
not defined
<50±5
Length of the carbon part of the sample (L3), mm
≤425 2)
not defined
Standard base condition
double layer of filter paper 3)
does not ignite 1)
tissue paper 4) does not ignite
and the wood board is not
heated
the tissue paper
does not ignite
Duration of self-combustion of the sample (taf), s
not defined
≤30
The number of samples that must withstand the
test, pcs. 4. 3. Methodology for studying the influence of the
characteristics of the bases on the assessment of resis
tance to the flame propagation of the cable 4. 3. Methodology for studying the influence of the
characteristics of the bases on the assessment of resis
tance to the flame propagation of the cable 1) blind cable ducting with transverse dimensions
50×20 mm, complying with EN 50085-1 [21]; 2) cable
trunking
with
transverse
dimensions
60×40 mm, which complies with EN 50085-1 [21]. To study the influence of the characteristics of the foun
dations under the sample on the assessment of resistance to 59 6/10 ( 114 ) 2021 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 5. Results of experimental studies on resistance to flame
propagation of long elements of the wiring system 5. Results of experimental studies on resistance to flame
propagation of long elements of the wiring system 1 or 2
additional
1 or 12
additional
1 or 3 or 2
additional
3
*See Fig. 1:
1) The criterion does not apply for cables covered by EN 13501-6 [8]. 2) The criterion applies only to cables that are subject to EN 13501-6 [8]. 3) Filter paper with a basis weight of (80±15) g/m2 and a square shape with a side of (300±10) mm. 4) Packing with surface density (21±9) g/m2. Characteristics of test methods and criteria for evaluating the resistance to flame propagation of
long elements of the wiring system 60 Ecology Table 2 Table 2 Table 2
Duration of application of a flame source of 1 kW to samples of long elements of the electrical wiring system
Cables [12, 13]
Cable conduits, liquid
tight sheathings [22, 23]
Outer diameter, mm
t, s
Wall thick
ness, mm
t, s
more
no more than
more
no
more
than
–
25
60
–
0.5
15
0.5
1.0
20
1.0
1.5
25
1.5
2.0
35
2.0
2.5
45
2.5
3.0
55
3.0
3.5
65
3.5
4.0
75
4.0
4.5
85
25
50
120
4.5
5.0
130
50
75
240
5.0
5.5
200
5.5
6.5
300
75
–
480
6.5
–
500
* For cables with a non-circular cross-section in which the ratio between the length of the longer and shorter axis
does not exceed 3, the nominal value of the length of the shorter axis is chosen as the outer diameter. For cables with
a non-circular cross-section, in which the ratio between the length of the longer and shorter axes is in the range from
3 to 16, the sum of the lengths of the longer and shorter axes, divided by 3.14 (π), is chosen as the outer diameter. For cables in which the ratio between the longer and shorter lengths exceeds 16, the test criteria are defined in the
product standards, and if they are not available, in the agreement between the manufacturer and the buyer. –
Tissue paper
Flame
Back
wall
Steel bar (for
conduits and liquid
tight sheathings)
Clamp
Sample located in the
center of the
horizontal plane
White pinewood board
10
45° ± 2°
100 ± 10
F
A
A
D
E
0
25
450
Fig. 1. Table 3 For all long elements of the wiring system, the distance
between the clamps is 550 mm (Table 1). Taking into ac
count the width of the clamps, a specimen length of 600 mm
is sufficient. Experimental data on the inflammatory capacity of
standard bases
Basis character
istics
Distance
between
cable and
base, m
The number of
burning parti
cles at which the
base ignited *
Duration from the
beginning of the
fall of the burning
particles to the base
ignition*, s
Double layer of
filter paper
150
6
3.7
200
7
5.5
600
9
7.9
White pinewood
board covered
with a layer of
tissue paper
150
2
1.1
200
2
1.5
600
3
2.6
*Average value over five experiments. Experimental data on the inflammatory capacity of
standard bases According to the Table 1, the distance between the
torch and the sample, which is not recommended in
IEC 60695-11-2 [27], is set for cables to ensure reproducibil
ity of results. The results of the study on the influence of this
factor on the assessment of resistance to flame propagation
of cables are presented in clause 5. 4. According to the Tables 1, 2 for long elements of the
wiring system, different durations of application of the flame
source are determined. The results of the study on the influ
ence of this factor on the assessment of resistance to flame
propagation of cables, cable conduits trunkings and ductings
are presented in clauses 5. 4 and 5. 5. For cables other than those intended for use in buildings,
the permissible length of the charred part of the sample can
be in the range from 425 to 490 mm (Table 1). At the same
time, for cables intended for use in buildings, the spread of
the flame downward from the point of application of the
flame source is not limited. The research results presented in Table 3 prove that: 1) standard white pinewood board base covered with a
layer of tissue paper is more flammable than a double layer
of filter paper; For other long elements of the wiring system, a stricter
permissible flame spread of 400 mm upward from the point
of application of the flame source is set than for cables. Table 3 2) ability of standard bases to ignite does not signifi
cantly decrease with an increase in the distance between
them and the source of the formation of burning particles
according to the distance between the source of formation
of burning particles and the base in the range from 150 mm
to 600 mm. A stricter permissible level of flame spread of 50 mm
downward from the point of application of the flame
source is established for liquid tight sheathings. When
this level is selected for all elements of the electrical
wiring system, with the permissible length of the carbon
part of 425 mm, there is an admissible level of flame prop
agation upward from the point of application of the flame
source of 375 mm. Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 Regarding the conditioning of the bases for assessing
the inflammatory capacity of particles separated from the
samples, such conditions are determined only for cables (Ta
ble 1). Moreover, such conditions for the base are not consis
tent with the conditions of sample conditioning. According to the Table 1 rules for deciding on the com
pliance of all three samples for cable conduits and liquid
tight sheathings with more stringent ones. Less stringent
rules are established for powertracks when a decision on
the test data of one or an additional sample is sufficient. At
the same time, more samples have been identified for cable
trunkings, ductings, trays and ladders for testing, which
contribute to obtaining more reliable results. To prevent the influence of extraneous air currents on
the combustion process, the cables are tested in a protective
chamber (Table 1). Also for these purposes, such a chamber
is used to test other electrical products in accordance with
fundamental standards [25, 26]. No such chamber is provid
ed for testing powertracks, cable trunkings, ductings, trays,
ladders and liquid tight sheathings. 5. 2. Influence of the characteristics of the bases
placed under the sample of long elements of the electrical
wiring system on their flammability 5. 2. Influence of the characteristics of the bases
placed under the sample of long elements of the electrical
wiring system on their flammability When testing all elements of the wiring system, the total
ambient temperature is (23±2) °С. At this temperature, it
is customary to test electrical products in accordance with
standards [25, 26]. According to these standards, the rela
tive humidity should not exceed 75 %. The data of experimental studies on the ability to ignite
standard bases for determining the igniting ability of parti
cles (drops) separated from a burning cable TRP 2×0.5 are
presented in Table 3. 5. Results of experimental studies on resistance to flame
propagation of long elements of the wiring system Scheme of testing long elements of the wiring system for flame propagation (legend – according to Table 1) Duration of application of a flame source of 1 kW to samples of long elements of the electrical wiring system Duration of application of a flame source of 1 kW to samples of long elements of the electrical wiring system * For cables with a non-circular cross-section in which the ratio between the length of the longer and shorter axis
does not exceed 3, the nominal value of the length of the shorter axis is chosen as the outer diameter. For cables with
a non-circular cross-section, in which the ratio between the length of the longer and shorter axes is in the range from
3 to 16, the sum of the lengths of the longer and shorter axes, divided by 3.14 (π), is chosen as the outer diameter. For cables in which the ratio between the longer and shorter lengths exceeds 16, the test criteria are defined in the
product standards, and if they are not available, in the agreement between the manufacturer and the buyer. Tissue paper
Flame
Back
wall
Steel bar (for
conduits and liquid
tight sheathings)
Clamp
Sample located in the
center of the
horizontal plane
White pinewood board
10
45° ± 2°
100 ± 10
F
A
A
D
E
0
25
450
Fig. 1. Scheme of testing long elements of the wiring system for flame propagation (legend – according to Table 1) Tissue paper
Flame
Back
wall
Steel bar (for
conduits and liquid
tight sheathings)
Clamp
Sample located in the
center of the
horizontal plane
White pinewood board
10
45° ± 2°
100 ± 10
F
A
A
D
E
0
25
450
Fig. 1. Scheme of testing long elements of the wiring system for flame propagation (legend – according to Table 1) Sample located in the
center of the
horizontal plane 100 ± 10 Flame Back
wall White pinewood board Tissue paper . 1. Scheme of testing long elements of the wiring system for flame propagation (legend – according to Table 1) 61 61 6/10 ( 114 ) 2021 6/10 ( 114 ) 2021 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 Table 4 5. 4. Influence of the conditions of use of a flame
source on the assessment of resistance to flame propaga
tion of cables The data of experimental studies on the influence of
the duration of the application of the flame source to the
sample and the distance between the burner and the sample
to assess the resistance to flame propagation of cables are
presented in Table 5. The duration of the application of the flame source to the
sample increases with an increase in the outer diameter of the
cable (Table 2). But it is constant for certain ranges of the
outer diameter of the cable. Therefore, to analyze the results, it
is better to consider the curves of the dependence of the resis
tance to flame propagation on the outer diameter of the cable. According to the Table 5, linear dependences of the aver
age values of the length of the charred part and the duration
of self-combustion of cables on their outer diameter (d) are
plotted, which are shown in Fig. 2, 3. 5. 3. Influence of the characteristics of the bases un
der the sample on the assessment of resistance to flame
propagation of the cable The data of experimental studies on the influence of the
characteristics of the bases under the sample on the assess
ment of resistance to flame propagation of the AVVG 4×240
cable are presented in Table 4. For cables and other elements of the electrical wiring
system, various bases have been determined for assess
ing the flammability of particles separated from samples
and (Table 1). Also, different distances are set between
the point of application of the ignition source and the base. The results of the study on the influence of these factors
on the flammability of the bases and the assessment of the
resistance to flame propagation of cables are presented in
clauses 5. 2 and 5. 3. The research results presented in Table 4 prove that
l
f
dd
l
f 1) application of an additional criterion for assessing re
sistance to flame propagation – the flammability of particles
separated from the sample increases the efficiency of detect
ing unsuitable cables; 2) standard white pinewood board backing covered with
a layer of tissue paper is more effective in locating unsuitable
cables than a double layer of filter paper; 2) standard white pinewood board backing covered with
a layer of tissue paper is more effective in locating unsuitable
cables than a double layer of filter paper; For long elements of the wiring system, in addition to
cables, such a criterion as the duration of self-combustion
of samples, not exceeding 30 s is used. To obtain additional
data on the fire hazardous properties of cables, such a crite
rion was applied during experimental studies. 3) assessment of the resistance of cables to flame propa
gation based on test data from only one sample may lead to
an incorrect decision on the conformity of the cables. 3) assessment of the resistance of cables to flame propa
gation based on test data from only one sample may lead to
an incorrect decision on the conformity of the cables. 5. 3. Influence of the characteristics of the bases un
der the sample on the assessment of resistance to flame
propagation of the cable 62 Ecology Table 4
Data of experimental studies on resistance to flame
propagation of AVVG 4×240 cable samples using various
standard bases
Sample number
L1/L2/L3, mm
taf, s
Combustion of the
standard base placed
under the sample
Double layer of filter paper
1
166/7/173
15
no
2
175/8/183
19
no
3
176/11/187
27
yes
White pinewood board covered with a layer of tissue paper
4
158/9/167
10
yes
5
170/7/177
26
yes
6
172/10/182
20
yes
Note: Legend – according to Table 1 Table 4 The convergence of the results of the duration of
self-combustion of the samples was influenced by the prop
erties of the insulating materials placed under the shell,
and the size of the zones of oxygen access to the place of
combustion of such materials formed as a result of damage
1
2
3
4
5
AVVG 4×240 cable
Blue cone of flame touches the sample surface
1
240
131/10/141
20.0
no
2
240
145/15/160
25.4
no
3
240
135/15/150
21.2
no
The distance between the torch and the sample, measured along the torch axis – 100 mm
4
240
151/11/162
25.8
no
5
240
155/15/170
24.2
no
6
240
148/12/160
21.4
no
Note: Legend – according to Table 1
Continuation of Table 5
2
1
L3 = 1,339d + 74
r = 0,969
L3 = 1,113d + 96,5
r = 0,985
0
20
40
60
80
100
120
140
160
180
0
10
20
30
40
50
60
70
L3, mm
d, mm
Fig. 2. Linear dependences of the length of the charred part of the cables
on their outer diameter (d): 1 – distance between the torch and the sample,
measured along the torch axis – 100 mm; 2 – blue cone of flame touches the
sample surface; r – the correlation coefficient
2
1
taf = 0,352d - 4,36
r = 0,801
taf = 0,426d - 1,985
r = 0,983
-5
0
5
10
15
20
25
30
0
10
20
30
40
50
60
70
taf, s
d, mm
Fig. 3. Linear dependences of the duration of self-combustion cables on
their outer diameter (d): 1 – distance between the burner and the sample,
measured along the axis of the burner – 100 mm; 2 – blue cone of flame
touches the sample surface; r – the correlation coefficient Also, at a distance of 100 mm, a better
correlation was obtained between the results
of testing different diameters of the AVVG
cable than when the blue cone touched the
sample surface. To assess the effect of the distance be
tween the burner and the sample on the
convergence of the results according to the
Table 5, the rms values of the length of the
charred part and the duration of self-com
bustion of the investigated AVVG cables are
calculated, presented in Table 6. Table 4 Table 6
The value of the standard deviation of the
length of the charred part and the duration
of self-combustion of cable samples of the
AVVG brand
1
2
3
4
5
AVVG 4×240 cable
Blue cone of flame touches the sample surface
1
240
131/10/141
20.0
no
2
240
145/15/160
25.4
no
3
240
135/15/150
21.2
no
The distance between the torch and the sample, measured along the torch axis – 100 mm
4
240
151/11/162
25.8
no
5
240
155/15/170
24.2
no
6
240
148/12/160
21.4
no
Note: Legend – according to Table 1
Continuation of Table 5
2
1
L3 = 1,339d + 74
r = 0,969
L3 = 1,113d + 96,5
r = 0,985
0
20
40
60
80
100
120
140
160
180
0
10
20
30
40
50
60
70
L3, mm
d, mm
Fig. 2. Linear dependences of the length of the charred part of the cables
on their outer diameter (d): 1 – distance between the torch and the sample,
measured along the torch axis – 100 mm; 2 – blue cone of flame touches the
sample surface; r – the correlation coefficient 1
2
3
4
5
AVVG 4×240 cable
Blue cone of flame touches the sample surface
1
240
131/10/141
20.0
no
2
240
145/15/160
25.4
no
3
240
135/15/150
21.2
no
The distance between the torch and the sample, measured along the torch axis – 100 mm
4
240
151/11/162
25.8
no
5
240
155/15/170
24.2
no
6
240
148/12/160
21.4
no
Note: Legend – according to Table 1
Continuation of Table 5 1
2
3
4
5
AVVG 4×240 cable
Blue cone of flame touches the sample surface
1
240
131/10/141
20.0
no
2
240
145/15/160
25.4
no
3
240
135/15/150
21.2
no
The distance between the torch and the sample, measured along the torch axis – 100 mm
4
240
151/11/162
25.8
no
5
240
155/15/170
24.2
no
6
240
148/12/160
21.4
no
Note: Legend – according to Table 1
Continuation of Table 5 2
1
L3 = 1,339d + 74
r = 0,969
L3 = 1,113d + 96,5
r = 0,985
0
20
40
60
80
100
120
140
160
180
0
10
20
30
40
50
60
70
L3, mm
d, mm
Fig. 2. Table 6 Fig. 2. Linear dependences of the length of the charred part of the cables
on their outer diameter (d): 1 – distance between the torch and the sample,
measured along the torch axis – 100 mm; 2 – blue cone of flame touches the
sample surface; r – the correlation coefficient The value of the standard deviation of the
length of the charred part and the duration
of self-combustion of cable samples of the
AVVG brand The value of the standard deviation of the
length of the charred part and the duration
of self-combustion of cable samples of the
AVVG brand Cable mark
size
RMS
В=62 mm
В=100 mm
L3, mm
taf, s
L3, mm
taf, s
AVVG 4×2,5
5.68
0.306
4.16
1.206
AVVG 4×16
3.46
0.231
2.52
0.529
AVVG 4×35
3.06
1.528
2.52
5.69
AVVG 4×120
1.629
3.64
3.89
2.5
AVVG 4×240
9.5
2.84
5.29
2.23
Note: Legend according to Table 1 2
1
taf = 0,352d - 4,36
r = 0,801
taf = 0,426d - 1,985
r = 0,983
-5
0
5
10
15
20
25
30
0
10
20
30
40
50
60
70
taf, s
d, mm
Fig. 3. Linear dependences of the duration of self-combustion cables on
their outer diameter (d): 1 – distance between the burner and the sample,
measured along the axis of the burner – 100 mm; 2 – blue cone of flame
touches the sample surface; r – the correlation coefficient From Table 6, there is in four out of five
cases better convergence of the results at
a distance of 100 mm than when the blue
flame cone touches the sample surface. As
for the standard deviation of the duration of
self-combustion of the samples, the best con
vergence at a distance of 100 mm between the
burner and the sample was obtained only in
two out of five cases. Fig. 3. Linear dependences of the duration of self-combustion cables on
their outer diameter (d): 1 – distance between the burner and the sample,
measured along the axis of the burner – 100 mm; 2 – blue cone of flame
touches the sample surface; r – the correlation coefficient In Fig. Table 4 Linear dependences of the length of the charred part of the cables
on their outer diameter (d): 1 – distance between the torch and the sample,
measured along the torch axis – 100 mm; 2 – blue cone of flame touches the
sample surface; r – the correlation coefficient Also, at a distance of 100 mm, a better
correlation was obtained between the results
of testing different diameters of the AVVG
cable than when the blue cone touched the
sample surface. To assess the effect of the distance be
tween the burner and the sample on the
convergence of the results according to the
Table 5, the rms values of the length of the
charred part and the duration of self-com
bustion of the investigated AVVG cables are
calculated, presented in Table 6. Table 6 Table 4 Table 5 Table 5 Experimental data on the influence of the duration of application of the flame source to the sample and the distance between
the burner and the sample on the assessment of the resistance to flame propagation of cables
Sample
number
Duration of the ignition source
type, s
L1/L2/L3, mm
taf, s
Combustion of the standard base
placed under the sample
1
2
3
4
5
AVVG 4×2,5 cable
Blue cone of flame touches the sample surface
1
60
60/12/72
3.6
no
2
60
65/15/80
4.2
no
3
60
70/13/83
4.0
no
The distance between the torch and the sample, measured along the torch axis – 100 mm
4
60
93/13/106
3.0
no
5
60
90/14/104
4.0
no
6
60
100/12/112
5.4
no
AVVG 4×16 cable
Blue cone of flame touches the sample surface
1
60
90/11/101
1.0
no
2
60
88/13/101
1.0
no
3
60
95/12/107
1.4
no
The distance between the torch and the sample, measured along the torch axis – 100 mm
4
60
110/11/121
3.0
no
5
60
109/14/123
4.0
no
6
60
105/13/118
3.8
no
AVVG 4×35 cable
Blue cone of flame touches the sample surface
1
60
99/15/114
1.0
no
2
60
100/18/118
4.0
no
3
60
103/17/120
3.6
no
The distance between the torch and the sample, measured along the torch axis – 100 mm мм
4
60
115/11/126
13.6
no
5
60
110/18/128
6.0
no
6
60
108/15/123
8.4
no
AVVG 4×120 cable
Blue cone of flame touches the sample surface
1
120
123/10/133
7.4
no
2
120
113/18/131
1.2
no
3
120
115/15/130
1.0
no
The distance between the torch and the sample, measured along the torch axis – 100 mm
4
120
125/14/139
14.4
no
5
120
115/16/131
15.0
no
6
120
129/13/142
19.0
no Experimental data on the influence of the duration of application of the flame source to the sample and the distance between
the burner and the sample on the assessment of the resistance to flame propagation of cables Experimental data on the influence of the duration of application of the flame source to the sample and the di
the burner and the sample on the assessment of the resistance to flame propagation of cables he influence of the duration of application of the flame source to the sample and the distance between
ner and the sample on the assessment of the resistance to flame propagation of cables 63 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 6/10 ( 114 ) 2021 In Fig. Table 4 2, 3 it is possible to see that with an increase in
the duration of application of the ignition source, the length
of the charred part and the duration of self-combustion of
the AVVG cable with an outer diameter from 10 mm to
60 mm increase At the same time the values of these in
Also, at a distance of 100 mm, a better
correlation was obtained between the results
of testing different diameters of the AVVG
cable than when the blue cone touched the
sample surface. To assess the effect of the distance be
tween the burner and the sample on the
convergence of the results according to the
Table 5, the rms values of the length of the
charred part and the duration of self-com
bustion of the investigated AVVG cables are
calculated, presented in Table 6. Table 6
The value of the standard deviation of the
length of the charred part and the duration
of self-combustion of cable samples of the
AVVG brand
Cable mark
size
RMS
В=62 mm
В=100 mm
L3, mm
taf, s
L3, mm
taf, s
AVVG 4×2,5
5.68
0.306
4.16
1.206
AVVG 4×16
3.46
0.231
2.52
0.529
AVVG 4×35
3.06
1.528
2.52
5.69
AVVG 4×120
1.629
3.64
3.89
2.5
AVVG 4×240
9.5
2.84
5.29
2.23
Note: Legend according to Table 1
From Table 6, there is in four out of five
cases better convergence of the results at
a distance of 100 mm than when the blue
flame cone touches the sample surface. As
for the standard deviation of the duration of
self-combustion of the samples, the best con
vergence at a distance of 100 mm between the
burner and the sample was obtained only in
two out of five cases. Table 7 Experimental data on resistance to flame propagation of
cable conduits trunkings and ductings
Sample
number
Duration of
the ignition
source
type, s
L1/L2/L3, mm
taf, s
Combustion of
the standard
base placed un
der the sample
Corrugated polyamide cable conduit with 16 mm outer diameter
and a 0.5 mm wall thickness
1
15
115/15/130
5
no
2
15
125/14/139
7
no
3
15
122/18/140
9
no
4
60
190/23/213
108
yes
5
60
154/21/175
84
yes
6
60
170/24/194
97
yes
Corrugated polypropylene cable conduit with 50 mm
outer diameter and 0.8 mm wall thickness
1
120
burned out
(Fig. 4)
≥30
yes
2
120
burned out
≥30
yes
3
120
burned out
≥30
yes
4
20
62/18/80
16
no
5
20
65/19/84
21
no
6
20
74/23/97
26
no
Rigid PVC cable conduit with 52 mm
outer diameter and 4.1 mm wall thickness
1
85
99/18/117
0
no
2
85
105/13/118
0
no
3
85
102/13/115
0
no
4
240
118/11/129
0
no
5
240
123/18/141
0
no
6
240
126/17/143
0
no
Corrugated polyethylene cable conduit with 62.5 mm
outer diameter and 1.4 mm wall thickness
1
25
136/28/164
22
no
2
25
129/27/156
18
no
3
25
138/29/167
24
no
4
240
burned out
(Fig. 5)
≥30
yes
5
240
burned out
≥30
yes
6
240
burned out
≥30
yes
Blind cable ducting with cross dimensions 50 mm×20 mm,
which complies with EN 50085-1 [19]
1
60
185/37/222
0
no
2
60
148/27/175
0
no
3
60
131/31/162
0
no
Cable trunking with cross dimensions 60mm×40mm, which com
plies with EN 50085-1 [19]
1
120
125/29/154
0
no
2
120
127/36/163
0
no
3
120
149/35/184
0
no
Note: Legend according to Table 1 Experimental data on resistance to flame propagation of
cable conduits trunkings and ductings
Sample
number
Duration of
the ignition
source
type, s
L1/L2/L3, mm
taf, s
Combustion of
the standard
base placed un
der the sample
Corrugated polyamide cable conduit with 16 mm outer diameter
and a 0.5 mm wall thickness Fig. 4. Corrugated polypropylene conduits that
spreads the flame Fig. 5. Corrugated polyethylene conduit that
spreads the flame Rigid PVC cable conduit with 52 mm Fig. 5. Table 6 2, 3 it is possible to see that with an increase in
the duration of application of the ignition source, the length
of the charred part and the duration of self-combustion of
the AVVG cable with an outer diameter from 10 mm to
60 mm increase. At the same time, the values of these in
dicators do not exceed the threshold values of 425 mm and
30 s, respectively. The convergence of the results of the duration of
self-combustion of the samples was influenced by the prop
erties of the insulating materials placed under the shell,
and the size of the zones of oxygen access to the place of
combustion of such materials, formed as a result of damage
to the shell. As a result, it can be considered that the use of the rec
ommended distance of 100 mm [2] will improve the conver
gence and reproducibility of the results of testing cables for
flame propagation. At a distance of 100 mm (curve 1), larger values of the
indicators were obtained than when the blue cone touched
the surface of the sample (curve 2). 64 Fig. 4. Corrugated polypropylene conduits that
spreads the flame 5. 5. Influence of the duration of the application of the
flame on the resistance to flame propagation of conduits
trunkings and ductings The data of experimental studies on the influence of the
duration of the application of the flame source to the sample
on the assessment of resistance to flame propagation of cable
conduits and ducts are presented in Table 7. 6. Discussion of research results on resistance to flame
propagation of long elements of electrical wiring systems duration of the application of the flame source to the samples
set for cables (Table 2). If flame-resistant materials are used, damage to such
elements occurs directly in the zone of influence of the ig
nition source. In this case, an increase in the duration of the
application of the flame source will only lead to the burnout
of materials in the zone of influence of this source. At the
same time, the duration of self-combustion can reach a peak
and further decrease. According to the principles set forth in [2], the condi
tions for the impact of a 1 kW flame source on long elements
of the electrical wiring system and the criteria for evaluating
the results, as far as possible, should be the same. According to the results presented in Tables 4, 5, it is
advisable to evaluate the resistance to flame propagation of
cables according to such criteria as the presence of particles
capable of ignition and the duration of self-combustion no
more than 30 s. Assessment by such indicators is necessary
for making a decision on the introduction of additional fire
protection for cables at facilities. Taking this into account, it is promising to evaluate the
long elements of the wiring system by the speed of flame
propagation. Also, according to the test results provided in Tables 5, 7
and Fig. 2, 3, it is advisable to choose for the cables the dis
tance between the torch and the sample recommended in [27]. This is due to the need to ensure the convergence and repro
ducibility of the results, since at a distance of about 100 mm,
the parameters of the 1 kW flame source are checked. The results are presented in Tables 3, 4, found that the
generally accepted basis for evaluating the flammability of
particles separated during the combustion of electrical prod
ucts is more effective than that selected for cables. Therefore,
the use of a base consisting of a white pine board covered
with tissue paper will help reduce the risk of making the
wrong decision about cable compliance. The permissible distance between the clamps for the
sample (Table 1) allows for all elements of the electrical
wiring system to use for testing samples with the same and
smaller length mm, adopted for cables. 6. Discussion of research results on resistance to flame
propagation of long elements of electrical wiring systems This makes it possi
ble to eliminate the influence of the effects associated with
the longitudinal heating of the samples under the influence
of the ignition source. The specified dimensional tolerance
will avoid the problems of fixing specimens due to the short
length. Also, the results presented in Table 3, enable all el
ements of the wiring system to establish the same dis
tance (200±5) mm between the point of application of the
flame source and the base. Since the thermal energy of burn
ing particles becomes less when falling from a greater height,
determining such a distance will help to reduce the risk of
making an incorrect decision in assessing the resistance to
flame propagation of these elements. It is provided that the length of the samples is the same,
eliminating the influence on the results of their transverse
dimensions allows to choose the optimal duration of appli
cation of the ignition source to the samples. Therefore, it is
promising to conduct relevant studies. It is not possible to determine the specified distance less
than the overall dimensions of the burner [27] and the estab
lished distance between it and the sample (Table 1). Also, reducing the specified distance allows to test all
the long elements of the wiring system in a metal fence with
the same height of 1200 mm (Table 1). This will help to min
imize the number of pieces of test equipment. The content of excess moisture in the samples and the
base, which is located under them, in excess of that which
may be contained in electrical products during use, can neg
atively affect the process of their combustion. Evaluation of the resistance of cables to flame propaga
tion only according to the test data of one sample can lead to
an incorrect decision about their compliance (Table 4). This
also applies to powertracks. For the elements of the electrical wiring system, the
conditions of sample conditioning are different (Table 1). To
eliminate the effect of this factor, for all elements of the wiring
system, equal conditioning conditions for the samples and the
base can be determined, established in the fundamental stan
dards [25, 26]. Such conditions are holding the samples and
the base for at least 48 hours at a temperature of (23±2) °С
and a relative humidity of (50±10) %. Table 7 Corrugated polyethylene conduit that
spreads the flame The results of experimental studies presented in Table 6
prove that the duration of the ignition source seems to be
a significant factor that affects the compliance of long ele
ments of the wiring system with the requirements for resis
tance to flame propagation. The duration of the operation of a flame source of the
type specified in IEC 61386-1 [23] may not ensure that the
conduits reach thermal energy sufficient to maintain stable
combustion and flame propagation. This was revealed using
the duration of application of the ignition source of the type
set for cables in IEC 60332-1-2 [13]. This also applies to the test method for flame propaga
tion of liquid tight sheathings [24]. For cable trunkings and ductings, an increase in the du
ration of the ignition source like this revealed their inconsis
tency. At the same time, additional testing of these products,
cable trays, ladders and powertracks with a flame source dura
tion of more than 60 s will provide confidence in the decision
on their proper resistance to flame propagation. 65 6/10 ( 114 ) 2021 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 duration of the application of the flame source to the samples
set for cables (Table 2). 6. Discussion of research results on resistance to flame
propagation of long elements of electrical wiring systems At the same time, for
safe and economic reasons, it is advisable to conduct exper
imental studies to determine the optimal duration of condi
tioning samples of long elements of the wiring system. To obtain reliable results, the decision rules established
for cable trunkings and ductings, trays and ladders can be
considered optimal (Table 1). At the same time, it is ad
visable to conduct experimental studies on the number of
samples exceeding six. With an increase in the duration of the application of the
flame source to the samples of long elements of the electrical
wiring system, an increase in the length of the charred part
and the duration of self-combustion of the samples was ob
tained (Tables 5, 7 and Fig. 2–5). This is due to the transfer
of more thermal energy from the ignition source to the sam
ples, which helps to maintain their combustion. The combustion processes of electrical products can be
affected by ambient temperature, relative humidity and at
mospheric pressure. In this regard, it is advisable to test long
elements of the wiring system under conditions that give the
worst result, but are limited by actual operating conditions. Therefore, it is promising to carry out studies to identify the
influence of such factors on the resistance to flame propa
gation of long elements of the wiring system. At the same
time, such studies are difficult in terms of maintaining stable
environmental parameters. At the same time, the levels of these indicators, as it is
known, depend on the properties of the used insulating ma
terials, their dimensions, the energy of the ignition source,
oxygen availability and environmental conditions. If materials that are not resistant to flame propagation
are used, the short duration of the application of the flame
source to them is not enough to initiate stable combustion. An increase in the duration of the application of the flame
source to the samples made it possible to identify combusti
ble conduits (Table 7). The combustion processes of samples of electrical prod
ucts can be influenced by air currents. To limit their impact,
protective chambers are used [25, 26]. Among the long ele
ments of the wiring system, such chambers are intended for
testing only cables (Table 1). 7. Conclusions 1. It was proposed to assess the resistance to flame propaga
tion of long elements of the wiring system to meet the require
ments of IEC 60695-1-30 [2], according to the same criteria: 1. It was proposed to assess the resistance to flame propaga
tion of long elements of the wiring system to meet the require
ments of IEC 60695-1-30 [2], according to the same criteria: [ ]
g
– the length of the carbon part up and down from the
point of application, the value of which does not exceed
375 mm and 50 mm, respectively; – the length of the carbon part up and down from the
point of application, the value of which does not exceed
375 mm and 50 mm, respectively; – the presence and absence of ignition of the base located
under the sample at a distance of (200±5) mm; – the duration of self-combustion, the value of which
does not exceed 30 s. 2. It was found that at a distance of 200 mm below the
source of the formation of burning particles, the base of a wood
en board covered with a layer of tissue paper with a surface den
sity of (21±9) g/m2 was occupied by 2 burning particles, and
the base was made of a double layer of filter paper with a surface
density (80±15) g/m2 – from 7 burning particles. At the speci
fied distance between the point of application of the 1 kW flame
source to the cable and the first base, its ignition was detected,
and when the second base was used, it did not occur. The use of
the first base, which is accepted for testing other elements of the
wiring system, increases the efficiency of detecting cables that
are not resistant to flame propagation. 2. It was found that at a distance of 200 mm below the
source of the formation of burning particles, the base of a wood
en board covered with a layer of tissue paper with a surface den
sity of (21±9) g/m2 was occupied by 2 burning particles, and
the base was made of a double layer of filter paper with a surface
density (80±15) g/m2 – from 7 burning particles. 6. Discussion of research results on resistance to flame
propagation of long elements of electrical wiring systems In this regard, for all elements of the wiring system, it
is advisable to take the most stringent requirements for the The disadvantage of using protective chambers is the de
pletion of oxygen during combustion of samples in a confined 66 Ecology space. This affects the course of the normal combustion pro
cess. Therefore, for the use of protective chambers for testing
elements of the wiring system, their internal dimensions
should be justified. criterion of the presence of a base fire located under the
cable. To reduce the risk of making an incorrect decision
on compliance, it is proposed to evaluate long elements of
the wiring system according to the rules established for
cable trunkings, ductings, trays and ladders in the stan
dards [21, 22]. The results obtained were discussed at the International
Scientific and Practical Conference [28]. 4. It was found for the AVVG cable in four out of five
cases a better convergence of the results along the length
of the charred part of the samples at a distance between the
burner and the sample, which is 100 mm along the burner
axis, than when the 1kW thief flame cone touches the sample
surface. But in three out of five cases, the best convergence
of the results of the duration of self-combustion was obtained
under the latter condition. Under the first condition, the
maximum values of the length of the carbon part of 170 mm
and the duration of self-combustion of 25.8 s were obtained,
and under the second condition, respectively, 160 mm and
25.4 s. Under the first condition, for the linear dependence of
these indicators on the diameter of the AVVG cable within
the outer diameter from 10 mm to 60 mm, the correlation co
efficients were 0.985 and 0.983, respectively, and under the
second condition, 0.969 and 0.801, respectively. Based on
the generalization of these results and the recommendations
of IEC 60695-11-2 [27], it was proposed to test cables under
the first condition. 7. Conclusions At the speci
fied distance between the point of application of the 1 kW flame
source to the cable and the first base, its ignition was detected,
and when the second base was used, it did not occur. The use of
the first base, which is accepted for testing other elements of the
wiring system, increases the efficiency of detecting cables that
are not resistant to flame propagation. 5. For two cable conduits that meet the requirements of
IEC 61386-1 [22], for the duration of the application of a
flame of 1 kW for 20 s and 25 s, it is found that they spread
the flame for the duration of the application of such a flame
source for 120 s and 240 s. To increase the efficiency of de
tecting long elements of the wiring system that are unstable
to flame propagation, it was proposed to test such elements
with a flame application duration of 1 kW, set for cables in
IEC 60332-1-2 [13]. 3. In one of three experiments, a cable was found that
was not resistant to flame propagation, based on the References 1. IEC 60695-1-10:2016 Fire hazard testing – Part 1-10: Guidance for assessing the fire hazard of electrotechnical products – General
guidelines (2016). Geneva: International Electrotechnical Commission, 50. 1. IEC 60695-1-10:2016 Fire hazard testing – Part 1-10: Guidance for assessing the fire hazard of electrotechnical products – General
guidelines (2016). Geneva: International Electrotechnical Commission, 50. 2. IEC 60695-1-30:2017 Fire hazard testing Part 1-30: Guidance for assessing the fire hazard of electrotechnical products –
Preselection testing process – General guidelines (2017). Geneva, International Electrotechnical Commission, 31. doi: http://
doi.org/10.3403/02683772u 2. IEC 60695-1-30:2017 Fire hazard testing Part 1-30: Guidance for assessing the fire hazard of electrotechnical products –
Preselection testing process – General guidelines (2017). Geneva, International Electrotechnical Commission, 31. doi: http://
doi.org/10.3403/02683772u g/
/
3. Huang, X., Zhu, H., He, L., Peng, L., Cheng, C., Chow, W. (2021). Improved model for estimating sidewall effect on the fire heat
release rate of horizontal cable tray. Process Safety and Environmental Protection, 149, 831–838. doi: http://doi.org/10.1016/
j.psep.2021.03.040 g/
/
3. Huang, X., Zhu, H., He, L., Peng, L., Cheng, C., Chow, W. (2021). Improved model for estimating sidewall effect on the fire heat
release rate of horizontal cable tray. Process Safety and Environmental Protection, 149, 831–838. doi: http://doi.org/10.1016/
j.psep.2021.03.040 3. 7. Conclusions Huang, X., Zhu, H., He, L., Peng, L., Cheng, C., Chow, W. (2021). Improved model for estimating sidewall effect on the fire heat
release rate of horizontal cable tray. Process Safety and Environmental Protection, 149, 831–838. doi: http://doi.org/10.1016/
j.psep.2021.03.040 j p
p
4. Li, L., Huang, X., Bi, K., Liu, X. (2016). An enhanced fire hazard assessment model and validation experiments for vertical cable
trays. Nuclear Engineering and Design, 301, 32–38. doi: http://doi.org/10.1016/j.nucengdes.2015.12.034 j p
p
4. Li, L., Huang, X., Bi, K., Liu, X. (2016). An enhanced fire hazard assessment model and validation experiments for vertical cable
trays. Nuclear Engineering and Design, 301, 32–38. doi: http://doi.org/10.1016/j.nucengdes.2015.12.034 5. An, W., Wang, T., Liang, K., Tang, Y., Wang, Z. (2020). Effects of interlayer distance and cable spacing on flame characteristics
and fire hazard of multilayer cables in utility tunnel. Case Studies in Thermal Engineering, 22. doi: http://doi.org/10.1016/
j.csite.2020.100784 6. An, W., Wang, X., Tang, Y., Wang, T., Lu, J. (2021). Influence of cable inclination angle and longitudinal ventilation on
temperature distribution during cable fire in utility tunnel. Case Studies in Thermal Engineering, 27. doi: http://doi.org/10.1016/
j.csite.2021.101304 7. Sundström, B., Axelsson, J., Van Hees, P. (2003). A proposal for fire testing and classification of cables for use in Europe. SP Swedish
National Testing and Research Institute, 38. 8. EN 13501-6:2018 Fire classification of construction products and building elements – Part 6: Classification using data from reaction
to fire tests on power, control and communication cables (2018). Brussels: European Committee for Standardization, 30. doi: http://
doi.org/10.3403/30348257 g/
/
9. Regulation (EU) No. 305/2011 of the European Parliament and of the Council of 9 March 2011 laying down harmonized conditions
for the marketing of construction products and repealing Council Directive 89/106/EEC (2011). OJ L 88, 4.4.2011, 5–43. 9. Regulation (EU) No. 305/2011 of the European Parliament and of the Council of 9 March 2011 laying down harmonized conditions
for the marketing of construction products and repealing Council Directive 89/106/EEC (2011). OJ L 88, 4.4.2011, 5–43. 10. Johansson, R., Post, J., Försth, M. (2015). Extended field of application (EXAP) for reaction-to-fire Euro-classification of optical
fibre cables. SP Technical Research Institute of Sweden. SP Report, 49. 10. Johansson, R., Post, J., Försth, M. (2015). Extended field of application (EXAP) for reaction-to-fire Euro-classification of optical
fibre cables. SP Technical Research Institute of Sweden. SP Report, 49. 7. Conclusions 67 67 6/10 ( 114 ) 2021 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 11. Kaczorek-Chrobak, K., Fangrat, J. (2019). Influence of Constructional-Material Parameters on the Fire Properties of Electric
Cables. Energies, 12 (23), 4569. doi: http://doi.org/10.3390/en12234569 12. Directive 2014/35/EU of the European Parliament and of the Council of 26 February 2014 on the harmonisation of the laws of the
Member States relating to the making available on the market of electrical equipment designed for use within certain voltage limits. OJ L 96, 29.3.2014. Р. 357–374. 13. IEC 60332-1-2:2004 Tests on electric and optical fibre cables under fire conditions – Part 1-2: Test for vertical flame propagation for
a single insulated wire or cable – Procedure for 1 kW pre-mixed flame (2004). Geneva: International Electrotechnical Commission,
21. doi: http://doi.org/10.3403/03171042 14. IEC 60332-1-3:2004 Tests on electric and optical fibre cables under fire conditions – Part 1-3: Test for vertical flame propagation
for a single insulated wire or cable – Procedure for determination of flaming droplets/particles (2004). Geneva: International
Electrotechnical Commission, 21. 15. IEC 60332-1-2:2004/AMD1:2015 Tests on electric and optical fibre cables under fire conditions – Part 1-2: Test for vertical
flame propagation for a single insulated wire or cable – Procedure for 1 kW pre-mixed flame (2015). Geneva, International
Electrotechnical Commission, 9. 16. IEC 60332-1-3:2004/AMD1:2015 Tests on electric and optical fibre cables under fire conditions – Part 1-3: Test for vertical flame
propagation for a single insulated wire or cable – Procedure for determination of flaming droplets/particles (2015). Geneva,
International Electrotechnical Commission, 9. doi: http://doi.org/10.3403/03172788 17. EN 60332-1-2:2004/A11:2016 Tests on electric and optical fibre cables under fire conditions – Part 1-2: Test for vertical flame
propagation for a single insulated wire or cable – Procedure for 1 kW pre-mixed flame (2016). Brussels: European Committee for
Electrotechnical Standardization, 4. 18. EN 60332-1-2:2004/A12:2020 Tests on electric and optical fibre cables under fire conditions – Part 1-2: Test for vertical flame
propagation for a single insulated wire or cable – Procedure for 1 kW pre-mixed flame (2020). Brussels: European Committee for
Electrotechnical Standardization, 4. doi: http://doi.org/10.3403/03171042 19. Fr Gyppaz, F. (2012). Flame spread behavior of electric cables: Round-robin characterization of the IEC/EN 60332-1 tests. Journal
of Fire Sciences, 30 (5), 404–412. doi: http://doi.org/10.1177/0734904112441918 20. IEC 61534-1:2011+AMD1:2014+AMD2:2020 Powertrack systems – Part 1: General requirements (2020). Geneva: International
Electrotechnical Commission, 285. 21. 7. Conclusions EN 50085-1:2005 Cable trunking systems and cable ducting systems for electrical installations – Part 1: General requirements
(2005). Brussels: European Committee for Electrotechnical Standardization, 43. 22. IEC 61537:2006 Cable management – Cable tray systems and cable ladder systems (2006). Geneva: International Electrotechnical
Commission, 161. 23. IEC 61386-1:2008+AMD1:2017 Conduit systems for cable management – Part 1: General requirements (2017). Geneva:
International Electrotechnical Commission, 159. 24. EN 50369:2005 Liquid tight sheathing systems for cable management (2
Standardization, 25. doi: http://doi.org/10.3403/30102733u 24. EN 50369:2005 Liquid tight sheathing systems for cable management (2005). Brussels: European Committee for Electrotechnical
Standardization, 25. doi: http://doi.org/10.3403/30102733u 25. IEC 60695-11-10:2013 Fire hazard testing – Part 11-10: Test flames – 50 W horizontal and vertical flame test meth
doi: http://doi.org/10.3403/01860485u 26. IEC 60695-11-20:2015 Fire hazard testing – Part 11-20: Test flames – 500 W flame test method. Brussels. doi: http://doi.org/
10.3403/01859086u 27. IEC 60695-11-2:2017 Fire hazard testing – Part 11-20: Test flames – 1 kW pre-mixed flame – Apparatus, confirmatory test
arrangement and guidance (2017). Geneva: International Electrotechnical Commission, 40. doi: http://doi.org/10.3403/30338608 27. IEC 60695 11 2:2017 Fire hazard testing
Part 11 20: Test flames
1 kW pre mixed flame
Apparatus, confirmatory test
arrangement and guidance (2017). Geneva: International Electrotechnical Commission, 40. doi: http://doi.org/10.3403/30338608
28. Kravchenko, R. I., Illiuchenko, P. O., Onyshchuk, A. Ye. (2021). Udoskonalennia metodiv vyprobuvannia ta kryteriivotsinky
stiikosti do poshyrennia polumia dovhykh elementiv systemy elektroprovodky. Problemy nadzvychainykh sytuatsii (PES-2021). Kharkiv: Natsionalnyi universytet tsyvilnoho zakhystu Ukrainy, 49–50. 28. Kravchenko, R. I., Illiuchenko, P. O., Onyshchuk, A. Ye. (2021). Udoskonalennia metodiv vyprobuvannia ta kryteriivotsinky
stiikosti do poshyrennia polumia dovhykh elementiv systemy elektroprovodky. Problemy nadzvychainykh sytuatsii (PES-2021). Kharkiv: Natsionalnyi universytet tsyvilnoho zakhystu Ukrainy, 49–50. 68 68
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Reference Intervals (RIs) in veterinary medicine
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ABSTRACT ABSTRACT
Reference Intervals (RIs) are necessary in veterinary clinical pathology to provide a data base in order to compare results obtained from
healthy versus diseased animals. Data are obtained from laboratory tests and, depending on whether they have a Gaussian or non-
Gaussian distribution, are processed through statistical tests to define the RIs. This process begins with a healthy reference population
made up of individuals who have been initially chosen based on inclusion or exclusion criteria. It is frequently challenging to have a large
number of healthy individuals on which to establish de novo RIs, especially in wild and exotic animals. However, the use of reference
intervals in daily clinical practice remains a fundamental instrument for therapeutic and diagnostic decisions, but it must always be
accompanied by clinical findings that can confirm the hypothesis. Received August 7, 2023; In final form August 7, 2023; Published March 2024 Copyright: This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 License, which per
distribution, and reproduction in any medium, provided the original author and source are credited. Corresponding author: Martina Quagliardi, e-mail: martina.quagliardi@unicam.it ACTA IMEKO
ISSN: 2221-870X
March 2024, Volume 13, Number 1, 1 - 5 March 2024 | Volume 13 | Number 1 | 1 Keywords: reference intervals; clinical pathology; veterinary medicine; laboratory data; statistical test Keywords: reference intervals; clinical pathology; veterinary medicine; laboratory data; statistical test Citation: M. Quagliardi, L. Galosi, G. Rossi, A. Roncarati, A. Gavazza, Reference Intervals (RIs) in veterinary medicine, Acta IMEKO
5. DOI: 10.21014/actaimeko.v13i1.1615 L. Galosi, G. Rossi, A. Roncarati, A. Gavazza, Reference Intervals (RIs) in veterinary medicine, Acta IMEKO, vol. 13 (2024) no. 1, pp. 1
eko.v13i1.1615 Section Editor: Leopoldo Angrisani, Università degli Studi di Napoli Federico II, Naples, Italy Received August 7, 2023; In final form August 7, 2023; Published March 2024 4. EXAMPLES Breed is another crucial parameter to consider within the
same species; numerous studies have been conducted to identify
the main hematobiochemical differences between different
breeds and standard RIs, with the majority of these studies
focusing on dogs. Greyhounds have been extensively studied in both veterinary
transfusion
medicine
and
clinical
pathology. Their
haematological and biochemical values are found to differ from
those of the general canine population, indicating distinct
physiological characteristics [15]. Numerous deviations have
been observed in this breed, including higher HCT, MCV,
MCHC, RBC counts and HGB concentrations. However, PLT
count and total white blood cell (WBC) counts tend to be lower
[15], [16]. Significant differences in values were also detected in
newborn Martina Franca donkeys in the first three weeks of life. Compared to horse foals, Martina Franca donkey foals exhibited
lower red blood cells (RBC) count, hematocrit (HCT) and
hemoglobin (HGB) levels in their first few days of life. Additionally,
mean
corpuscular
volume
(MCV),
mean
corpuscular hemoglobin (MCH), mean corpuscular hemoglobin
concentration (MCHC) also resulted lower. These findings
underline that RIs should be made to particular breeds species
during the newborn stage [8]. There are hematological differences even among sighthounds
of the same category, such as Whippet, Greyhound, Italian
Greyhound, Sloughi, Saluki, Borzoi, Pharaoh Hound and
Azawakh. In a study of 2013, it was documented that Whippets
had the laboratory profile that matched Greyhounds the closest
and, with the exception of Pharaoh Hounds, Italian Greyhounds
demonstrated significantly higher alanine aminotransferase
(ALT) activity than the other sighthound breeds [17]. Calves are another species in which hematological values have
been observed to deviate from adult RIs. Both neonatal Holstein
and Norwegian Red Breed calves present higher RBC and PLT
counts during the first period of life. However, at the same time,
MCV and MCHC values decrease [9], [10]. Recent studies on sighthounds have revealed that most RIs
available in veterinary textbooks cannot be confirmed for Italian
Greyhounds (Piccolo Levriero Italiano-PLI). Only a handful of
these variants can be interpreted as peculiarities unique to this
breed, perhaps as a result of its predisposition for racing. 3. METHODS OF DETERMINATION Generally, a RI of an analyte for a given population could be
determined in three ways: calculated de novo, transferred from a
previous RI when a method or instrument is changed or
validated from a previously established or transferred RI [3]. In contrast to calves, during the early stages of life, dogs tend
to have lower RBC count, HCT, total proteins and creatinine
levels; conversely, alkaline phosphatase (ALP), creatine kinase
(CK) and MCV increase [11], [12]. The ASVCP (American Society of Veterinary Clinical
Pathology) published the reference interval guidelines in order to
determine de novo RI in veterinary species, based on the CLSI
(Clinical Laboratory and Standards Institutes) recommendations
of 2008 [5], [6]. In addition to the influence of age, it is important that the
animals are non-treated with drugs that can affect test results,
such as corticosteroids that increase neutrophil count and liver
enzymes; because of their action, their use is an exclusion
criterion for reference individuals in order to determine de novo
RIs [5]. De novo determination of RIs is the most used and frequent
procedure in veterinary medicine; an "a priori" method is advised
for determining inclusion or exclusion criteria for the starting
population, whereas a "a posteriori" approach is utilised when
pre-existing data have not yet been mined to generate reference
values. The reference individuals chosen form the reference
population, are described as “an undefined number of
individuals that represent the demographic for which the
reference intervals will be used” [5]. Moreover, if the animals are females, it is essential that they
are not late pregnant or in early lactations; these physiologic
states are uncontrollable pre-analytical variables, such as age. It
is known from published studies that this state could affect
clinical pathology tests. For example, in Standardbred mares during peripartum (last
month of gestation and first week after parturition), a reduction
of HGB, HCT and WBC has been found, as well as in many
other serum analytes; it is important to place the attention in
these temporary haematological changes, which greatly
differentiate the mares in perinatal from those not in lactation
and not pregnant [13]. The samples must be carried out from animals of a particular
species, using a mixture of breeds, ages and gender. The animals
of the population must be adults, clinically and serological testing
healthy, since the age is a physiological pre-analytical variable that
could induce different results in clinical pathology tests. 1. INTRODUCTION Firstly, these intervals are used to help identify abnormalities
in a sick patient, compared to known values from healthy
patients. It is important for a RI to represent a large,
heterogeneous and appropriate demographic population in order
to undertake a precise comparison [2]. Reference Intervals (RIs) in veterinary medicine have a
fundamental role in the everyday medical practices and
application. This topic could be strictly associated to the field of
clinical pathology, but, at the same time they are implicitly used
by everyone that processes and interprets laboratory data. RIs of laboratory tests are calculated from a group or
population of healthy adult animals (hence they are called
population-based reference intervals). In fact, without a RI it
would be very difficult to determine if the results of a laboratory
test are altered or normal; other terms that could be used for RI
include normal, normal values and reference range [1]. In fact, reference values are used to describe the dispersion of
variables in healthy individuals, comprising 95 % of the healthy
population. In addition, they describe fluctuations observed in
healthy populations or individuals that make the definition of
health and health status. For this, it remains a fundamental tool
in order to make clinical decisions for the patients [3]. The terms "range" and "interval" have rather different
meanings and should not be used interchangeably. A "range"
refers to the difference between two values, the highest and
lowest observations, and is a single whole number, whereas a
"interval" only includes the values between two reference limits
and the reference limits themselves. On the other hand, the term
“normal value” is also discouraged, since it is difficult to define
the concept of “normality”, often many variations may appear
“abnormal” due to age, physiology, diet, environment or other
non-pathological factors [1], [2]. This emphasises the first significant drawback of the
reference intervals, which is that they differ when the
composition of the group of healthy animals used to construct
them varies. The most significant influencing factors are race,
age, sex, environment, diet, and lifestyle choices; as a result, each
laboratory should establish its own reference ranges based on the
population of animals that visit the facility. However, this is
frequently impractical, with the exception of large facilities that
receive a large volume of samples [4]. 1. INTRODUCTION March 2024 | Volume 13 | Number 1 | 1 ACTA IMEKO | www.imeko.org puppies has identified three different RIs, corresponding to 3
phases of 15 days, starting from 16th day after birth [12]. 3. METHODS OF DETERMINATION For
instance, hematocrit and protein concentration are lower and
lymphocyte count is higher in younger animals compared to
adults. The impact of age has been observed in a variety of
species, including dogs, cats, horses, calves, and donkeys, thus
this selection criterion needs to be handled with extreme caution. A similar study was carried out in Holstein dairy cows,
covering the first month after calving; actually 39 analytes out of
52 tested were found significantly different due to lactation
period. These differences due to the lactation phase can be
statistically validated and biologically demonstrated, but they can
also be affected by pre-analytic factors, such as herds, parity
groups and management, which must be considered [14]. The majority of age-related changes are primarily associated
to the growth and differentiation that are unique to young
animals, such as higher metabolism and grater tissue maturation. A study conducted on Dutch warmblood foals that were raised
on pasture with free movement demonstrated that age has
significant effects on several hematological values, with the
exception of leucocytes (band-shaped, eosinophilic, basophilic
and
monocytes),
platelets
(PLT),
creatinine,
creatine
phosphokinase (CPK), pCO2 and sodium, potassium and
calcium. Moreover, it appeared that raising conditions may
impact other characteristics, although the clinical significance of
these effects may be minimal [7]. ACTA IMEKO | www.imeko.org 4. EXAMPLES In fact,
they present higher MCH, MCHC, cell haemoglobin
concentration mean (CHCM), albumin, calcium and iron, while In clinical practice of small animals, it is important for
clinicians to be aware of the temporal variations in biochemical
and hematologic values for dog puppies during the first two
months of life to prevent misinterpretation of findings [11]. During this period, haematological parameters change
significantly, and a study conducted on 101 clinically healthy dog March 2024 | Volume 13 | Number 1 | 2 ACTA IMEKO | www.imeko.org large unstained cells (LUC) is lower. At the same time RIs for
total proteins and cholesterol resulted wider and the morphology
of RBC and reticulocytes different [18]. reference intervals. In most cases, the breed-specific data
overlapped with the standard reference intervals, suggesting
minimal impact on clinical interpretation [31]. However, certain
breeds exhibited unique characteristics, such as the Birman,
which has been found to have physiologically elevated levels of
creatinine and serum symmetric dimethylarginine (SDMA)
concentrations [32]. Standard RIs were also compared with populations of other
dog breeds, underlining the presence of breed-specific variation
in blood values. For example, Bernese Mountain dogs were
found to require new RIs for 7 analytes (eosinophils, MCHC,
ALP, γ-glutamyltransferase (GGT), total bilirubin, amylase, and
cholesterol) [19], while in Shetland sheepdogs, cholesterol and
glucose resulted to need specific RIs because of differences
depending on breed peculiarities [20]. As mentioned above, in veterinary medicine it is often
challenging to obtain the numbers suggested by guidelines,
particularly in wild species or exotic animals. As a result,
practitioners often have to make do with limited data sets where
one has to make do with small numbers of samples that have
been managed to collect. Concerning the French mastiff Dogue de Bordeaux (DDB),
age was found to significantly affect RIs for HGB, HCT, MCHC,
WBC,
neutrophil,
lymphocyte,
and
monocyte
counts. Additionally, DDBs have higher RIs for HGB, HCT, MCV,
MCHC and mean platelet volume (MPV), while lower RIs for
reticulocytes counts, platelets by impedance (PLT-I) and optical
count (PLT-O) and plateletcrit. For this reason, new RIs for RBC
and PLT have been recommended to avoid pathological
suspicions of polycythaemia and thrombocytopenia in this breed
[21]. 5. STATISTICAL METHODS The hematologic profile of Miniature Dachshunds has been
found to be like Greyhounds, with higher RBC count, packed
cell volume (PCV) and hemoglobin concentration [26]. On the
other hand, a 1995 Australian study reported that bile acids
measurement may not be very useful in Maltese dogs, as they
may have "artifactually" elevated serum bile acids due to
unknown reacting substances [27]. According to the guidelines, the nonparametric ranking
approach with 90 % confidence intervals should be used to
determine new Reference Intervals from a minimum of 120
Reference Individuals (CI) [6]. Based on the number of individuals, it is necessary to test if
data follow a Gaussian distribution. Then, in order to determine
reference limits and intervals, methods are used to remove
outliers and select the central 95 % of reference values [1], [37]. The Japanese breeds, such the Akita or Shiba Inu and
Hokkaido, are perhaps the ones with the most well-known
hematological changes related to the breed in daily clinical
practice. It has been observed in clinically healthy dogs that they
have physiological microcytosis (red blood cells smaller than
normal), resulting in lower MCV, MCH, MCHC, higher red cell
distribution width (RDW) and significant anisocytosis (variation
in red cell size) on smear exam [28]. When data do not follow a Gaussian distribution, a possible
solution is applying a simple nonparametric method like
percentile rank. This is called rank-percentile method, and it is
used when there are more than 40 individuals not normally
distributed [2]. Then, parametric (mean ± 2sd) or non-parametric
(percentiles) methods can be used in order to establish reference
limits [1]. By the way, veterinary transfusion centers and related
databases can often be an excellent source for creating de novo
RIs and studying breed differences for hematological and
biochemical values. This is due to donor dogs being par
excellence a healthy animal and must respect well-defined
characteristics [29] and to the large number of samples
processed. A recent study carried out through the blood donor
database of the EMOVET-UNIPG blood bank and transfusion
unit found significant differences in 5 hunting dog breeds
(Ariégeois, Bleu de Gascogne, Bracco Italiano, Segugio Italiano,
Briquet Griffon Vandeen) for 12 significant differences in
hematologic and serum biochemical analytes; new RIs for HCT,
MCH, MCHC, RDW, PLT, monocytes, eosinophils, albumin,
urea, creatinine, aspartate aminotransferase (AST) and alanine
aminotransferase (ALT) were supplied for at least one breed [30]. 4. EXAMPLES For example, for the 50th EAAM (European Association for
Aquatic Mammals) Annual Symposium of 2023 that took place
in Valencia in March, two posters were presented regarding the
RIs of hematological and biochemical parameters in a particular
species of dolphin, Risso’s dolphin (Grampus griseus) [33], [34]. As
for other exotic species, also in this case the bibliography
available is very little numerous and the number of individuals
involved in the studies is very small [35], [36]. Despite the small number of individuals (9 prepuberal
females), the study on the RIs of hematological and biochemical
parameters in Risso’s dolphins presented at the 50th EAAM
Annual Symposium in Valencia in March 2023 is noteworthy for
the high number of samples under analysis (818 blood samples). This represents the first investigation on a large number of
samples in this species and may serve as a preliminary study for
future research on age and sex differences. There are also breed-specific hematologic peculiarities
observed in small dog breeds. For instance, hereditary poodle
macrocytosis, a clinically silent condition, has been reported in
some Miniature and Toy Poodles [22]. In Cairn and Norfolk
Terriers,
a
condition
known
as
hereditary
macrothrombocytopenia has been identified, which is caused by
mutations in the beta1-tubulin gene [23]. This condition is
prevalent in the Cavalier King Charles Spaniel (CKCS) [24], that
have been demonstrated to have a significantly lower whose
platelet count than the general RI [25]. 6. CONCLUSIONS DOI: 10.1016/j.theriogenology.2014.04.004 Laboratories should report the type of RIs used and how it
was calculated, since incorrect interpretation of laboratory data
can lead to clinical errors and is often underestimated. This is due
to the fact that the reference intervals are "statistical artifacts"
and the percentage of probability that a healthy animal taken at
random from the population has an anomalous value is 5 % [4]. [9]
H. C. Brun-Hansen, A. H. Kampen, A. Lund, Hematologic values
in calves during the first 6 months of life, Vet. Clin. Pathol. 35(2)
(2006), pp. 182-187. DOI: 10.1111/j.1939-165X.2006.tb00111.x DOI: 10.1111/j.1939-165X.2006.tb00111.x [10] N. Panousis, N. Siachos, G. Kitkas, E. Kalaitzakis, M. Kritsepi-
Konstantinou, G. E. Valergakis, Hematology reference intervals
for neonatal Holstein calves, Res Vet Sci. 118 (2018), pp. 1-10. DOI: 10.1016/j.rvsc.2018.01.002 As a matter of fact, when multiple analytes are measured, the
more analytes measured, the greater the risk of interpreting
values as pathological when they are actually due to chance. On
the other hand, just as a value falling within the reference limits
does not mean that the presence of underlying pathologies can
be excluded, meanwhile the mere fact that a value falls within the
reference limits does not imply that the patient is healthy [4]. [11] E. Rosset, B. Rannou, G. Casseleux, K. Chalvet-Monfray, S. Buff,
Age-related changes in biochemical and hematologic variables in
Borzoi and Beagle puppies from birth to 8 weeks, Vet. Clin. Pathol. 41(2) (2012), pp. 272-282. DOI: 10.1111/j.1939-165X.2012.00415.x DOI: 10.1111/j.1939-165X.2012.00415.x [12] R. Rørtveit, B. K. Saevik, A. V. Eggertsdóttir, E. Skancke,
F. Lingaas, S. I. Thoresen, J. H. Jansen, Age-related changes in
hematologic and serum biochemical variables in dogs aged 16-60
days, Vet. Clin. Pathol. 44(1) (2015), pp. 47-57. DOI: 10.1111/vcp.12220 However, the importance of reference intervals remains
fundamental in clinical practice and must always be accompanied
by concrete clinical findings on the patient. In conclusion, this paper highlights the importance of
reference intervals (RIs) in clinical pathology. While RIs are used
in other areas of veterinary medicine that involve measurements
such as diagnostic imaging, with other units of measurements
and values, the focus of this discussion has been on the
importance of RIs in interpreting laboratory test results. [13] J. Mariella, A. Pirrone, F. Gentilini, C. Castagnetti, Hematologic
and biochemical profiles in Standardbred mares during
peripartum, Theriogenology 81(4) (2014), pp. 526-534. DOI: 10.1016/j.theriogenology.2013.11.001 [14] P. Moretti, S. Paltrinieri, E. Trevisi, M. Probo, A. Ferrari,
A. 5. STATISTICAL METHODS In the reference interval guidelines of ASVCP there is a table
that recommends procedures for establishing RIs based on
reference sample size and distribution; for example, when
reference samples are ≥ 20 and < 40, if the distribution is
Gaussian, it could be used a parametric method, while, if non-
Gaussian, a robust method [5]. This large number of required data are often impossible to
achieve in veterinary clinical pathology, but at the same time it is
stated that “the smaller the sample is, the higher is the degree of
uncertainty in the estimation of reference limits” [5]. Consequently, for small samples, it is better to report them
graphically in histograms or dot plot and subsequently use
different methods in order to see which one could fit better [38]. This large number of required data are often impossible to
achieve in veterinary clinical pathology, but at the same time it is
stated that “the smaller the sample is, the higher is the degree of
uncertainty in the estimation of reference limits” [5]. Consequently, for small samples, it is better to report them
graphically in histograms or dot plot and subsequently use
different methods in order to see which one could fit better [38]. This was tested in a study that randomly selected a small
sample from a large sample group, showing that the RI estimated
from small samples was highly variable and dissimilar from the This was tested in a study that randomly selected a small
sample from a large sample group, showing that the RI estimated
from small samples was highly variable and dissimilar from the Several feline breeds have been evaluated for hematological
and biochemical measurements in comparison to standard March 2024 | Volume 13 | Number 1 | 3 ACTA IMEKO | www.imeko.org conditions from birth to 5 months of age, Vet Q. 23(2) (2001),
pp. 92-95. / conditions from birth to 5 months of age, Vet Q. 23(2) (2001),
pp. 92-95. RI of the large whole sample. In conclusion, when there are small
samples, the bias and not normality of distribution increase [38]. This is why reporting all values and forgoing calculations when
there are less than 20 samples is preferable [39]. pp. 92-95. DOI: 10.1080/01652176.2001.9695090 DOI: 10.1080/01652176.2001.9695090 [8] [8]
M. C. Veronesi, A. Gloria, S. Panzani, M. P. Sfirro, A. Carluccio,
A. REFERENCES [1]
S. L. Stockham, M. A. Scott, Introductory concepts, in:
Fundamentals of Veterinary Clinical Pathology 2nd ed., Ames
Iowa, Blackwell Pub, 2008, pp 16-20, ISBN 9780813800769 [15] S. Zaldívar-López, L. M. Marín, M. C. Iazbik, N. Westendorf-
Stingle, S. Hensley, C. G. Couto, Clinical pathology of
Greyhounds and other sighthounds, Vet. Clin. Pathol. 40(4)
(2011), pp. 414-425. DOI: 10.1111/j.1939-165X.2011.00360.x [2]
K. S. Latimer, J. R. Duncan, Generating and interpreting test
results: test validity, quality control, reference values, and basic
epidemiology, in: Duncan & Prasse's Veterinary Laboratory
Medicine: Clinical Pathology, 5th ed. Chichester West Sussex UK,
Wiley-Blackwell, 2011, pp. 369-373, ISBN 9780813820149. [16] C. Campora, K. P. Freeman, F. I. Lewis, G. Gibson, F. Sacchini,
M. J. Sanchez-Vazquez,
Determination
of
haematological
reference intervals in healthy adult greyhounds, J. Small Anim. Pract. 52(6) (2011), pp. 301-309. DOI: 10.1111/j.1748-5827.2011.01070.x y
pp
[3]
A. Geffré, K. Friedrichs, K. Harr, D. Concordet, C. Trumel, J.P. Braun, Reference values: a review, Vet. Clin. Pathol. 38(3) (2009),
pp. 288-298. DOI: 10.1111/j.1939-165X.2009.00179.x DOI: 10.1111/j.1748-5827.2011.01070.x [17] I. Uhríková, A. Lačňáková, K. Tandlerová, V. Kuchařová,
K. Řeháková, E. Jánová, J. Doubek, Haematological and
biochemical
variations
among
eight
sighthound
breeds,
Aust. Vet. J. 91(11) (2013), pp. 452-459. DOI: 10.1111/avj.12117 [4]
S. Paltrinieri, W. Bertazzolo, A. Giordano, Come fare e intepretare
correttamente un esame: errori preanalitici e interpretativi - How
to do and interpret correctly examination: analytical and
interpretative errors, in: Patologia clinica del cane e del gatto:
approccio pratico alla diagnostica di laboratorio - Clinical
pathology of the dog and cat: practical approach to laboratory
diagnostics, Italia, ed. Elsevier Health Sciences Italy, 2015, pp. 18.20, ISBN 8821431592 [In Italian] [18] P. Scarpa, B. Ruggerone, S. Gironi, T. Vitiello, S. Paltrinieri,
Haematological and biochemical reference intervals in healthy
racing and retired Italian Greyhounds, Acta Vet. Hung. 68(1)
(2020), pp. 71-78. DOI: 10.1556/004.2020.00006 [5]
K. R. Friedrichs, K. E. Harr, K. P. Freeman, B. Szladovits, R. M. Walton, K. F Barnhart, J. Blanco-Chavez; American Society for
Veterinary Clinical Pathology, ASVCP reference interval
guidelines: determination of de novo reference intervals in
veterinary species and other related topics, Vet. Clin. Pathol. 41(4)
(2012), pp. 441-453. DOI: 10.1111/vcp.12006 [19] L. Nielsen, M. Kjelgaard-Hansen, A. L. Jensen, A. T. Kristensen,
Breed-specific variation of hematologic and biochemical analytes
in healthy adult Bernese Mountain dogs, Vet. Clin. Pathol. 39(1)
(2010), pp. 20-28. DOI: 10.1111/j.1939-165X.2009.00186.x [20] B. Ruggerone, M. Giraldi, S. Paltrinieri, P. 5. STATISTICAL METHODS Contri, Blood analysis in newborn donkeys: hematology,
biochemistry, and blood gases analysis, Theriogenology 82(2)
(2014), pp. 294-303. 6. CONCLUSIONS Minuti, A. Giordano, Reference intervals for hematological and
biochemical parameters, acute phase proteins and markers of
oxidation in Holstein dairy cows around 3 and 30 days after
calving, Res. Vet. Sci. 114 (2017), pp. 322-331. DOI: 10 1016/j rvsc 2017 06 012 DOI: 10.3390/ani10071212 [31] S. Paltrinieri, F. Ibba, G. Rossi, Haematological and biochemical
reference intervals of four feline breeds, J. Feline Med. Surg. 16(2)
(2014), pp. 125-136. DOI: 10.1177/1098612X13499337 [23] M. E. Gelain, W. Bertazzolo, G. Tutino, E. Pogliani, F. Cian, M. K. Boudreaux, A novel point mutation in the β1-tubulin gene in
asymptomatic macrothrombocytopenic Norfolk and Cairn
Terriers, Vet. Clin. Pathol. 43(3) (2014), pp. 317-321. DOI: 10.1111/vcp.12168 [32] S. Paltrinieri, M. Giraldi, A. Prolo, P. Scarpa, E. Piseddu,
M. Beccati, B. Graziani, S. Bo, Serum symmetric dimethylarginine
and creatinine in Birman cats compared with cats of other breeds,
J. Feline Med. Surg. 20(10) (2018), pp. 905-912. DOI: 10.1177/1098612X17734066 [24] H. D. Pedersen, J. Häggstrom, L. H. Olsen, K. Christensen, A. Selin, M. L. Burmeister, H. Larsen, Idiopathic asymptomatic
thrombocytopenia in Cavalier King Charles Spaniels is an
autosomal recessive trait, J. Vet. Intern. Med. 16(2) (2002),
pp. 169-173. DOI: 10.1111/j.1939-1676.2002.tb02350.x [33] A. Gavazza, M. Quagliardi, G. Rossi, I. Jen, K. Mu, A. Roncarati,
L. Galosi, B. Biancani. Hematological parameters in subadult
female Risso’s dolphins (Grampus griseus), Proc. of the EAAM 51st
annual symposium, Oceanografic-Valencia, Spain, 7-11 March
2023, pp. 43. [25] J. Kelley, L. C. Sharkey, P. W. Christopherson, A. Rendahl, Platelet
count and plateletcrit in Cavalier King Charles Spaniels and
Greyhounds using the Advia 120 and 2120, Vet. Clin. Pathol. 43(1)
(2014), pp. 43-49. pp
[34] B. Biancani, M. Quagliardi, G. Rossi, I. Jen, K. Mu, A. Roncarati,
L. Galosi, A. Gavazza. Biochemical parameters in subadult female
Risso’s dolphins (Grampus griseus), Proc. of the EAAM 51st annual
symposium, Oceanografic-Valencia, Spain, 7-11 March 2023, pp. 41. [26] A. R. Torres, S. E. Cassle, M. Haymore, R. C. Hill, Hematologic
differences between Dachshunds and mixed breed dogs, Vet. Clin. Pathol. 43(4) (2014), pp. 519-524. DOI: 10.1111/vcp.12178 [35] P. Natchtigall, J. L. Pawloski, J. P. Schroeder, S. Sinclair, Successful
maintenance and research with a formerly stranded Risso’s
dolphin (Grampus griseus), Aquatic Mammals 16(1) (1990), pp. 8-
13. p
[27] P. L. Tisdall, G. B. Hunt, G. Tsoukalas, R. Malik, Post-prandial
serum bile acid concentrations and ammonia tolerance in Maltese
dogs with and without hepatic vascular anomalies, Aust. Vet. J. 72(4) (1995), pp. 121-126. DOI: 10.1111/j.1751-0813.1995.tb15029.x [36] K. Shirai, T. Sataki, Haematological findings in captive dolphins
and whales, Aust. Vet. J. 75(7) (1997), pp. 512-514. DOI: 10.1111/j.1751-0813.1997.tb14384.x [28] O. Aniołek, A. Barc, A. Jarosińska, G. March 2024 | Volume 13 | Number 1 | 5 REFERENCES Scarpa, Hematologic
and biochemical reference intervals in Shetland Sheepdogs, Vet. Clin. Pathol. 47(4) (2018), pp. 617-624. DOI: 10.1111/vcp.12665 [6]
Clinical and Laboratory Standards Institute (CLSI) document
EP28-A3c, Defining, Establishing, and Verifying Reference
Intervals in the Clinical Laboratory; Approved Guideline-Third
Edition. Wayne, PA, 2008, ISBN 978-1-56238-682-5. [21] R. Lavoué, A. Geffré, J. P. Braun, D. Peeters, F. Granat,
N. Bourgès-Abella, C. Trumel, Breed-specific hematologic
reference intervals in healthy adult Dogues de Bordeaux, Vet. Clin. Pathol. 43(3) (2014), pp. 352-361. DOI: 10.1111/vcp.12151 y
[7]
H. Brommer, M. M. Sloet van Oldruitenborgh-Oosterbaan,
B. Kessels, Haematological and blood biochemical characteristics
of Dutch warmblood foals managed under three different rearing March 2024 | Volume 13 | Number 1 | 4 ACTA IMEKO | www.imeko.org [22] O. W. Schalm, Erythrocyte macrocytosis in miniature and toy
poodles, Canine Practice 3 (1976), pp. 55-57. DOI: 10.3390/ani10071212 DOI: 10.3390/ani10071212 Zdzislaw, Evaluation of
frequency and intensity of asymptomatic anisocytosis in the
Japanese dog breeds Shiba, Akita, and Hokkaido, Acta Veterinaria
Brno 86 (2017), pp. 385-391. DOI: 10 2754/avb201786040385 [37] P. S. Horn, L. Feng, Y. Li, A. J. Pesce, Effect of outliers and
nonhealthy individuals on reference interval estimation, Clin. Chem. 47(12) (2001), pp. 2137-2145. DOI: 10.1093/clinchem/47.12.2137 [38] A. Geffré, J. P. Braun, C. Trumel, D. Concordet, Estimation of
reference intervals from small samples: an example using canine
plasma creatinine, Vet Clin Pathol. 38(4) (2009), pp. 477-484. DOI: 10.1111/j.1939-165X.2009.00155.x [29] Gazzetta Ufficiale della Repubblica Italiana, Linea guida relativa
all’esercizio delle attività sanitarie riguardanti la medicina
trasfusionale in campo veterinario - Guideline on the practice of
transfusion medical health activities in the veterinary field, n.25, 1
February 2016. Online [Accessed 15 February 2024]
https://www.fnovi.it/sites/default/files/LineeGuidaMedicinaTr
asfus_0.pdf [In Italian] [39] J. P. Braun, D. Concordet, A. Geffré, N. Bourges Abella,
C. Trumel, Confidence intervals of reference limits in small
reference sample groups, Vet. Clin. Pathol. 42(3) (2013), pp. 395-
398. / DOI: 10.1111/vcp.12065 [30] A. Miglio, A. Gavazza, D. Siepi, F. Bagaglia, A. Misia,
M. T. Antognoni, Hematological and Biochemical Reference
Intervals for 5 Adult Hunting Dog Breeds Using a Blood Donor
Database, Animals 10(7) (2020), art. No. 1212. March 2024 | Volume 13 | Number 1 | 5 ACTA IMEKO | www.imeko.org
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Which factors determine adaptation to drought amongst farmers in Northern Thailand? Investigating farmers’ appraisals of risk and adaptation and their exposure to drought information communications as determinants of their adaptive responses
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Mitigation and adaptation strategies for global change
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Mitig Adapt Strateg Glob Change (2024) 29:6
https://doi.org/10.1007/s11027-023-10099-w Mitig Adapt Strateg Glob Change (2024) 29:6
https://doi.org/10.1007/s11027-023-10099-w ORIGINAL ARTICLE Abstract Drought communications constitute an important source of learning about climate risks
and responses that can assist adaptation decision-making amongst those whose livelihoods
are threatened by drought. This paper applies Protection Motivation Theory to explore
associations between drought communications and attitudes towards drought risk and adap-
tation amongst farmers in Northern Thailand. The analysis reveals links between drought
communications, farmers’ adaptation appraisal, and their adaptation decisions, whilst
links with risk appraisal are minimal. The results highlight positive feedbacks between
adaptation experience and appraisal and reveal a weak negative relationship between risk
appraisal and adaptation appraisal. The findings imply benefits to framing drought com-
munications in terms of the efficacy and attainability of suitable adaptations, rather than
simply highlighting drought risks or providing drought warnings, to best enable farmers to
build drought resilience. Keywords Protection Motivation Theory · Drought risk perceptions · Climate change
resilience · Thailand Chloe Sutcliffe1,2 · Ian Holman1 · Daniel Goodwin1,3 · Gloria Salmoral1 ·
Liwa Pardthaisong4 · Supattra Visessri5,6 · Chaiwat Ekkawatpanit7 · Dolores Rey1 Received: 3 February 2022 / Accepted: 19 December 2023 / Published online: 11 January 2024
© The Author(s) 2024 Extended author information available on the last page of the article Which factors determine adaptation to drought
amongst farmers in Northern Thailand? Investigating
farmers’ appraisals of risk and adaptation and their exposure
to drought information communications as determinants
of their adaptive responses Chloe Sutcliffe1,2 · Ian Holman1 · Daniel Goodwin1,3 · Gloria Salmoral1 ·
Liwa Pardthaisong4 · Supattra Visessri5,6 · Chaiwat Ekkawatpanit7 · Dolores Rey1 Chloe Sutcliffe1,2 · Ian Holman1 · Daniel Goodwin1,3 · Gloria Salmoral1 ·
Liwa Pardthaisong4 · Supattra Visessri5,6 · Chaiwat Ekkawatpanit7 · Dolores Rey1 Chloe Sutcliffe1,2 · Ian Holman1 · Daniel Goodwin1,3 · Gloria Salmoral1 ·
Liwa Pardthaisong4 · Supattra Visessri5,6 · Chaiwat Ekkawatpanit7 · Dolores Rey1 1 Introduction Agriculture is sensitive to water availability, with climatic extremes leading to substantial
yield losses (Lesk et al. 2016). Droughts constrain crop productivity and pose an ongoing
threat to global food production which is likely to increase due to climate change, popula-
tion growth and pressure on water resources (Daryanto et al. 2017; Leng and Hall 2019). Building agricultural drought resilience is of crucial importance to global food security and
human well-being (Carrão et al. 2016; Challinor et al. 2014). However, many factors limit
effective adaptation, including informational, attitudinal and behavioural barriers (Howden
et al. 2007). Understanding how to overcome these barriers and increase the adaptive 23451 56789)
3 Mitig Adapt Strateg Glob Change (2024) 29:6 6
Page 2 of 21 6 capacity of farmers must be prioritised to support agricultural development, crisis preven-
tion and vulnerability reduction (Lipper et al. 2014). As such, research has increasingly
focused on how farmers’ understandings and perceptions of climate change determine their
adaptation decisions (Deressa et al. 2011; Mertz et al. 2009; Sutcliffe et al. 2016), with
studies addressing the cognitive processes that link climate perceptions with decisions to
implement action (Grothmann and Patt 2005; Truelove et al. 2015). The attitudinal factors that determine decisions to employ a protective measure in
response to a threat (i.e. adaptation) have been modelled using the Protection Motivation
Theory (PMT) conceptual framework. The PMT framework identifies two cognitive path-
ways which are believed to determine whether individuals decide to employ protective
behaviour. These pathways are risk appraisal and adaptation appraisal. Risk appraisal com-
bines an assessment of the potential severity of a negative impact with the likelihood that
such an impact will occur. Adaptation appraisal combines an assessment of how effectively
a proposed response would curtail the threat of the negative impact (response efficacy), with
an assessment of the capacity of the individual to employ that particular response (self-effi-
cacy), along with an assessment of the potential costs of doing so (Milne et al. 2000).i PMT was first employed in the 1970s within social psychology studies of health
behaviours (Rogers 1975) and has since been highly influential across a range of disci-
plinary areas including business studies, computing and environmental science. It has
been applied in relation to a wide array of different threats, including information secu-
rity (Haag et al. 2021; Herath and Rao 2009), pollution risks (Wang et al. Mitig Adapt Strateg Glob Change (2024) 29:6 Page 3 of 21 6 Key questions: 1. To what extent do PMT and drought communication variables influence adaptation
outcome variables?f 1. To what extent do PMT and drought communication variables influence adaptation
outcome variables?f 2. Which determinants affect adaption and risk appraisal? f
3. What role do socioeconomic factors (age, education and wealth) play in determining
access to drought communications? f
3. What role do socioeconomic factors (age, education and wealth) play in determining
access to drought communications? Based on these questions, we discuss the extent to which PMT variables predict adapta-
tion outcome variables and how institutional drought communications influence farmers’
motivations and actions to protect their production activities from the risk of drought. By
using the PMT framework to understand the relative importance of farmers’ appraisals of
risk and of adaptive responses within their agricultural decision-making and illustrating
the influence of drought communications, the paper provides communication design rec-
ommendations that should increase farmers’ implementation of adaptations to reduce the
negative impacts of drought on agricultural livelihoods. 1 Introduction 2019) and the
threat of extreme weather and climate change (Bubeck et al. 2012; Kuruppu and Liv-
erman 2011; Grothmann and Patt 2005; Grothmann and Reusswig 2006). Most studies
related to extreme weather have concentrated on flood protection, often in a European
context (e.g. Babcicky and Seebauer 2017; Bamberg et al. 2017; Bradford et al. 2012;
Poussin et al. 2014). Few studies have used PMT to explore drought adaptation in the
Global South, although a body of research is building in Asian (e.g. Keshavarz and Kar-
ami 2016; Truelove et al. 2015) and African (e.g. Gebrehiwot and van der Veen 2015;
Tabe-Ojong et al. 2020; Wens et al. 2021) contexts. Knowledge and understanding of climate change are positively associated with farm-
ers’ adaptation intentions (Ngo et al. 2020). Studies have noted the influence of different
sources of information for risk and adaptation appraisal (Milne et al. 2000), including
social networks (Babcicky and Seebauer 2017; Haer et al. 2016) and public institutions
(Grothmann and Reusswig 2006), and the role of institutions in communicating climate
information to influence local level responses (Dorward et al. 2020; Haer et al. 2016; Stey-
nor et al. 2021). The provision of drought warnings, forecasts and advice can effectively
build resilience to drought by mitigating impacts and leading to faster recoveries within
farming communities in developing countries (Ewbank et al. 2019). Optimising the provi-
sion of this information is an increasingly key necessity for policy makers, climate services
and agricultural development practitioners. This paper aims to build understanding of the processes that determine adaptation to
drought amongst farmers in Northern Thailand. In it, we explore and compare the roles
that risk appraisal and adaptation appraisal play as determinants of past adaptations and
their perceived success and as determinants of responsiveness to official adaptation recom-
mendations and the desire to implement further adaptations in future. We also investigate
the extent to which exposure to and perceptions of both formal and informal drought com-
munications are associated with these adaptation outcomes. 1 3 1 Mitig Adapt Strateg Glob Change (2024) 29:6 2.3.1 Adaptation outcome variables The first set of questions within the questionnaire collected data from respondents on
their adaptation actions and perceptions. In particular, respondents provided informa-
tion about the number of adaptations they had undertaken in the past (Ai), whether or
not they considered these adaptations to have successfully reduced their longer term
vulnerability to drought (Aii), whether they had changed anything about their agricul-
tural production activities in response to having a received a drought warning (Aiii) and
whether or not they had a desire to implement any further adaptations that they had not
yet been able to (Aiv) (see Table 1 for further details). 2.2 Sampling and data collection The research team purposively selected a sample of villages exposed to different lev-
els of drought over the last decade. Villages both within and outside of formal irriga-
tion zones were selected to represent a range of typical agricultural production profiles
(such as rice, longan and livestock production). According to the customary context,
the village headperson (a village elder who acts as the community leader) in each vil-
lage was contacted initially, and the research team requested their permission to con-
duct the research. The village headperson then invited farm household heads to partici-
pate. As opposed to random sampling of households, this method may have resulted in
a degree of sampling bias, meaning that the sample should not be interpreted as fully
representative. In particular, readers should bear in mind the possibility that due to
the selection of participants by the village headperson, older, wealthier farmers may
be over-represented, whilst poorer, younger farmers and female-headed farms may be
under-represented. However, this sampling approach was considered the most appropri-
ate given the local socio-cultural context, and the validity of the analysis reported herein
is not contingent on a perfectly representative sample. Trained enumerators undertook
data collection after obtaining informed consent. In total, 176 questionnaires were com-
pleted in January 2020 with a close to even distribution of respondents coming from the
provinces of Chiang Mai (n = 41), Lamphun (n = 45), Kamphaeng Phet (n = 45) and
Tak (n = 45). The structured questionnaire collected socioeconomic, agricultural, risk
appraisal, adaptation appraisal, adaptation behaviour and drought communications data
from farmers. The questionnaire was originally written in English, translated into Thai
and administered before translation back to English for analysis. Questionnaires were
anonymised, and the questionnaire and dataset can be found at https://doi.org/10.17862/
cranfield.rd.16553136. 2.1 Case study As Southeast Asia’s second largest economy, Thailand’s development significantly affects
its neighbours, and the country plays an important role in policy-making within the wider
region (Kiguchi et al. 2021). Agricultural production in Thailand, like other countries in
Southeast Asia, is important for global food supply, with the region as a whole contribut-
ing 40% of global rice exports (Yuan et al 2022). However, rice yields have stagnated in
Thailand in recent decades, as they have in several neighbouring countries, with drought
having played a major role in crop losses in Thailand, Cambodia and Myanmar since 2015
(Venkatappa et al. 2021). Across Southeast Asia, there is an increasing risk of agricultural
drought in the near term (Amnuaylojaroen and Chanvichit 2019), with projections sug-
gesting countries across the region will experience longer, more intense and more severe
droughts prior to 2050 due to the impacts of climate change under a range of probable
emission scenarios (Supharatid and Nafung 2021). Identifying strategies to support farmers
to stabilise and increase yields whilst reducing their vulnerability to drought is therefore a
key concern for policy makers across the region that may look to Thailand as an example. Drought has already had major impacts on the availability of water for irrigation in
Thailand in recent years (Kiguchi et al. 2021), with this problem set to grow in the near
future (Chang et al. 2021; Singhrattna and Babel 2011). Due to the recent incidence and
severity of agricultural drought impacts, the Ping River catchment (a major tributary of
Thailand’s main interior river, the Chao Phraya) was chosen for data collection. Signifi-
cant regional droughts took place in 2015–2016 and 2019–2020 (Khan and Gilani 2021;
Pak-uthai 2019), with reports that the timing of government drought warnings and advice
hampered effective farm responses (Khan and Gilani 2021). Despite the major agricultural
impacts and increasing risk of drought in Thailand, few studies have focused on climate
risk communications (Arunrat et al. 2017; Lebel et al. 2018). As such, Northern Thailand
provides a suitable context for investigating how institutional drought communications 3 3 Mitig Adapt Strateg Glob Change (2024) 29:6 6
Page 4 of 21 6 6 currently support farmers to build resilience to drought and for identifying strategies to
optimise such support in future. 2.3.2 PMT variables Respondents were then asked to indicate agreement (measured on a five-point scale
from strongly agree to strongly disagree) with statements constructed to investigate 1 3 Page 5 of 21
6 Mitig Adapt Strateg Glob Change (2024) 29:6 6 1 3
Table 1 Description and questionnaire numbers for key adaptation, PMT and drought communication variables
Variable name
Variable type
Variable composition and explanation
Total adaptation count (Ai)
Continuous
Total count of number of past adaptations indicated by the respondent
Proportion adaptations successful (Aii)
Continuous
Total count of adaptations perceived to reduce vulnerability to drought impacts in future divided by
grand total of adaptations
Adapted in response to drought warning (Aiii) Dichotomous (0, 1) Yes/no response to questions, “During previous droughts was your household informed that a drought
was coming? Did your household farm change anything as a result of receiving this information?”
Desires to adapt further (Aiv)
Dichotomous (0, 1) Yes/no response to question, “To reduce the impacts of droughts in the long-term, are there any adapta-
tions you would like to undertake but did not yet?”
Adaptation appraisal
Continuous
Sum of adaptation cost scores subtracted from sum of adaptation and self-efficacy statement scores. 2.3.2 PMT variables (“Appropriate adaptation strategies exist for protecting farms like mine from drought” + “I feel confi-
dent implementing adaptation strategies for protecting my household farm from drought” + “I consider
myself well informed about drought”) minus (“I work hard every day, so I feel too tired to implement
the necessary adaptation strategies” + “Farm work is too busy, so there is no extra time to implement
the necessary adaptation strategies” + “I have not got enough money to implement the necessary
adaptation strategies”)
Risk appraisal
Continuous
Combined scale (sum) consisting of three statements
“My household farm is likely to be affected by drought in the next five years” + “The impacts of drought
on my farm are likely to be severe” + “The impacts of drought on my household farm are worse than
before”
Drought information types count
Continuous
Sum of drought information types received by respondent
Drought information channels count
Continuous
Sum of information channels through which drought information was received
Drought information efficacy scores
Continuous
Agreement (using a five-point scale) that drought information had: helped them prepare for drought
(“helped prepare”); arrived early enough in the season to be of use (“early enough”); was relevant to
the household’s farming activities (“relevant”); was accurate (“accurate”); was in the correct format
(“best format”); and whether the organisation providing the information was receptive to receiving
farmer drought and adaptation information back (“receptive”). These measures were combined to form
a single variable
Number of other farmers drought communica-
tions were received from
Ordinal
Number of farmers (ranked groups) that had informed household a drought was coming and what to do Mitig Adapt Strateg Glob Change (2024) 29:6 6
Page 6 of 21 6 their levels of adaptation appraisal and risk appraisal (Milne et al. 2000) (see Table 1). An overall adaptation appraisal score was calculated, based on the sum of perceived
self-efficacy and perceived response efficacy scores, less the sum of perceived response
costs scores. Similarly, an overall risk appraisal score was based on the sum of per-
ceived vulnerability and perceived severity scores. 2.3.4 Land and asset scores In contexts where agricultural households use some of their produce for subsistence, mon-
etary income may not accurately reflect household wealth. This necessitates assessing farm
household wealth in terms of asset and land ownership. Scores for land and asset owner-
ship were calculated following Cordova (2009) and Filmer and Pritchett (2001), whereby
standardised scores for a range of culturally prevalent assets are derived from the frequency
of their ownership (represented using a dummy variable as either 0 or 1) amongst respond-
ent households in the dataset. In the case of land ownership, since this was a continuous
variable, farm sizes were split into five bands (at cut points of 95%, 75%, 50%, 25% and 5%
of the sample) represented by dummy variables. Standardised scores were then calculated
based on frequencies for each band, and scores for household land ownership were made
up by adding these scores cumulatively, so that those with land in the highest band accu-
mulated the scores for the band they were in as well as all the lower bands. 2.3.3 Drought communications The questionnaire then collected information on whether respondents had received drought
warnings, which types of drought monitoring information they had received (for example,
weather forecasts, river discharge information and irrigation availability forecasts) and
which channels drought information was received through, in addition to the perceived effi-
cacy of that information. These were converted into counts of drought information types
and channels and a perceived drought information efficacy score (Table 1). 3 Results In the “3” section, RQ1 presents the factors influencing the four adaptation outcome
variables using individual regression models. In each case, the influence of the PMT and
drought communication variables on the adaptation outcome variables is described, and
other influential variables are identified. RQ2 explores factors affecting adaptation and risk
appraisal, and RQ3 identifies relationships between adaptation, drought communications
and socioeconomic characteristics (age, education and wealth). 3.1.1 Total adaptation count (regression model Ai) Model Ai was significant (F(8) = 12.232, p = .000) and accounted for 37% of the variance
in the total adaptation count scores (with an adjusted R2 of .37). Of the PMT variables,
only adaptation appraisal significantly predicted total adaptation count, risk appraisal did
not. Of the adaptation appraisal components, the statement “Appropriate adaptation strat-
egies exist for protecting my household farm from drought” was a stronger determinant
than the aggregate “adaptation appraisal” variable; hence, only the “Appropriate…” state-
ment variable was selected for inclusion in the model. Of the socioeconomic factors, only
age was found to contribute significantly, with a negative relationship evident between age
and the number of adaptations employed. In terms of drought communication variables,
the count of types of drought information received significantly predicted total adapta-
tion counts, as did agreement with the information efficacy statements, “early enough”,
“accurate”, “relevant” and “best format”, and the number of farmers that information had
been received from. Whilst the relationship between total adaptation count and the state-
ments “relevant” and “accurate” were positive, the relationships with the statements “early
enough” and “best format” were negative, suggesting that as the number of adaptations
undertaken increased, agreement with these particular statements declined (see Table 2). 2.3.5 Statistical analysis Relationships between the four adaptation outcome variables (Ai–Aiv), PMT variables
and drought communications variables, in addition to socioeconomic (age, education and
wealth), and agricultural and policy factors (formal irrigation support and drought compen-
sation provision) were investigated using multiple regression analyses and, where relevant,
t-tests, ANOVA, chi-square, Fisher’s exact test and Pearson’s and Spearman’s correlation
coefficients. Linear regression was used to identify significant determinants of the two con-
tinuous adaptation outcome variables Ai and Aii (“total adaptation count” and “propor-
tion adaptations successful”, respectively). Binary logistic regression was used to construct
models for the two dichotomous adaptation outcome variables Aiii and Aiv (“adapted in
response to drought warning” and “desires further adaptation”, respectively), with interac-
tion terms investigated for potential effects (Field 2018). f
The regression analysis sought to investigate relationships between the PMT variables
(adaptation appraisal and risk appraisal), the drought information variables and the four adap-
tation outcome variables (Ai–Aiv). Alternative regression analyses were run in turn incorpo-
rating either the compound adaptation and risk appraisal scores or their disaggregated com-
ponent variables (such as, for example, agreement scores with the statements representing Mitig Adapt Strateg Glob Change (2024) 29:6 Page 7 of 21 6 response efficacy and self-efficacy). This was done in order to compare results and determine
whether the compound scores or the disaggregated scores were more powerful predictors of
the independent adaptation variable (Babazadeh et al. 2017). Where the separate component
factors proved to have stronger predictive power than the compound variables, these were
included preferentially within the regression models presented in the results.i response efficacy and self-efficacy). This was done in order to compare results and determine
whether the compound scores or the disaggregated scores were more powerful predictors of
the independent adaptation variable (Babazadeh et al. 2017). Where the separate component
factors proved to have stronger predictive power than the compound variables, these were
included preferentially within the regression models presented in the results.i Following a principle of parsimony (Field 2018), variables found not to significantly
predict the outcome variable were excluded (Braun and Oswald 2011). Variables were
entered into the models hierarchically, with those expected to have the largest influence
entered first. 3.1.2 Proportion of adaptations perceived as successful for longer term drought
vulnerability reduction (regression model Aii) R2 .21 (Cox & Snell) and .285 (Nagelkerke). Model
χ2 (5) = 36.573, p = .000)i Table 4 Binary logistic regression results showing predictors of whether the household changed anything in
response to receiving a drought warning (model Aiii). R2 .21 (Cox & Snell) and .285 (Nagelkerke). Model
χ2 (5) = 36.573, p = .000)
95% confidence
interval
B
Standard error Wald statistic Sig. Odds ratio
Lower
Upper
(Constant)
−0.562
0.366
2.36
0.124 0.57
Received financial drought
compensation × fruit
production
−1.182
0.433
7.454
0.006 0.307
0.131
0.716
Instances of drought in last
10 years
0.346
0.149
5.411
0.02
1.413
1.056
1.891
Adaptation appraisal
0.44
0.196
5.055
0.025 1.552
1.058
2.277
Received weather forecast
information
1.148
0.52
4.874
0.027 3.153
1.138
8.74 adaptation appraisal, animal husbandry and land within a supported irrigation zone, was
significant (F(5) = 5.132, p = .000) and accounted for 16% of the variance in the propor-
tion of adaptations perceived as successful. All included variables significantly determined
the proportion of adaptations perceived as successful, with negative relationships pertain-
ing with wealth and animal husbandry, indicating that wealthier farmers and those that
raised livestock tended to perceive a lower proportion of their past adaptations as success-
ful than other farmers (see Table 3). 3.1.2 Proportion of adaptations perceived as successful for longer term drought
vulnerability reduction (regression model Aii) Analysis showed that the drought communications variables did not improve model Aii,
and this group of variables was excluded. The final model, which incorporated age, wealth, 3 3 Mitig Adapt Strateg Glob Change (2024) 29:6 6
Page 8 of 21 Table 2 Linear model of predictors of total adaptation count (model Ai, adjusted R2 = .37, F(8) = 12.232, p =.000)
Unstandardised
coefficients
Standard-
ised coef-
ficients
t
Sig. 95.0% confidence interval
for B
B
Std. error Beta
Lower bound Upper bound
(Constant)
1.140
1.394
0.818
0.415 −1.615
3.896
“Appropriate adaptation strategies exist for protecting my household farm from drought”
0.839
0.145
0.386
5.774
0.000 0.552
1.126
Age group
−0.712 0.230
−0.206
−3.089 0.002 −1.167
−0.256
Count of different drought monitoring information types received
0.478
0.138
0.254
3.453
0.001 0.204
0.751
“The information provided arrives early enough in the season to be useful to my household
farm”
−0.527 0.168
−0.233
−3.140 0.002 −0.859
−0.195
“The information provided is usually relevant to the farming in the household farm activi-
ties”
0.605
0.253
0.207
2.393
0.018 0.105
1.105
“I am confident that the information provided is accurate”
0.722
0.224
0.243
3.217
0.002 0.278
1.165
“The information is provided in the best format for farmers to access and understand it
easily”
−0.845 0.261
−0.273
−3.240 0.001 −1.360
−0.329
Information was received from how many farmers
0.171
0.066
0.173
2.572
0.011 0.040
0.302 Mitig Adapt Strateg Glob Change (2024) 29:6 Page 9 of 21 Page 9 of 21 6 Table 3 Linear model of predictors of proportion of adaptations perceived to successfully reduce vulner-
ability to future droughts (model Aii, adjusted R2 = .16, F(5) = 5.132, p = .000)
Unstandardised coef-
ficients
Standardised
coefficients
Sig. 95.0% confidence interval
for B
B
Std. error
Beta
Lower bound
Upper bound
(Constant)
0.231
0.174
0.186
−0.112
0.575
Age
0.136
0.047
0.221
0.004
0.044
0.228
Wealth
−0.013
0.005
−0.182
0.017
−0.023
−0.002
Adaptation appraisal
0.075
0.026
0.222
0.004
0.025
0.126
Household raises animals
−0.135
0.054
−0.184
0.014
−0.242
−0.028
Household has land in
official irrigation zone
0.14
0.057
0.179
0.016
0.027
0.252 le 3 Linear model of predictors of proportion of adaptations perceived to successfully reduce vulner-
ity to future droughts (model Aii, adjusted R2 = .16, F(5) = 5.132, p = .000) Table 4 Binary logistic regression results showing predictors of whether the household changed anything in
response to receiving a drought warning (model Aiii). 3.1.4 Respondent indicated a desire to implement further adaptations (regression
model Aiv) Model Aiv was significant (χ2 (4) = 26.164, p = .000) and accounted for 23% of the vari-
ance. Within the model, the odds that a farmer would indicate wanting to make further
drought adaptations were significantly increased (by 1.44) for each additional drought
adaptation that they had previously implemented (Table 5). The odds also increased if
they indicated lacking money for implementing adaptations, feeling well informed about
drought risks or perceiving drought information to be in the right format for farmers. The
odds were lowered amongst farmers that indicated stronger agreement that the drought
information received had been relevant (Table 5). Whilst this last finding was unexpected, a
potential explanation could be that a perceived lack of relevant drought information caused
farmers to delay implementing their desired adaptations.i Whilst risk appraisal was not a significant predictor of a desire to implement further
adaptations in this model, a t-test revealed that farmers that indicated a desire to adapt fur-
ther had significantly higher risk appraisal scores than farmers that indicated that they did
not wish to implement any further changes (x ̅ = 11.9 vs x ̅ = 11.2, t = 1.985, p = 0.049). 3.1.3 Respondent adapted their agricultural practice in response to receiving
a drought warning (regression model Aiii) Model Aiii, which used binary logistic regression to investigate decisions to adapt in
response to official advice, was significant (χ2 (5) = 36.573, p = .000) and accounted for
29% of the variance. Both fruit production and rice production were found to interact sig-
nificantly with having received financial drought compensation, although when both fruit
and rice were included in the same model, the significance of the rice × compensation 3 3 Mitig Adapt Strateg Glob Change (2024) 29:6 Page 10 of 21 6 interaction was lost (Table 4). Nevertheless, this result points to a potentially important
relationship between past receipt of drought compensation and behavioural responses to
drought information amongst producers of these crops, which reduced the likelihood that
households would have changed something about their agricultural or livelihood activities
in response to receiving drought information. However, given the cross-sectional nature of
our data, we cannot discern whether failing to adapt in response to advice came before or
after receiving compensation. The other significant determinants (all of which increased
the likelihood that households would indicate having changed something in response to
the information they received) were the number of droughts that the household had experi-
enced over the last 10 years, the respondent’s adaptation appraisal score and if the respond-
ent had received weather forecast information (Table 4). Amongst these variables, having
received a weather forecast had an odds ratio of 3.15, meaning that the odds of households
adapting in response to official advice were 3.15 times greater amongst households that
had received weather forecast information than amongst those that had not. For every addi-
tional instance of drought the household reported experiencing in the last decade, the odds
that they had adapted in response to a drought warning were increased by 1.41, and for
every unit increase in adaptation appraisal, the odds increased by 1.56.i Although not found to contribute significantly to the logistic regression, growers that
indicated having received information via a social network messaging group were statisti-
cally more likely to have changed something in response to official advice (χ2 = 3.991, p =
.046), as were growers that had been educated to high school level or above (χ2 = 7.780, p
= .005). 3.2.1 Adaptation appraisal Multiple significant associations were found using Pearson’s correlation coefficient
between adaptation appraisal and the drought information communications variables. In 1 3 Page 11 of 21
6 Mitig Adapt Strateg Glob Change (2024) 29:6 Table 5 Binary logistic regression results showing predictors of “Are there any further changes you would like to implement on the farm, but that you did not yet?” (model
Aiv). R2 .17 (Cox & Snell) and .229 (Nagelkerke). Model χ2 (4) = 26.164, p = .000)
95% C.I. for
EXP (B)
B
Standard error
Wald statistic
Sig. Odds ratio
Lower
Upper
Constant
−5.852
2.027
8.334
0.004
0.003
“I have not got enough money to implement the necessary adaptation strategies”
0.626
0.212
8.689
0.003
1.869
1.233
2.833
Total number of adaptations
0.365
0.111
10.869
0.001
1.44
1.159
1.789
“I consider myself to be well informed about drought risks”
0.633
0.264
5.746
0.017
1.883
1.122
3.158
“The information provided is usually relevant to the farming in the household farm activities”
−0.959
0.409
5.512
0.019
0.383
0.172
0.853
“The information is provided in the best format for farmers to access and understand it easily”
1.037
0.394
6.935
0.008
2.82
1.304
6.102 1 3 Mitig Adapt Strateg Glob Change (2024) 29:6 6
Page 12 of 21 6 particular, confidence in implementing adaptation strategies correlated with the number of
types of climate information that had been received (r = .23, p = .04), the perception that
climate information had helped the farmer prepare in the past (r = .40, p = .001) and that
the information was provided in the best format for farmers (r = .36, p =.001).i Using multiple regression, adaptation appraisal scores were significantly determined by
education (Beta .271, p = .001), the total number of adaptations that had been undertaken
in the past (Beta .220, p = .005), the proportion of past adaptations perceived as successful
(Beta .213, p = .007) and the drought information efficacy score (Beta .199, p = .013). The
model was significant (F, 10.478; p = .000), and these four variables accounted for a fifth
of the variance in adaptation appraisal (adjusted R2 = .21).i Wealth correlated significantly with adaptation appraisal (r = .207, p = .006), but did
not contribute significantly to the regression model due to high semi-partial correlation
with education. 3.2.1 Adaptation appraisal Older farmers (aged over 60) were also found to have significantly lower
adaptation appraisal scores (mean = 0.27 vs 0.63 for younger farmers, t = −2.161, p =
.032). Adaptation appraisal was lower if a respondent perceived there to be higher costs asso-
ciated with undertaking adaptations. Respondents’ perceptions of adaptation costs were
measured via their scores for the availability of time, energy and finance for implementing
adaptations. Whilst correlation between the availability of time and energy for implement-
ing adaptations was high (.61, p = .000), there were no significant associations between
financial capacity to adapt and these other factors, suggesting that financial constraints
operate independently for a large proportion of the respondents.ii Spearman’s rank tests identified significant weak positive correlations between age and
agreement that the respondent could not implement adaptations due to insufficient energy
and time (ρ (rho) =.20, p = .009 and ρ (rho) = .16, p = .032, respectively). Conversely, edu-
cation correlated negatively with all three types of perceived adaptation cost: energy (ρ (rho)
= −.25, p =.001), time (ρ (rho) = −.32, p = .000) and money (ρ (rho) = −.24, p = .002). 1 3 3.3 RQ3: What role do socioeconomic factors (age, education and wealth) play
in determining access to drought communications? Statistical tests demonstrated the role of socioeconomic factors in determining access to
drought warnings. Older farmers reported receiving drought communications through
significantly fewer channels (mean = 1.0 vs 1.3 for farmers aged under 60, t = −2.883,
p = .004) and tended to report receiving drought communications from a smaller num-
ber of other farmers (although p = .059). Respondents in older age groups were less
likely to have received drought warnings that were accompanied by advice on what to
do (Fisher’s exact test = 14.998, p = .003), as were respondents with lower education
levels (Fisher’s exact test = 33.219, p = .000). Wealth was also a factor, with the highest mean household asset and land wealth scores
found within households that received both a drought warning and advice on what to do
(10.0), compared to lower scores amongst those that only received warnings (8.9), and those
that received neither (5.4), F(2172) = 5.416, p = .005. Adaptation appraisal scores followed the same pattern, with the lowest mean scores in
households that received no warning or advice (−0.85), mean scores of 0.36 in households
that only received the drought warning and mean scores of 0.73 in households that received
both the warning and advice, F(2169) = 15.419, p = .000. Respondents that received
drought warnings accompanied by adaptation advice were significantly more likely to have
changed something as a result, than those that received the drought warning alone (67.5%
compared to only 25.0%, χ2 = 24.696, p = .000). 3.2.2 Risk appraisal Farmers’ risk appraisal was not statistically associated with any of the climate information
variables, apart from a weak positive association with how many other farmers the infor-
mation was received from (r = .183, p = .016). Additionally, those that received informa-
tion at a village meeting had significantly stronger mean agreement that their household
was likely to be negatively affected by drought in the next 5 years, than those who received
the information by other means (mean = 3.98 vs 3.59, t = 2.655, p = .021). Growers that experienced drought in the most recent 3 years before the survey (in Janu-
ary 2020) displayed significantly higher agreement with the risk statements “The impacts
of drought on my household farm are likely to be severe” (mean = 4.02, t = 2.951, p =
.004) and “The impacts of drought on my farm are worse than before” (mean = 4.25, t =
5.316, p = .000) than those that did not have such recent experience of drought (means =
3.44 and 3.29, respectively). A positive correlation was observed with the total number
of economic and crop drought impacts indicated by the farmer (r = .262, p = .000), and
a weak negative correlation was observed between risk appraisal and adaptation appraisal
(r = −.169, p = .026). These findings suggest that direct experience of drought influences
risk appraisal, with more recent experiences having a more pronounced effect. f
The three variables, years since last drought experience (Beta −.216, p = .011), number
of farmers climate information was received from (Beta .185, p = .027) and household 1 3 Page 13 of 21 Mitig Adapt Strateg Glob Change (2024) 29:6 6 engages in animal husbandry (Beta .139, p = .096), resulted in a significant model (F =
5.721, p = .001) with an R2 of .096. 4.1 Conceptual diagram Figure 1 provides a conceptual overview of the main relationships reported in the results
above, with the routes connecting the four adaptation outcome variables with risk appraisal
and adaptation appraisal illustrated. Also shown are other sociodemographic and policy
factors found to have significant effects on either adaptation outcomes or the cognitive
pathways associated with adaptation appraisal and/or risk appraisal. Being in a formal irri-
gation zone is indicated as a policy intervention since farmers based in these areas benefit
from irrigation zone policies which provide additional government-funded support. The
results synthesised in Fig. 1 show that adaptation appraisal interacted directly with all four
of the adaptation outcome variables whilst only a weak association was found between
risk appraisal and one of the adaptation outcome variables. This lends weight to the idea
(reported in the context of flooding by Babcicky and Seebauer 2019) that, although there
may be two separate PMT pathways, only the one operating through adaptation or cop-
ing appraisal leads to the implementation of protective measures. As such, the results sup-
port Babcicky and Seebauer (2019), suggesting that the traditional interpretation of PMT,
where high risk appraisal and high adaptation appraisal combined lead to the implementa-
tion of protective measures, may not be empirically accurate in this kind of context. 1 3 Mitig Adapt Strateg Glob Change (2024) 29:6 6
Page 14 of 21 6 Fig. 1 The relationships represented are those uncovered using regression analysis, but the diagram addi-
tionally includes two relationships identified using other statistical tests: a negative correlation between
adaptation and risk appraisal and a difference in mean risk appraisal relating to desire to implement further
adaptations (both represented by dashed lines). These additions are considered relevant to the discussion. Arrows represent the direction of influence that was modelled, with line thickness representing the strength
of the relationship and colour representing whether the relationship was positive (green) or negative (red). For simplicity, symbols are used to represent groups of variables as described in the key. Where the model-
ling identified relationships with sub-components of these variable groups, the individual variable is identi-
fied using a label at the middle of the relationship line 1 3 4.3 To what extent do institutional drought communications influence farmers’
motivations and actions to protect their production activities from the risk
of drought? Whilst direct personal experience of climate impacts strongly shapes adaptation motiva-
tions (Niles et al. 2015), climate and adaptation information received through personal and
institutional networks also affects adaptation decision-making (Babcicky and Seebauer
2017). Further, contrary to the suggestion that top-down campaigns may have limited
capacity to affect the uptake of protective measures against extreme climate events (Haer
et al. 2016), our results indicate that institutional drought communications (via commu-
nity, private or government institutions) interact significantly with adaptation attitudes and
actions amongst agricultural producers. The diamonds in Fig. 1 illustrate where drought
communications or direct policy interventions (the provision of drought compensation or
official irrigation support) significantly predicted adaptation outcome variables. It is evi-
dent that both the size of a farmer’s local drought information network and the nature of the
drought information they receive via institutional channels interact significantly with adap-
tation intention and behaviour. The study has shown that receiving more different types of
drought information as well as the perceived efficacy of the information received are both
positively associated with higher confidence in implementing adaptations and with the
number of adaptations farmers have employed in the past. Additionally, our findings sup-
port the importance that drought information is accompanied by practical advice on what
to do to limit negative impacts (Ewbank et al. 2019; Haer et al. 2016; Koerth et al. 2013).i Although the institutional drought communication measures significantly contributed to
adaptation outcome variables (namely, adaptation appraisal scores, past adaptation counts
and whether respondents reported having adapted in response to official advice), they
appeared not to contribute to respondents’ drought risk appraisal. Instead, risk appraisal was
influenced by local communication factors, such as the number of farmers drought informa-
tion was received from, and whether information was received at a village meeting. This
supports findings that local and informal risk communications are more likely to alter risk
perceptions (Binh et al. 2020). Additionally, risk appraisal was influenced by direct personal
experience of drought, supporting the suggestion that factors which are more psychologi-
cally close (either in time, space or socially) determine risk perceptions more strongly than
official communications (Binh et al. 2020; Niles et al. 2015; Steynor et al. 4.2 To what extent do PMT variables predict adaptation outcomes? Looking at the relationship between PMT variables and the adaptation outcome variables
analysed, this study reveals a much more pronounced connection between adaptation and
adaptation appraisal than between adaptation and risk appraisal, which reflects findings from
many previous PMT studies exploring a range of different risks and contexts (Bubeck et al. 2012; Milne et al. 2000; Poussin et al. 2014; Sheeran et al. 2014; Truelove et al. 2015). It has
been argued that risk appraisal could be higher prior to an adaptation being undertaken, but
then decline in light of adaptation success (Bamberg et al. 2017). Our finding of significantly
higher mean risk appraisal scores amongst respondents that indicated wanting to implement
further adaptations supports this possibility. However, since no significant associations
were found between adaptation counts and other more stable factors that correlate with risk
appraisal (for example, the number of droughts and drought impacts respondents reported
having experienced), the authors argue that the findings suggest adaptation appraisal is the
more robust behavioural predictor of the two PMT constructs (Milne et al. 2000). The finding of a weak negative correlation between adaptation appraisal and risk
appraisal illustrates that, rather than being purely additive, it is possible that high levels
of risk perception may lower perceived capacity to adapt, or vice versa. The conceptual
diagram in Fig. 1 depicts positive feedbacks between adaptation and adaptation appraisal,
showing that direct experience of implementing adaptation and perceived adaptation
success may increase cognitive readiness to adapt in future. These findings suggest that
direct experience, observation and learning about adaptation and drought risk continually
reshape perceptions, illustrating processes of adaptation as continuous rather than finite 1 3 Mitig Adapt Strateg Glob Change (2024) 29:6 Page 15 of 21 6 and suggesting that adaptation experience and a pro-adaptive mindset are mutually rein-
forcing. This identification of positive adaptation feedbacks mirrors those identified by
Noll et al. (2022) in the context of flood adaptation. On the basis that adaptation actions
may trigger further adaptation intentions, drought communications and adaptation cam-
paigns should not be regarded as standalone responses, but rather as elements within long-
term support programmes geared towards developing cultures of resilience and capacity-
building (Ewbank et al. 2019). This finding is of particular relevance for countries where
agricultural impacts from drought are projected to increase in coming decades. 4.3 To what extent do institutional drought communications influence farmers’
motivations and actions to protect their production activities from the risk
of drought? 2021).iil fi
The finding of a significant influence of local farmer communications on risk appraisal
and past adaptation points to the role of social connectedness in facilitating access to infor-
mation and as an important determinant of adaptation responses. Moreover, the appar-
ent role of smartphone messaging group membership in enhancing responsiveness to
official advice should not be overlooked. Overall, these findings highlight the benefits of
strengthening opportunities for all farmers to participate in local and institutional drought 1 3 3 Mitig Adapt Strateg Glob Change (2024) 29:6 6
Page 16 of 21 6 communications networks via locally appropriate and accessible means, particularly in
agricultural development contexts similar to the case study presented here. communications networks via locally appropriate and accessible means, particularly in
agricultural development contexts similar to the case study presented here. 4.4 Which types of farmers are less likely to implement successful drought
adaptations and why? Demographic variables such as age and education are important determinants of disaster
warning response, with evidence that different segments of at risk populations may com-
prehend information differently (Mayhorn and McLaughlin 2014). Our findings reflect
socio-economic patterns well-established by diffusion of innovations scholars (E. M. Rogers
2003), namely that education, age and wealth determine capacity to implement new behav-
iours. The study found that older farmers were more likely to indicate adaptation constraints,
such as a lack of time or energy. Older farmers were also likely to receive drought informa-
tion from fewer local farmers and via fewer communication channels. As such, this study
highlights that older farmers may be less able to adapt to drought and supports findings
that older individuals may have different communication needs and support should be tai-
lored accordingly (Walkling and Haworth 2020). As drought information communications
move online in future, the need may grow to implement additional communication strategies
which ensure that older and poorer population segments do not end up missing out.i The study findings also suggest that livestock producers experience drought risks and
impacts differently, resulting in measurably higher risk appraisal scores and lower per-
ceived success rates for past adaptations. This indicates that households which focus on
livestock production may likewise benefit from specific targeted drought support, reflecting
findings from different regional contexts that drought responses within the livestock sector
are different to other agricultural sectors (Salmoral et al. 2020). 4.5 How might drought communications and policy be improved to support
adaptation? The absence of a strong link between risk appraisal and adaptation behaviour (aligning
with many other PMT studies) suggests that seeking to heighten risk appraisal by incor-
porating fear appeals within official drought communications is unlikely to enhance
adaptation behaviour amongst farmers and signals the possibility that fear appeals could
be counterproductive. Whilst this study supports the communication of targeted drought
information to farmers, emphasis should be placed upon accompanying drought informa-
tion with positive messages about household coping capacity and practical tailored advice
about adaptation strategies which enhance farmers’ perceptions of both self- and adapta-
tion efficacy (Binh et al. 2020; Ong and Araral 2021).i fi
Although the findings do not support risk appraisal directly determining adaptation
actions, higher risk appraisal levels may promote adaptation desire and/or intention (Milne
et al. 2000). The findings indicated that risk appraisal levels may be higher following a
direct experience of drought, but then decline thereafter. Drought communications should
be targeted to take advantage of potential synergies between aspects of risk and adaptation
appraisal (Sheeran et al. 2014), by ensuring that drought adaptation information and physical/
financial assistance are made available soon after farming households have directly experi-
enced drought, since this may lead to greater uptake than if only provided at a later stage.i The findings indicate that the constraint of not having enough money to implement the nec-
essary adaptations may be delaying farmers from implementing desired further adaptations. 1 3 Mitig Adapt Strateg Glob Change (2024) 29:6 Page 17 of 21 6 Given the lack of a correlation between financial and the other adaptation constraints meas-
ured (time and energy), providing financial support to farmers who indicate an inclination to
adapt may boost the uptake of drought adaptation measures. In some instances, pursuing this
proactive approach to building drought resilience may be preferable to providing drought com-
pensation after losses have already been incurred, particularly given the negative relationship
between compensation for major crops such as fruit and rice and responsiveness to official
drought warnings and adaptation advice. This finding reflects Tabe-Ojong et al. (2020), who
show that relief assistance may negatively influence household resilience strategies for coping
with extreme weather events. 4.5 How might drought communications and policy be improved to support
adaptation? However, given that our study is cross-sectional, it is not pos-
sible to establish whether compensation arrived before or after farmers failed to take action
in response to official drought advice; hence, longitudinal research on this issue is warranted. 5 Conclusion The study supports protective behavioural changes (in this case, implementation of drought
adaptations) being more closely linked to adaptation appraisal than risk appraisal. Further,
it has indicated that an inverse relationship can pertain between these PMT constructs. Both
institutional and local drought communications contribute in important ways to adaptation
actions and perceptions, but institutional communications appear to operate more through
pathways leading to adaptation via adaptation appraisal. Conversely, local communications
are of greater significance for risk appraisal, although the study has not found evidence
to support a direct pathway from risk appraisal to adaptation. Institutional drought com-
munications should include adaptation advice to promote adaptation behaviour amongst
agricultural producers, with drought communications framed in positive terms, emphasis-
ing the efficacy of adaptation recommendations and the feasibility of implementing them
for the producers themselves. Policy makers should employ measures that support farmer
participation in both local and institutional drought communication networks. The study shows that farmers who are older, less educated or poorer are less likely to
adapt to drought due to perceived costs and reduced exposure to and assimilation of drought
information. More research is needed on how to shape and supply institutional drought and
adaptation communications to meet the needs of these segments of agricultural populations
more effectively. Whilst financial support may sufficiently address adaptation constraints
for some, evidence of time and energy constraints for, particularly, older farmers indicate a
need for different targeting of adaptation support for these farmers, including the provision of
social safety nets in cases where adaptation is improbable. Evidence that those practicing ani-
mal husbandry perceive greater levels of drought risk and lower levels of adaptation success
than other agricultural producers also suggests a need for specific targeted support.i i
Understanding adaptation as a continuous (rather than finite) process, characterised by
feedbacks between experience and appraisal, means that efforts to build drought resilience
must go beyond providing assistance only when drought is forecast or causing measur-
able impacts. Efforts to reduce drought vulnerability should provide long-duration, con-
tinuous institutional support for resilience building. Such support needs to engender pro-
adaptive mindsets across all sections of agricultural communities by improving social and
institutional drought communication networks, ensuring that organisations are receptive to
climate and adaptation information fed back by farmers and developing habits across the
board of engaging in ongoing climate, drought and adaptation dialogue. Declarations Ethics approval The study gained ethical approval through the Cranfield University Research Ethics System
(CURES) CURES/9419/2019. Competing interests The authors declare no competing interests. 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 Com-
mons 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/. 5 Conclusion 3 1 Mitig Adapt Strateg Glob Change (2024) 29:6 6
Page 18 of 21 6 Acknowledgements We wish to thank the wider members of the STAR project team, the CMU students
who performed the interviews and the participant farmers for their time. Author contribution DR, CS and IH acquired the funding. CS, DR, IH and GS developed the survey instru-
ment and conceived the original scope of the article. LP, SV and CE coordinated the fieldwork and transla-
tions. CS developed the statistical analysis. CS drafted the manuscript. All authors reviewed, edited and
approved the manuscript. Funding This project is funded through a NERC (Natural Environment Research Council, UK) grant for the
STAR project (Strengthening Thailand’s Agricultural drought Resilience) grant number NE/S003223/1 and
Thailand Science Research and Innovation (TSRI) grant number is RDG6130017. Data availability The dataset and questionnaire for this study can be found in the Cranfield Online Resea
Data (CORD) 10.17862/cranfield.rd.16553136. References https://doi.org/10.1016/j.accre.2021.03.001 62–171. https://doi.org/10.1016/j.accre.2021.03.001 p
g
j
Cordova A (2009) Methodological note: measuring relative wealth using household asset indicators. In:
AmericasBarometer Insights Series, vol 6. https://www.vanderbilt.edu/lapop/insights/I0806en_v2.
pdf. Accessed 02/02/2024 p
Daryanto S, Wang L, Jacinthe PA (2017) Global synthesis of drought effects on cereal, legume, tuber and root
crops production: a review. Agric Water Manag 179:18–33. https://doi.org/10.1016/j.agwat.2016.04.022 Deressa TT, Hassan RM, Ringler C (2011) Perception of and adaptation to climate change by farmers in the
Nile basin of Ethiopia. J Agric Sci 149(1):23–31. https://doi.org/10.1017/S0021859610000687f Dorward P, Osbahr H, Sutcliffe C, Mbeche R (2020) Supporting climate change adaptation using histori
climate analysis. Clim Dev 12(5):469–480. https://doi.org/10.1080/17565529.2019.1642177 Ewbank R, Perez C, Cornish H, Worku M, Woldetsadik S (2019) Building resilience to El Niño-related
drought: experiences in early warning and early action from Nicaragua and Ethiopia. Disasters
43(S3):S345–S367. https://doi.org/10.1111/disa.12340 (
)
p
g
Field A (2018) Discovering Statistics Using IBM SPSS Statistics. Sage Publications Ltd.f Filmer D, Pritchett LH (2001) Estimating wealth effects without expenditure data - or tears: an application to
educational enrollments in states of India. Demography 38(1):115–132. https://doi.org/10.2307/3088292 mer D, Pritchett LH (2001) Estimating wealth effects without expenditure data - or tears: an application to
educational enrollments in states of India. Demography 38(1):115–132. https://doi.org/10.2307/3088292 Gebrehiwot T, van der Veen A (2015) Farmers prone to drought risk: why some farmers undertake farm-
level risk-reduction measures while others not? Environ Manag 55(3):588–602. https://doi.org/10.
1007/s00267-014-0415-7 Gebrehiwot T, van der Veen A (2015) Farmers prone to drought risk: why some farmers undertake farm-
level risk-reduction measures while others not? Environ Manag 55(3):588–602. https://doi.org/10.
1007/s00267-014-0415-7 Grothmann T, Patt A (2005) Adaptive capacity and human cognition: the process of individual adaptation to
climate change. Glob Environ Change 15(3):199–213. https://doi.org/10.1016/j.gloenvcha.2005.01.002l Grothmann T, Patt A (2005) Adaptive capacity and human cognition: the process of individual adaptation to
climate change. Glob Environ Change 15(3):199–213. https://doi.org/10.1016/j.gloenvcha.2005.01.002l Grothmann T, Reusswig F (2006) People at risk of flooding: why some residents take precautionary action
while others do not. Nat Hazards 38(1–2):101–120. https://doi.org/10.1007/s11069-005-8604-6 Grothmann T, Reusswig F (2006) People at risk of flooding: why some residents take precautionary action
while others do not. Nat Hazards 38(1–2):101–120. https://doi.org/10.1007/s11069-005-8604-6 Grothmann T, Reusswig F (2006) People at risk of flooding: why some residents take precautionary action
while others do not. Nat Hazards 38(1–2):101–120. References Amnuaylojaroen T, Chanvichit P (2019) Projection of near-future climate change and agricultural drought
in Mainland Southeast Asia under RCP8.5. Clim Chang 155(2):175–193. https://doi.org/10.1007/
s10584-019-02442-5 Arunrat N, Wang C, Pumijumnong N, Sereenonchai S, Cai W (2017) Farmers’ intention and decision to
adapt to climate change: a case study in the Yom and Nan basins, Phichit province of Thailand. J Clean
Prod 143:672–685. https://doi.org/10.1016/j.jclepro.2016.12.058 Babazadeh T, Nadrian H, Banayejeddi M, Rezapour B (2017) Determinants of skin cancer preventive
behaviors among rural farmers in Iran: an application of protection motivation theory. J Cancer Educ
32(3):604–612. https://doi.org/10.1007/s13187-016-1004-7lf Babcicky P, Seebauer S (2017) The two faces of social capital in private flood mitigation: opposing effects
on risk perception, self-efficacy and coping capacity. J Risk Res 20(8):1017–1037. https://doi.org/10.
1080/13669877.2016.1147489 Babcicky P, Seebauer S (2019) Unpacking protection motivation theory: evidence for a separate protective
and non-protective route in private flood mitigation behavior. J Risk Res 22(12):1503–1521. https://
doi.org/10.1080/13669877.2018.1485175l g
Bamberg S, Masson T, Brewitt K, Nemetschek N (2017) Threat, coping and flood prevention – a me
analysis. J Environ Psychol 54:116–126. https://doi.org/10.1016/j.jenvp.2017.08.001 Binh PT, Zhu X, Groeneveld RA, van Ierland EC (2020) Risk communication, women’s participation and
flood mitigation in Vietnam: an experimental study. Land Use Policy 95:104436. https://doi.org/10.
1016/j.landusepol.2019.104436 Bradford RA, O’Sullivan JJ, Van Der Craats IM, Krywkow J, Rotko P, Aaltonen J, Bonaiuto M, De Domini-
cis S, Waylen K, Schelfaut K (2012) Risk perception - issues for flood management in Europe. Nat
Hazards Earth Syst Sci 12(7):2299–2309. https://doi.org/10.5194/nhess-12-2299-2012 Braun MT, Oswald FL (2011) Exploratory regression analysis: a tool for selecting models and determining
predictor importance. Behav Res Methods 43(2):331–339. https://doi.org/10.3758/s13428-010-0046-8 1 1 3 Mitig Adapt Strateg Glob Change (2024) 29:6 Page 19 of 21 6 Bubeck P, Botzen WJW, Aerts JCJH (2012) A review of risk perceptions and other factors that influence flood
mitigation behavior. Risk Anal 32(9):1481–1495. https://doi.org/10.1111/j.1539-6924.2011.01783.x Carrão H, Naumann G, Barbosa P (2016) Mapping global patterns of drought risk: an empirical framework
based on sub-national estimates of hazard, exposure and vulnerability. Glob Environ Chang 39:108–
124. https://doi.org/10.1016/j.gloenvcha.2016.04.012 p
g
j g
Challinor AJ, Watson J, Lobell DB, Howden SM, Smith DR, Chhetri N (2014) A meta-analysis of crop yi
under climate change and adaptation. Nat Clim Chang 4(4):287–291. https://doi.org/10.1038/nclimate21 Chang S, Zi-Niu X, Nguyen M (2021) Projection on precipitation frequency of different intensities and
precipitation amount in the Lancang-Mekong River basin in the 21st century. Adv Clim Chang Res
12(2):162–171. References https://doi.org/10.1007/s11069-005-8604-6 p
g
Haag S, Siponen M, Liu F (2021) Protection motivation theory in information systems security research: a
review of the past and a road map for the future. Data Base Adv Inf Systems 52(2):25–67. https://doi.
org/10.1145/3462766.3462770fl Haag S, Siponen M, Liu F (2021) Protection motivation theory in information systems security research: a
review of the past and a road map for the future. Data Base Adv Inf Systems 52(2):25–67. https://doi.
org/10.1145/3462766.3462770fl Haer T, Botzen WJW, Aerts JCJH (2016) The effectiveness of flood risk communication strategies and the
influence of social networks-insights from an agent-based model. Environ Sci Pol 60:44–52. https://
doi.org/10.1016/j.envsci.2016.03.006 Haer T, Botzen WJW, Aerts JCJH (2016) The effectiveness of flood risk communication strategies and the
influence of social networks-insights from an agent-based model. Environ Sci Pol 60:44–52. https://
doi.org/10.1016/j.envsci.2016.03.006 g
j
Herath T, Rao HR (2009) Protection motivation and deterrence: a framework for security policycompliance
in organisations. Eur J Inf Syst 18(2):106–125. https://doi.org/10.1057/ejis.2009.6 g
j
Herath T, Rao HR (2009) Protection motivation and deterrence: a framework for security policycompliance
in organisations. Eur J Inf Syst 18(2):106–125. https://doi.org/10.1057/ejis.2009.6 g
y
p
g
j
Howden SM, Soussana JF, Tubiello FN, Chhetri N, Dunlop M, Meinke H (2007) Adapting agriculture to cli-
mate change. Proc Natl Acad Sci U S A 104(50):19691–19696. https://doi.org/10.1073/pnas.0701890104 Howden SM, Soussana JF, Tubiello FN, Chhetri N, Dunlop M, Meinke H (2007) Adapting agriculture to cli-
mate change. Proc Natl Acad Sci U S A 104(50):19691–19696. https://doi.org/10.1073/pnas.0701890104
Keshavarz M, Karami E (2016) Farmers’ pro-environmental behavior under drought: application of protection Howden SM, Soussana JF, Tubiello FN, Chhetri N, Dunlop M, Meinke H (2007) Adapting agriculture to cli-
mate change. Proc Natl Acad Sci U S A 104(50):19691–19696. https://doi.org/10.1073/pnas.0701890104 Keshavarz M, Karami E (2016) Farmers’ pro-environmental behavior under drought: application of protection
motivation theory. J Arid Environ 127:128–136. https://doi.org/10.1016/j.jaridenv.2015.11.010 Keshavarz M, Karami E (2016) Farmers’ pro-environmental behavior under drought: application of protection
motivation theory. J Arid Environ 127:128–136. https://doi.org/10.1016/j.jaridenv.2015.11.010 y
g
j j
Khan R, Gilani H (2021) Global drought monitoring with big geospatial datasets using Google Earth
Engine. Environ Sci Pollut Res 28(14):17244–17264. https://doi.org/10.1007/s11356-020-12023-0 Khan R, Gilani H (2021) Global drought monitoring with big geospatial datasets using Google Earth
Engine. Environ Sci Pollut Res 28(14):17244–17264. y
p
1111/j.1559-1816.2000.tb02308.x https://doi.org/10.1037/a0033065 Sheeran P, Harris PR, Epton T (2014) Does heightening risk appraisals change people’s intentions and behavi
A meta-analysis of experimental studies. Psychol Bull 140(2):511–543. https://doi.org/10.1037/a003306 y
p
y
( )
p
g
Singhrattna N, Babel MS (2011) Changes in summer monsoon rainfall in the upper Chao Phraya River
Basin, Thailand. Clim Res 49(2):155–168. https://doi.org/10.3354/cr01015 y
p
y
( )
p
g
Singhrattna N, Babel MS (2011) Changes in summer monsoon rainfall in the upper Chao Phraya River
Basin, Thailand. Clim Res 49(2):155–168. https://doi.org/10.3354/cr01015 Steynor A, Pasquini L, Thatcher A, Hewitson B (2021) Understanding the links between climate change
risk perceptions and the action response to inform climate services interventions. Risk Anal. https://doi.org/10.1111/risa.13683 Steynor A, Pasquini L, Thatcher A, Hewitson B (2021) Understanding the links between climate change
risk perceptions and the action response to inform climate services interventions. Risk Anal. https://doi.org/10.1111/risa.13683 Supharatid S, Nafung J (2021) Projected drought conditions by CMIP6 multimodel ensemble over
SoutheastAsia. J Water Clim Change 12(7, SI):3330–3354. https://doi.org/10.2166/wcc.2021.308f Supharatid S, Nafung J (2021) Projected drought conditions by CMIP6 multimodel ensemble over
SoutheastAsia. J Water Clim Change 12(7, SI):3330–3354. https://doi.org/10.2166/wcc.2021.308f g
p
g
Sutcliffe C, Dougill AJ, Quinn CH (2016) Evidence and perceptions of rainfall change in Malawi: do
maize cultivar choices enhance climate change adaptation in sub-Saharan Africa? Reg Environ
Chang 16(4):1215–1224. https://doi.org/10.1007/s10113-015-0842-xl Sutcliffe C, Dougill AJ, Quinn CH (2016) Evidence and perceptions of rainfall change in Malawi: do
maize cultivar choices enhance climate change adaptation in sub-Saharan Africa? Reg Environ
Chang 16(4):1215–1224. https://doi.org/10.1007/s10113-015-0842-xl g
p
g
Tabe-Ojong MPJ, Boakye JA, Muliro M (2020) Mitigating the impacts of floods using adaptive and resil-
ient coping strategies: the role of the emergency Livelihood Empowerment Against Poverty program
(LEAP) in Ghana. J Environ Manag 270:110809. https://doi.org/10.1016/j.jenvman.2020.110809 Tabe-Ojong MPJ, Boakye JA, Muliro M (2020) Mitigating the impacts of floods using adaptive and resil-
ient coping strategies: the role of the emergency Livelihood Empowerment Against Poverty program
(LEAP) in Ghana. J Environ Manag 270:110809. https://doi.org/10.1016/j.jenvman.2020.110809 (
)
g
p
g
j j
Truelove HB, Carrico AR, Thabrew L (2015) A socio-psychological model for analyzing climate change
adaptation: a case study of Sri Lankan paddy farmers. Glob Environ Change 31:85–97. https://doi.org/
10.1016/j.gloenvcha.2014.12.010l (
)
g
p
g
j j
Truelove HB, Carrico AR, Thabrew L (2015) A socio-psychological model for analyzing climate change
adaptation: a case study of Sri Lankan paddy farmers. Glob Environ Change 31:85–97. References https://doi.org/10.1007/s11356-020-12023-0 g
p
g
Kiguchi M, Takata K, Hanasaki N, Archevarahuprok B, Champathong A, Ikoma E, Jaikaeo C, Kaew g
p
g
uchi M, Takata K, Hanasaki N, Archevarahuprok B, Champathong A, Ikoma E, Jaikaeo C, Kaewrueng Kiguchi M, Takata K, Hanasaki N, Archevarahuprok B, Champathong A, Ikoma E, Jaikaeo C, Kaewrueng
S, Kanae S, Kazama S, Kuraji K, Matsumoto K, Nakamura S, Nguyen-Le D, Noda K, Piamsa-Nga
N, Raksapatcharawong M, Rangsiwanichpong P, Ritphring S et al (2021) A review of climate-change
impact and adaptation studies for the water sector in Thailand. Environ Res Lett 16(2). https://doi.org/
10.1088/1748-9326/abce80 g
,
,
,
p
,
p
g
,
,
,
g
S, Kanae S, Kazama S, Kuraji K, Matsumoto K, Nakamura S, Nguyen-Le D, Noda K, Piamsa-Nga
N, Raksapatcharawong M, Rangsiwanichpong P, Ritphring S et al (2021) A review of climate-change
impact and adaptation studies for the water sector in Thailand. Environ Res Lett 16(2). https://doi.org/
10.1088/1748-9326/abce80 Koerth J, Vafeidis AT, Hinkel J, Sterr H (2013) What motivates coastal households to adapt pro-actively to
sea-level rise and increasing flood risk? Reg Environ Chang 13(4):897–909. https://doi.org/10.1007/
s10113-012-0399-x Koerth J, Vafeidis AT, Hinkel J, Sterr H (2013) What motivates coastal households to adapt pro-actively to
sea-level rise and increasing flood risk? Reg Environ Chang 13(4):897–909. https://doi.org/10.1007/
s10113-012-0399-x Kuruppu N, Liverman D (2011) Mental preparation for climate adaptation: the role of cognition and culture
in enhancing adaptive capacity of water management in Kiribati. Glob Environ Change 21(2):657–
669. https://doi.org/10.1016/j.gloenvcha.2010.12.002f Kuruppu N, Liverman D (2011) Mental preparation for climate adaptation: the role of cognition and culture
in enhancing adaptive capacity of water management in Kiribati. Glob Environ Change 21(2):657–
669. https://doi.org/10.1016/j.gloenvcha.2010.12.002f Lebel L, Lebel P, Lebel B, Uppanunchai A, Duangsuwan C (2018) The effects of tactical message inserts
on risk communication with fish farmers in Northern Thailand. Reg Environ Chang 18(8):2471–
2481. https://doi.org/10.1007/s10113-018-1367-x Lebel L, Lebel P, Lebel B, Uppanunchai A, Duangsuwan C (2018) The effects of tactical message inserts
on risk communication with fish farmers in Northern Thailand. Reg Environ Chang 18(8):2471–
2481. https://doi.org/10.1007/s10113-018-1367-x 1 3 Mitig Adapt Strateg Glob Change (2024) 29:6 Page 20 of 21 6 Leng G, Hall J (2019) Crop yield sensitivity of global major agricultural countries to droughts and the projected
changes in the future. Sci Total Environ 654:811–821. y
p
1111/j.1559-1816.2000.tb02308.x Ngo CC, Poortvliet PM, Feindt PH (2020) Drivers of flood and climate change risk perceptions and
intention to adapt: an explorative survey in coastal and delta Vietnam. J Risk Res 23(4):424–446. https://doi.org/10.1080/13669877.2019.1591484 p
g
Niles MT, Lubell M, Brown M (2015) How limiting factors drive agricultural adaptation to climate
change. Agric Ecosyst Environ 200:178–185. https://doi.org/10.1016/j.agee.2014.11.010 g
g
y
p
g
j g
Noll B, Filatova T, Need A (2022) One and done? Exploring linkages between households’ intended adap-
tations to climate-induced floods. Risk Anal 42(12):2781–2799. https://doi.org/10.1111/risa.13897 l
(
)
p
g
Ong C, Araral E (2021) Using a serious digital game to communicate drought risk in singapore: an
experimental study. Environ Behav. 54(2):450–486. https://doi.org/10.1177/00139165211031197 Pak-uthai S (2019) Coping with drought: farmers’ actions and public policies in Suphanburi Province,
Thailand. Dissertation, Asian Institute of Technology, Thailand. https://www.researchgate.net/
publication/333948940_Coping_with_Drought_Farmers’_Actions_and_Public_Policies_in_Supha
nburi_Province_Thailand. Accessed 02/01/2024ll ssin JK, Botzen WJW, Aerts JCJH (2014) Factors of influence on flood damage mitigation behaviour by
households. Environ Sci Policy 40:69–77. https://doi.org/10.1016/j.envsci.2014.01.013f Poussin JK, Botzen WJW, Aerts JCJH (2014) Factors of influence on flood damage mitigation behaviour
households. Environ Sci Policy 40:69–77. https://doi.org/10.1016/j.envsci.2014.01.013f Rogers EM (2003) Diffusion of innovations, Fifth edn. Free Press, New York, London, Toronto, Sydney
Rogers RW (1975) Protection motivation theory of fear appeals and attitude-change. J Psychol 91(1):93– Rogers EM (2003) Diffusion of innovations, Fifth edn. Free Press, New York, London, Toronto, Sydney
Rogers RW (1975) Protection motivation theory of fear appeals and attitude-change. J Psychol 91(1):93–
114. https://doi.org/10.1080/00223980.1975.9915803 Rogers EM (2003) Diffusion of innovations, Fifth edn. Free Press, New York, London, Toronto, Sydney
Rogers RW (1975) Protection motivation theory of fear appeals and attitude-change. J Psychol 91(1):93–
114. https://doi.org/10.1080/00223980.1975.9915803 f
Rogers RW (1975) Protection motivation theory of fear appeals and attitude-change. J Psychol 91(1):93–
114. https://doi.org/10.1080/00223980.1975.9915803 p
g
Salmoral G, Ababio B, Holman IP (2020) Drought impacts, coping responses and adaptation in the UK outdoor
livestock sector: insights to increase drought resilience. LAND 9(6). https://doi.org/10.3390/land9060202
Sh
P H
i PR E
T (2014) D
h i h
i
i k
i l
h
l ’ i
i
d b h
i ? Salmoral G, Ababio B, Holman IP (2020) Drought impacts, coping responses and adaptation in the UK outdoor
livestock sector: insights to increase drought resilience. LAND 9(6). https://doi.org/10.3390/land9060202 eran P, Harris PR, Epton T (2014) Does heightening risk appraisals change people’s intentions and behavior? A meta-analysis of experimental studies. Psychol Bull 140(2):511–543. References https://doi.org/10.1016/j.scitotenv.2018.10.434l Leng G, Hall J (2019) Crop yield sensitivity of global major agricultural countries to droughts and the projected
changes in the future. Sci Total Environ 654:811–821. https://doi.org/10.1016/j.scitotenv.2018.10.434
Lesk C, Rowhani P, Ramankutty N (2016) Influence of extreme weather disasters on global crop production. Nature 529(7584):84–87. https://doi.org/10.1038/nature16467 g
p
g
j
Lesk C, Rowhani P, Ramankutty N (2016) Influence of extreme weather disasters on global crop production. Nature 529(7584):84–87. https://doi.org/10.1038/nature16467 g
p
Lesk C, Rowhani P, Ramankutty N (2016) Influence of extreme w
Nature 529(7584):84–87. https://doi.org/10.1038/nature16467 Lipper L, Thornton P, Campbell BM, Baedeker T, Braimoh A, Bwalya M, Caron P, Cattaneo A, Gar-
rity D, Henry K, Hottle R, Jackson L, Jarvis A, Kossam F, Mann W, McCarthy N, Meybeck A,
Neufeldt H, Remington T et al (2014) Climate-smart agriculture for food security. Nat Clim Chang
4(12):1068–1072. https://doi.org/10.1038/nclimate2437 Mayhorn CB, McLaughlin AC (2014) Warning the world of extreme events: a global perspective on risk commu-
nication for natural and technological disaster. Saf Sci 61:43–50. https://doi.org/10.1016/j.ssci.2012.04.014 Mertz O, Mbow C, Reenberg A, Diouf A (2009) Farmers’ perceptions of climate change and agricul-
tural adaptation strategies in rural Sahel. Environ Manag 43(5):804–816. https://doi.org/10.1007/
s00267-008-9197-0 Milne S, Sheeran P, Orbell S (2000) Prediction and intervention in health-related behavior: a meta-ana-
lytic review of protection motivation theory. J Appl Soc Psychol 30(1):106–143. https://doi.org/10.
1111/j.1559-1816.2000.tb02308.xl y
p
1111/j.1559-1816.2000.tb02308.x https://doi.org/10.1016/j.ijdrr.2021.102435 p
g
j j
Yuan S, Stuart AM, Laborte AG, Edreira JIR, Dobermann A, Kien LVN, Thuy LT, Paothong K, Traesang P,
Tint KM, San SS, Villafuerte MQ, Quicho ED, Pame ARP, Then R, Flor RJ, Thon N, Agus F, Agus-
tiani N, Deng NY, Li T, Grassini P (2022) Southeast Asia must narrow down the yield gap to continue
to be a major rice bowl. Nat Food 3(3):217–226. https://doi.org/10.1038/s43016-022-00477-z Yuan S, Stuart AM, Laborte AG, Edreira JIR, Dobermann A, Kien LVN, Thuy LT, Paothong K, Traesang P,
Tint KM, San SS, Villafuerte MQ, Quicho ED, Pame ARP, Then R, Flor RJ, Thon N, Agus F, Agus-
tiani N, Deng NY, Li T, Grassini P (2022) Southeast Asia must narrow down the yield gap to continue
to be a major rice bowl. Nat Food 3(3):217–226. https://doi.org/10.1038/s43016-022-00477-z Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. y
p
1111/j.1559-1816.2000.tb02308.x https://doi.org/
10.1016/j.gloenvcha.2014.12.010l j g
Venkatappa M, Sasaki N, Han P, Abe I (2021) Impacts of droughts and floods on croplands and crop pro-
duction inSoutheast Asia-an application of Google Earth Engine. Sci Total Environ 795. https://doi.
org/10.1016/j.scitotenv.2021.148829 Venkatappa M, Sasaki N, Han P, Abe I (2021) Impacts of droughts and floods on croplands and crop pro-
duction inSoutheast Asia-an application of Google Earth Engine. Sci Total Environ 795. https://doi.
org/10.1016/j.scitotenv.2021.148829 Venkatappa M, Sasaki N, Han P, Abe I (2021) Impacts of droughts and floods on croplands and crop pro-
duction inSoutheast Asia-an application of Google Earth Engine. Sci Total Environ 795. https://doi.
org/10.1016/j.scitotenv.2021.148829 g
j
Walkling B, Haworth BT (2020) Flood risk perceptions and coping capacities among the retired population,
with implications for risk communication: a study of residents in a north Wales coastal town, UK. Int J
Disaster Risk Reduct 51(August):101793. https://doi.org/10.1016/j.ijdrr.2020.101793 Walkling B, Haworth BT (2020) Flood risk perceptions and coping capacities among the retired population,
with implications for risk communication: a study of residents in a north Wales coastal town, UK. Int J
Disaster Risk Reduct 51(August):101793. https://doi.org/10.1016/j.ijdrr.2020.101793 1 3 Mitig Adapt Strateg Glob Change (2024) 29:6 Page 21 of 21 6 Wang Y, Liang J, Yang J, Ma X, Li X, Wu J, Yang G, Ren G, Feng Y (2019) Analysis of the environmen-
tal behavior of farmers for non-point source pollution control and management: an integration of the
theory of planned behavior and the protection motivation theory. J Environ Manag 237:15–23. https://
doi.org/10.1016/j.jenvman.2019.02.070 Wang Y, Liang J, Yang J, Ma X, Li X, Wu J, Yang G, Ren G, Feng Y (2019) Analysis of the environmen-
tal behavior of farmers for non-point source pollution control and management: an integration of the
theory of planned behavior and the protection motivation theory. J Environ Manag 237:15–23. https://
doi.org/10.1016/j.jenvman.2019.02.070 Wens MLK, Mwangi MN, van Loon AF, Aerts JCJH (2021) Complexities of drought adaptive behav-
iour: linking theory to data on smallholder farmer adaptation decisions. Int. J. Disaster Risk Reduct
63:102435. https://doi.org/10.1016/j.ijdrr.2021.102435 Wens MLK, Mwangi MN, van Loon AF, Aerts JCJH (2021) Complexities of drought adaptive behav-
iour: linking theory to data on smallholder farmer adaptation decisions. Int. J. Disaster Risk Reduct
63:102435. Wang Y, Liang J, Yang J, Ma X, Li X, Wu J, Yang G, Ren G, Feng Y (2019) Analysis of the environmen-
tal behavior of farmers for non-point source pollution control and management: an integration of the
theory of planned behavior and the protection motivation theory. J Environ Manag 237:15–23. https://
doi.org/10.1016/j.jenvman.2019.02.070 Chloe Sutcliffe1,2 · Ian Holman1 · Daniel Goodwin1,3 · Gloria Salmoral1 ·
Liwa Pardthaisong4 · Supattra Visessri5,6 · Chaiwat Ekkawatpanit7 · Dolores Rey1 Chloe Sutcliffe1,2 · Ian Holman1 · Daniel Goodwin1,3 · Gloria Salmoral1 ·
Liwa Pardthaisong4 · Supattra Visessri5,6 · Chaiwat Ekkawatpanit7 · Dolores Rey1 Chloe Sutcliffe1,2 · Ian Holman1 · Daniel Goodwin1,3 · Gloria Salmoral1 ·
Liwa Pardthaisong4 · Supattra Visessri5,6 · Chaiwat Ekkawatpanit7 · Dolores Rey1 * Ian Holman
i.holman@cranfield.ac.uk
Chloe Sutcliffe
chloesutcliffe@rhs.org.uk
Daniel Goodwin
daniel.goodwin@utas.edu.au
Gloria Salmoral
gsalmoral@icatalist.eu
Liwa Pardthaisong
liwa.p@cmu.ac.th
Supattra Visessri
supattra.vi@chula.ac.th
Chaiwat Ekkawatpanit
chaiwat.ekk@kmutt.ac.th
Dolores Rey
dolores.rey-vicario@ec.europa.eu * Ian Holman
i.holman@cranfield.ac.uk
Chloe Sutcliffe
chloesutcliffe@rhs.org.uk
Daniel Goodwin
daniel.goodwin@utas.edu.au
Gloria Salmoral
gsalmoral@icatalist.eu
Liwa Pardthaisong
liwa.p@cmu.ac.th
Supattra Visessri
supattra.vi@chula.ac.th
Chaiwat Ekkawatpanit
chaiwat.ekk@kmutt.ac.th
Dolores Rey
dolores.rey-vicario@ec.europa.eu
1
Centre for Water, Environment and D
Bedfordshire MK43 0AL, UK
2
Environmental Horticulture Departm
3
School of Social Sciences, Universit Chaiwat Ekkawatpanit
chaiwat.ekk@kmutt.ac.th Dolores Rey
dolores.rey-vicario@ec.europa.eu 1
Centre for Water, Environment and Development, Cranfield University, College Road, Cranfield,
Bedfordshire MK43 0AL, UK 2
Environmental Horticulture Department, Royal Horticultural Society, Wisley GU23 6BQ, UK 3
School of Social Sciences, University of Tasmania, Hobart, Australia 4
Department of Geography, Faculty of Social Sciences, Chiang Mai University, Chiang Mai,
Thailand 5
Department of Water Resources Engineering, Faculty of Engineering, Chulalongkorn University,
Bangkok, Thailand 6
Disaster and Risk Management Information Systems Research Unit, Chulalongkorn University,
Bangkok, Thailand 7
Department of Civil Engineering, King Mongkut’s University of Technology Thonburi, Bangkok,
Thailand 1 3
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Rapid Adsorption Enthalpy Surface Sampling (RAESS) to Characterize Nanoporous Materials
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Rapid adsorption enthalpy surface sampling (RAESS) to
characterize nanoporous materials
Emmanuel Ren François-Xavier Coudert Emmanuel Ren, François-Xavier Coudert To cite this version: Emmanuel
Ren,
François-Xavier
Coudert. Rapid
adsorption
enthalpy
surface
sampling
(RAESS) to characterize nanoporous materials. Chemical Science, 2023, 14 (7), pp.1797-1807. 10.1039/D2SC05810C. hal-03955453v2 Emmanuel
Ren,
François-Xavier
Coudert. Rapid
adsorption
enthalpy
surface
sampling
(RAESS) to characterize nanoporous materials. Chemical Science, 2023, 14 (7), pp.1797-1807. 10.1039/D2SC05810C. hal-03955453v2 Distributed under a Creative Commons Attribution 4.0 International License 1
Introduction associated physical and chemical properties. These databases
can be computationally screened to retrieve key structure–
property relationships, potential theoretical limitations and
promising new structures.7,8 This is particularly true in the case
of nanoporous materials, which have been extensively studied
in high-throughput screening methodologies9 for the identi-
cation of top-performing materials for applications such as
electrocatalysis,10 photocatalysis,11 heterogeneous catalysis,12
membrane separation,13 adsorptive separation,14 adsorptive
storage,15 mechanical behaviors,16 etc. 1 Molecular adsorption has many large-scale industrial applica-
tions in our society, including uid storage, molecular separa-
tion, and purication, and is therefore a very active area of
research in both chemistry and materials science. Improvement
in the performance of adsorption-based processes could reduce
the environmental impact of separation and purication
applications by replacing energy-intensive processes such as
cryogenic distillation.1,2 In the energy industry, the use of
nanoporous solids is a promising low-energy alternative to
pressurized gas tanks for the storage of fuel such as H2 or CH4.3
Adsorption is also foreseen as a crucial component in the next
generation of carbon capture and storage systems.4 To further
unfold the potential of this technology, the design of materials
for targeted applications needs to be rened. Because high-throughput screening is performed on the scale
of hundreds of thousands or even millions of structures, there is
a need for faster computational methods to predict the materials'
properties, to be able to analyze larger and larger databases. In the
eld of adsorption, several measures have been proposed in order
to study the performance of a material for the adsorption of
a specic guest molecule. The most accurate, but also the most
computationally expensive one, is the grand canonical Monte
Carlo (GCMC) simulation. On the other end of the scale, the use of
simple geometric descriptors (pore size, pore volume, and surface
area) has also been proposed as proxies for various adsorption-
related metrics. As the eld of computational chemistry is
turning more and more towards machine learning (ML), the
development of a wide range of rapidly calculable descriptors17 is
an exciting research focus especially to speed up computational
screenings. Although the nal steps of this design process can only be
achieved by experimentation and engineering, computational
simulations can play a key role in guiding the design process
and speeding up the discovery of materials for targeted appli-
cations. aCEA, DES, ISEC, DMRC, Univ. Montpellier, Marcoule, France
bChimie ParisTech, PSL University, CNRS, Institut de Recherche de Chimie Paris,
75005 Paris, France. E-mail: fx.coudert@chimieparistech.psl.eu
† Electronic supplementary information (ESI) available: Additional information is
available in the supporting information le, raw data are available online at
https://github.com/fxcoudert/citable-data and the RAESS code is available at
https://github.com/coudertlab/RAESS.
See
DOI:
https://doi.org/10.1039/d2sc05810c HAL Id: hal-03955453
https://hal.science/hal-03955453v2
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Accepted 2nd January 2023
DOI: 10.1039/d2sc05810c
rsc.li/chemical-science Received 19th October 2022
Accepted 2nd January 2023 Accepted 2nd January 2023
DOI: 10.1039/d2sc05810c
rsc.li/chemical-science DOI: 10.1039/d2sc05810c
rsc.li/chemical-science Chemical
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View Journal | View Issue Rapid adsorption enthalpy surface sampling
(RAESS) to characterize nanoporous materials† Cite this: Chem. Sci., 2023, 14, 1797
All publication charges for this article
have been paid for by the Royal Society
of Chemistry Cite this: Chem. Sci., 2023, 14, 1797 This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. rticle is licensed under a Creative Commons Attribution 3.0 Unported Licence. nder a Creative Commons Attribution 3.0 Unported Licence. Emmanuel Ren
ab and François-Xavier Coudert
*b All publication charges for this article
have been paid for by the Royal Society
of Chemistry Molecular adsorption in nanoporous materials has many large-scale industrial applications ranging from
separation to storage. To design the best materials, computational simulations are key to guiding the
experimentation and engineering processes. Because nanoporous materials exist in a plethora of forms,
we need to speed up the existing simulation tools to be able to screen databases of hundreds of
thousands of structures. Here, we describe a new algorithm that quickly calculates adsorption enthalpies
by sampling the surface of the material instead of the whole porous space. This surface sampling has
been tested on the CoRE MOF 2019 database and has been proven to be more than 2 orders of
magnitude faster than the gold standard method (Widom insertion), with an acceptable level of error on
an enthalpy value of 0.34 kJ mol−1, and is therefore proposed as a valuable addition to the high-
throughput screening toolbox. This article is licensed under a Creative Commons Attribution 3 All simulations are performed for xenon adsorption on struc-
tures of the CoRE MOF 2019 database at innite dilution and at
298 K. Krypton adsorption has also been considered in order to see
the viability of the method in prediction of Xe/Kr selective mate-
rials (see full details in ESI† Section S4). Adsorption at another
temperature (600 K) was tested using the nal algorithm, and the
results are presented in ESI† Section S2. Other databases have also
been briey explored to test the robustness of the proposed algo-
rithm. A subset of a hypothetical MOF database, the ToBaCCo
database,24 has been screened (Section S6.1†), because it contains
very different MOFs according to the diversity analysis of Moosavi
et al.25 (for instance, the pores are larger). An amorphous material
database (containing 205 structures) has also been screened and
the algorithm identied some top materials for xenon adsorption
(Section S6.2†). One such biased sampling methodology was recently
proposed and used in a computational screening study of
adsorptive separation of xenon from krypton. Simon et al. used
a machine learning model to screen over 670 000 structures
based on geometrical descriptors and one energy descriptor
that mainly explains the accuracy of the nal model. To calcu-
late this descriptor, the authors used the average of the inter-
action energy over the Voronoi network to account for the
exponential contribution of the energy in the Henry constant. According to the authors, this approach can sample the most
favorable sites of the structure without wasting computational
time on unfavorable ones. However, because the sampled
points are at the center of the cages, they may not always be the
most attractive sites especially for large pores. This approximate
approach
makes
this
sampling
interesting
for
quickly
producing an energy-based ML descriptor, but it could not
replace a Widom insertion for assessing the adsorption
performance. Other biased sampling methods that calculate the
integrals of the adsorption enthalpy and the Henry constant
could be applied, instead. In this work we propose one way of
exploiting prior chemical knowledge of adsorption—the fact
that it occurs near the surface of the pores—to efficiently
sample the nanoporous space. Widom insertion19 is implemented in RASPA2. It consists in
randomly inserting a single molecule inside an existing molecular
system to measure an interaction energy. 1
Introduction Thanks to initiatives such as the Materials Genome
project,5,6 we now have databases of hundreds of thousands of
structures at our disposal, including both experimentally
known and theoretically predicted structures, along with their For low-pressure adsorption, a widely used characterization
parameter is the adsorption enthalpy DHads, which describes the
affinity of an adsorbate molecule with the internal surface of
a porous framework. In theory,18 this value is derived from the
continuous Boltzmann average of the interaction energies Eint of
the adsorbate with the framework over the entire porous space, Chem. Sci., 2023, 14, 1797–1807 | 1797 © 2023 The Author(s). Published by the Royal Society of Chemistry Edge Article
View Article Online Edge Article
View Article Online Chemical Science Chemical Science and this integral is calculated using a discrete summation over
a set of well-chosen points i (note that the −RT term comes from
the ideal gas approximation): and this integral is calculated using a discrete summation over
a set of well-chosen points i (note that the −RT term comes from
the ideal gas approximation): © 2023 The Author(s). Published by the Royal Society of Chemistry 2.1
Benchmark Before describing the core components of our surface sampling
algorithm, we briey present the other simulation tools used in
the article, for comparison and benchmarking purposes. We
used the RASPA2 soware for calculations such as Widom
insertion and surface area calculations.20 For the Voronoi
sampling technique, we used the Zeo++ soware to determine
the positions of the Voronoi nodes.21 All the calculations were
performed by considering the CoRE MOF 2019 (ref. 22) struc-
tures as rigid. In order to speed up the surface sampling, our
algorithm exploits the symmetry of the material, looping over
all symmetry-inequivalent atoms. Since the space groups (and
symmetry operations) of the nanoporous structures in the CoRE
MOF database were not specied, we used a Python script based
on the Spglib library to determine them.23 The unique atoms are
dened according to the symmetry determined by the afore-
mentioned Python script. DHads ¼
Ð
EintðrÞeEintðrÞ=RTdr
Ð eEintðrÞ=RTdr
RTx
P
i
Eiexpð Ei=RTÞ
P
i
expð Ei=RTÞ RT
(1) (1) This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. However, in molecular simulations, a complete sampling of
the free volume can be extremely cumbersome. Therefore,
random samplings are used to reduce simulation time, for
example in the Widom insertion method.19 Still, convergence of
the random sampling of space requires a large number of
samples, and most of the points may not contribute signi-
cantly to the Boltzmann average (i.e., they might have high
energy). It is possible to reduce the computational cost further
by reducing the number of sampled points, to try and capture
only those with the highest contribution to the actual value of
enthalpy. But, how can we choose these points a priori, without
rst calculating their energy? 3.1
Beyond Widom insertion Widom insertion is a standard calculation method that consists in
randomly inserting a single molecule inside an existing molecular
system by randomly choosing its center and its rotation angle.19 By
measuring the interaction energy of the molecule inserted, one can
obtain the excess free energy DFexc difference associated with its
insertion into the framework, i.e., the species chemical potential
mi. In the context of adsorption, this method has been used to
randomly insert a molecule in the empty porous framework: aer
many cycles, the simulation has generated a diverse enough
sample of points with different interaction energies Eint, yielding
the adsorption free energy DFads = −RT ln(hexp(−Eint/RT)i), the
Henry constant KH, and the adsorption enthalpy, DHads (eqn (1)),
which has the opposite sign of the zero-loading isosteric heat of
adsorption Q0
st. The Henry constant18,41,42 KH (in mol kg−1 Pa−1)
associated with the adsorption inside a crystalline framework of
mass density rf at temperature T can be derived using the
following eqn (2): This article is licensed under a Creative Commons Attribution 3.0 Unpo To model the van der Waals interactions, we used Lennard-Jones
(LJ) truncated and shied potentials with a 12 Å cut-offwithout
tail corrections. The atoms of the structures were modeled using
the LJ parameters from the universal force eld UFF.28 For xenon
we used the following LJ parameters:29 3Xe = 221.0 K and sXe =
4.100 Å.30 To determine cross interaction parameters between
xenon and the host atoms, we used the Lorentz–Berthelot combi-
nation rules.31 Throughout the article, all interaction energies are
calculated with these same parameters, and while the exact results
in terms of adsorption enthalpies depend on the force eld
chosen, the goal of this article is the comparison of different
methodologies. We want to stress that the sampling method
proposed herein could be used with any other force eld. Other
analytical forms could be used, or other LJ parameters could be
chosen; for example, it is standard to mix Dreiding32 for the
organic part and UFF for the inorganic part of the MOF structures. This article is licensed under a Creative Commons Attribution 3 In adsorption simula-
tions, these interaction energies of the randomly inserted adsor-
bate are typically used to determine values of the Henry constant
KH and of adsorption enthalpy DHads at the zero-loading limit. The surface area calculation implemented in RASPA2 follows
a very standard “rolling ball” algorithm26 based on hard spheres
with sizes determined by the sigma value of the Lennard-Jones
potential. First, a probe-molecule samples the spherical surface of
the atoms of the framework. A portion of the sphere is excluded,
because the probe overlaps with another atom. Each framework
atom has an area of adsorbable surface, and the sum of all the
areas gives the total adsorbable surface area. The values given by
these simulations are then compared to the values given by our
algorithm to check the consistency of our implementation. We propose a novel algorithm for rapid adsorption enthalpy
characterization, based on the reduction of the sampling space
from 3D to 2D. This method is more accurate than the previous
Voronoi sampling strategy. Moreover, the dimension reduction
coupled with symmetric properties makes it faster than the stan-
dard Widom insertion method. This algorithm has been tested on
xenon and is directly applicable to any spherical adsorbate model;
and it can then be adapted to polyatomic adsorbate molecules as
well. This algorithm can be used in the future to speed up the
calculation of adsorption properties in regular or ML-assisted
high-throughput
computational
screenings
of
nanoporous
materials. In mathematics, a tessellation of a given space corresponds
to a partition into non overlapping sub-spaces. In the Voronoi
tessellation,
named
aer
Georgy
Feodosevich
Voronoy
, a set of points (seeds) are
associated with a tessellation of regions (Voronoi cells) so that
each seed has a cell that is closer to it than any other seeds.27 © 2023 The Author(s). Published by the Royal Society of Chemistry 1798 | Chem. Sci., 2023, 14, 1797–1807 Chemical Science
View Article Online Chemical Science
View Article Online 2.3
Simulation box To design a versatile simulation tool, we use periodic boundary
conditions to create a rectangular simulation box for structures
with non-rectangular unit cells. An extended neighbor list is
created from the atoms of the translated rectangular boxes
within the chosen cutoff. When looping over the unique atoms
of the framework in the surface sampling, this neighbor list is
restrained to a shorter neighbor list to be used in the interaction
energy calculation, like in most molecular simulation algo-
rithms.33 To evaluate the effect of the neighbor list, we tested the
nal implementation without the implementation of the
neighbor list, and the simulation ran for 12.6 s instead of 0.34 s
(37 times slower) without altering the accuracy. This shows that
the implementation of an efficient neighbor list is a key point in
our algorithm. 3 2.2
Force eld p
y
p
g
The method we found to be the most efficient (slightly faster)
for the typical values of n that we consider (between 100 and 300
000) consists in wrapping a string of points around the sphere in
a spiral manner. This technique is closer to laying a uniform grid
over the surface than a “random sampling” of the surface, which
avoids redundant sampling points. The height h = r cos f (where
(r, q, f) are the spherical coordinates) of the sphere is uniformly
divided into n points; for each of these heights we chose an angle q
in the orthogonal plane space so that the difference between two
consecutive angles is the golden number.40 This method is referred
to as the spherical Fibonacci mapping. While it is not the optimal
solution to the Thomson problem, its uniformity is rather good for
our purposes (we have checked that its inuence on the calculated
properties is negligible) and the computational cost is lower. It also
gives a convergence for values of n smaller than the random
distribution methods described above, allowing us to use smaller
sampling sizes, which is why we used this method for the rest of
the surface sampling simulations presented in this article. © 2023 The Author(s). Published by the Royal Society of Chemistry Edge Article Applied in materials science, the Voronoi cells associated with
each atom of the framework can be used to determine key
geometrical descriptors (void volume, accessible surface area,
and pore sizes). At the vertices of each cell, there are Voronoi
nodes that were used in the Voronoi energy calculation pre-
sented by Simon et al.14 To compare our algorithm to a Voronoi
sampling, we used the interaction energy values at the Voronoi
nodes to calculate a Boltzmann average. These proxies for the
adsorption enthalpy are then indirectly compared to the
adsorption enthalpies calculated by our surface sampling. n. To generate random 3D unit vectors, one approach is to draw
random vectors in the corresponding cube, rejecting the points
that are not inside the sphere. A simple normalization of the
remaining vectors gives a random sampling of the sphere
surface.36 The same result can also be achieved by generating
three normally distributed random values and normalizing the
vector obtained by these numbers.36,37 This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Another technique to obtain a uniform distribution is to
imagine using a simulation of n charged points on the surface
of a sphere and minimize their electrostatic repulsion. This
method, based on the Thomson problem,38 relies on numerical
optimization and can become very expensive if n is high.39 2.4
Sphere sampling algorithm In our proposed method as in the surface area calculation
algorithm,26,34 we rely heavily on the use of a uniform sampling
of n points on the surface of a sphere, but this problem can be
quite challenging in and of itself. In fact, except for very specic
values of n, there is not a general analytical solution to the
problem, only numerical approximations. During the develop-
ment of our algorithm, we tested several existing methodolo-
gies35 to achieve this sampling. KH ¼ hexpð Eint=RTÞi
rfRT
¼
1
rfRT
1
Nsample
X
i˛sample
expð Ei=RTÞ The rst technique is to rely on random sampling, with no
guarantee of uniformity, but which should converge for a large (2) Chem. Sci., 2023, 14, 1797–1807 | 1799 © 2023 The Author(s). Published by the Royal Society of Chemistry Edge Article
View Article Online Edge Article
View Article Online Chemical Science where i represents the point of a sample used in practice to
calculate the integral. approximation of the adsorption enthalpy. However, this sampling
assumes that the nodes are close to the real, most favorable,
adsorption sites. Or to put it differently, the adsorption sites need
to be at the center of the pores, which is only true for structures
with pore sizes close to the adsorbate size. If the free volume has been thoroughly explored, the Boltzmann
average of the host/guest interaction energies converges to the
adsorption enthalpy at innite dilution. The Widom insertion
method is very accurate, meaning that it converges to the “perfect”
value of the adsorption enthalpy—for a given choice of interaction
parameters—in the limit of innite sampling. However, it is
computationally expensive, and a lot of computational resources
are wasted during this sampling in the calculation of interaction
energies that have a negligible contribution to the overall Boltz-
mann average (points of high energy). Therefore, we can improve
this method if we manage to sample preferentially points with the
most negative interaction energies. To achieve this improvement
we need to identify the characteristics of the adsorption sites that
will have the highest weight (“count the most”) in the nal average,
while avoiding sampling parts of space where points will have
marginal contribution. To check the accuracy of this sampling technique, we compared
it to our reference sampling, the Widom insertion with 100 000
cycles. Fig. This article is licensed under a Creative Commons Attribution 3. The Voronoi sampling and the surface sampling presented in
this article are examples of biased sampling methods, that follow
this idea. The change in the sampling technique can dramatically
improve the computation time required, and even a slight
improvement in computational efficiency can have a non-
negligible impact when dealing with datasets containing thou-
sands of structures. This article focuses on biased sampling tech-
niques to speed up adsorption enthalpy calculations. As a proof of
concept, we only consider monoatomic adsorbates (in our tests, we
used xenon) or adsorbates that can be modeled as a sphere (which
is common in molecular simulations of species such as CH4). However, the methodology can be adapted to rigid polyatomic
adsorbates, where the algorithm would need to be adapted by
sampling the rotational degree of freedom of the adsorbed
molecule. The root mean square error (RMSE) and the mean absolute
error (MAE) for Voronoi sampling are respectively 6.78 kJ mol−1
and 2.01 kJ mol−1, if we consider all structures in our set, which
seem too high to be useful for screening purposes. However, non-
porous materials would be screened out a priori in any high- Fig. 1
Scatter-plot of the enthalpies calculated by a Voronoi sampling
compared to the enthalpies calculated by a 100k-step Widom inser-
tion simulation of xenon in the structures of CoRE MOF 2019. The
points are labeled according to the largest cavity diameter (LCD)
belonging to one of the intervals. In all the comparisons in this paper, we chose to take a Widom
insertion simulation with 100 000 cycles (a very large number) as
a ground truth or reference for the adsorption enthalpy values of
every structure of the CoRE MOF 2019 database.22 2.4
Sphere sampling algorithm 1 compares the enthalpy computed in the Voronoi
sampling with the reference adsorption enthalpy (ground truth)—
showing at the same time the largest cavity diameter for each
porous framework. The correlation between the values of enthalpy
is very good only for a restricted number of structures with
enthalpy of around −50 kJ mol−1. For structures with higher
enthalpy, the correlation starts to degrade, and becomes very poor
for small-pore structures. For the points in purple, the largest
cavity diameter is lower than the kinetic diameter of a xenon,
where the sampling of the Voronoi nodes is clearly insufficient. In
addition, the accuracy loss at the other points (larger pores) can be
explained by the fact that the pores are slightly bigger and the
center of the pore is not a good approximation of adsorption site
position anymore: the adsorption sites are actually closer to the
pore surface than to the center of the pore. This conclusion is what
prompted us to propose a new sampling scheme based on the
molecular surface of the pore space, which we will detail in the
next sections. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This is reinforced by the very low computational cost of the
method. The Voronoi tessellation performed using the Zeo++
soware is extremely quick and can output the positions of the
Voronoi nodes in 0.28 s (measured as an average over all the
structures of the CoRE MOF 2019 database), on a typical
workstation (a single Intel Xeon Platinum 8168 core at 2.7 GHz). While a simple Python code for the energy calculation took
around 27 s per structure, we benchmarked that a C++ opti-
mized implementation can perform the Voronoi sampling in
around 0.4 s. We only need to remember that this method takes
a few hundred milliseconds per structure, while a Widom
insertion needs approximately hundreds of seconds per struc-
ture. A Voronoi sampling is therefore 2 to 3 orders of magnitude
quicker than a full sampling of the pore space. 3.3.1
Initial implementation. We present here our initial
implementation of the surface sampling algorithm, and its
basic principles. This rst implementation is a relatively basic
one and already performs well compared to the other methods. In the next sections, we rene it with two additional features
that will improve its accuracy and its speed. This initial implementation speeds up the calculation of
adsorption enthalpy in nanoporous materials by sampling
interaction energies only near the surface. It is illustrated in
Fig. 2. For this purpose, a loop over all unique atoms (as dened
by crystalline symmetry) is performed. And for each atom,
a sphere around its position is sampled using a uniform distri-
bution around it; these points will be called sampling points and
we can change the number of sampling points. The default
radius chosen for the sampling spheres is the distance rmin = 21/
6sij to the minimum of the LJ potential between atoms of type i
(belonging to the framework) and j (the guest), corresponding to
the strongest possible pair interaction (although the neighboring
atoms will of course have an inuence). Aer calculating the
interaction energy at each of the sampled points, a Boltzmann
average of these energies corresponds to a biased adsorption
enthalpy, as described by eqn (1). This preliminary study identied a fast method for adsorption
enthalpy calculations that can be widely used in screening proce-
dures, but has limited accuracy for quantitative prediction. 3.2
Voronoi sampling The use of the Voronoi decomposition of the pore space of mate-
rials for their geometric characterization has been widely
employed in computational studies in the last decade,21 in
particular since it was made easily available as part of the Zeo++
soware package.43 Its use was extended recently to implement
a novel sampling scheme, in a study proposing the ML-assisted
screening of nanoporous materials for xenon/krypton separation. In this article, Simon et al.14 relied on a Voronoi tessellation of the
nanoporous materials and assigned the potential adsorption sites
(i.e., the sampling points) at the nodes of this decomposition. The
Voronoi tessellation identies the vertices of polygons that corre-
spond to the closest regions of each atom of the structure. These
vertices (or Voronoi nodes) are the points equidistant to at least
four atoms of the structure, and they can be associated with
adsorption sites since they are positioned near the center of the
pores. It is possible to calculate the host/guest interaction energies
at
every
Voronoi
node,
and
average
them
to
obtain
an Fig. 1
Scatter-plot of the enthalpies calculated by a Voronoi sampling
compared to the enthalpies calculated by a 100k-step Widom inser-
tion simulation of xenon in the structures of CoRE MOF 2019. The
points are labeled according to the largest cavity diameter (LCD)
belonging to one of the intervals. © 2023 The Author(s). Published by the Royal Society of Chemistry 1800 | Chem. Sci., 2023, 14, 1797–1807 Chemical Science
View Article Online Chemical Science
View Article Online This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. It
raised important questions on the importance of selecting
sampling points within the pore space of materials, and we wanted
to develop an intermediate technique that is both fast and accurate
for the prediction of adsorption enthalpy. For this purpose, we
developed and optimized a new sampling technique that focuses
the sampling on the surface of the material, which is expected to
make up for the main aws of the Voronoi sampling. Edge Article Voronoi sampling and faster than Widom insertion. Our initial
idea is based on a series of theoretical considerations: (1) the
strong adsorption sites are near the surface of the material; (2) by
changing the problem from 3D to 2D sampling we can reduce the
complexity; and (3) the algorithm can scale with the number of
unique atoms in the structure (and not with the size of the unit
cell), which is efficient because many porous frameworks have
high symmetry. The rst consideration ensures that this method
will be more accurate than a Voronoi sampling, and the last two
made us think that a well-optimized code would be fast. To
conrm these hypotheses, we will analyze both the accuracy and
the speed of this new algorithm and compare them to those of
existing methods. throughput workow, as they would not be of interest. We can only
consider the structures with large enough cavities, larger than 3.7 Å
(a bit lower than 3.96 Å Xe kinetic diameter). Thereby, the RMSE
and MAE drop respectively to 2.11 kJ mol−1 and 1.55 kJ mol−1,
which can be considered acceptable for a quick estimation of the
guest–host affinity, but not for an accurate adsorption enthalpy
calculation. Voronoi sampling and faster than Widom insertion. Our initial
idea is based on a series of theoretical considerations: (1) the
strong adsorption sites are near the surface of the material; (2) by
changing the problem from 3D to 2D sampling we can reduce the
complexity; and (3) the algorithm can scale with the number of
unique atoms in the structure (and not with the size of the unit
cell), which is efficient because many porous frameworks have
high symmetry. The rst consideration ensures that this method
will be more accurate than a Voronoi sampling, and the last two
made us think that a well-optimized code would be fast. To
conrm these hypotheses, we will analyze both the accuracy and
the speed of this new algorithm and compare them to those of
existing methods. This article is licensed under a Creative Commons Attribution 3.0 Fig. 3
RMSE convergence of our algorithm (left) compared to a 100k-
step Widom insertion simulation (right) for xenon adsorption in all the
structures of the CoRE MOF 2019 database. In order to validate the accuracy of the approximation made
using this sampling, we applied this algorithm with 300 000
sampling points per unique atom. The results are illustrated in Fig. S1 and S2 and Table S1 of the ESI.† There is a good numerical
agreement with the reference calculations; the RMSE and MAE are
only around 0.90 kJ mol−1 and 0.66 kJ mol−1 considering all the
structures from the database. Moreover, there is no noticeable
difference in RMSE when considering the structures with a pore
size above 3.7 Å (as determined by the largest cavity diameter, or
LCD). Unlike Voronoi sampling, this method gives a consistent
accuracy across all the structures of the database with a lower
error. The fact that the RMSE error is below 1 kJ mol−1 is quite
promising, and validates our intuition that this new sampling
technique can be an intermediate between to the two previous
methods (Voronoi and Widom). Because we have no physical model that would predict the
optimal value of the sampling sphere, we followed a statistical
approach. We studied the inuence of the l parameter on both
the accuracy and the computation time, and the results are
presented in Fig. 4. The RMSE turns out to be relatively high at
around 0.90 kJ mol−1 for a radius of the sphere lower than the
rmin, and it then decreases for larger values of radius to reach
a plateau at around 0.35 kJ mol−1. We conrm that by
increasing the sampling sphere radius we can improve the
accuracy of our algorithm, and nd that for values of l higher
than 1.6, the accuracy is stabilized. We also nd that increasing
the sphere radius negatively impacts the computational effi-
ciency, since it increases the number of neighbors considered in
the energy calculation. Aer proving the good accuracy of the method, we are now
exploring the computation time required. We see in Fig. 3 that the
method reaches an RMSE below 1.0 kJ mol−1 very quickly for an
average CPU time of 1.2 s (Table S1†), corresponding to 2000
sampling points per atom. © 2023 The Author(s). Published by the Royal Society of Chemistry 3.3
Construction of a surface sampling algorithm In this section we describe the development of our surface
sampling algorithm, with the goal of being more accurate than Fig. 2
Schematic description of our surface sampling based on the three main steps of the algorithm: the loop over the unique atoms, the spiral
sampling around each atom, and the energy averaging. The adsorbate is represented by the point i and is moved across all the points around the
unique atoms of the structure. Fig. 2
Schematic description of our surface sampling based on the three main steps of the algorithm: the loop over the unique atoms, the spiral
sampling around each atom, and the energy averaging. The adsorbate is represented by the point i and is moved across all the points around the
unique atoms of the structure. Chem. Sci., 2023, 14, 1797–1807 | 1801 © 2023 The Author(s). Published by the Royal Society of Chemistry Edge Article
View Article Online Edge Article
View Article Online Fig. 3
RMSE convergence of our algorithm (left) compared to a 100k-
step Widom insertion simulation (right) for xenon adsorption in all the
structures of the CoRE MOF 2019 database. Chemical Science Chemical Science Chemical Science and adopting a fast rejection criterion. The rejection of high-
energy points with little contribution to the nal enthalpy
value can reduce the simulation time, whereas the size of the
sampling sphere can improve the accuracy. The initially chosen
sphere size only takes into account the interaction with the
closest atom; we therefore chose to set it at the minimum of
Lennard-Jones potential. However, the interaction with the
neighboring atoms can further stabilize the adsorbate, so
sampling further from this minimum could in consequence
increase the accuracy of our surface sampling method. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 3.3.2
Size of the sampling sphere. The validity of the initial
algorithm is based on the assumption that the adsorption site is
at the minimum of the Lennard-Jones potential. It will only
perform well if the closest atom contributes to almost all the
interactions, but in real frameworks other neighboring atoms
contribute to the host/guest interaction as well. We have found
that in the vast majority of materials, the adsorption sites are
located farther apart compared to the LJ potential minimum, in
order to maximize the contribution of all atoms—and because
of the dissymmetry of the interaction potential well. In order to
see if this could be introduced in our algorithm, we imple-
mented a parameter l, and the sampling sphere radius is now
dened using Rl = ls, where s is the distance at which the LJ
potential is zero. If l = 21/6, we fall back to our initial denition
of the sampling sphere, and the adsorbent is at the minimum of
the LJ potential of the atom. If l = 1, the sampling sphere is at
the zero of the LJ potential, and by increasing this parameter,
we can check if our intuition was right. This article is licensed under a Creative Commons Attribution 3.0 can optimize the parameter on a small diverse sample of the
unseen data. strong and points with non negligible contribution would end
up being rejected. This rejection condition is schematically
represented in Fig. 5. 3.3.3
Rejection conditions. As shown above, our algorithm
has better accuracy than Voronoi sampling, but its initial
implementation was several times slower, which could make it
unsuitable for screening applications in high-throughput
workows, where the number of structures to be screened can
reach one million or more. To reduce the computational
expense, we thought of rejecting the points with little contri-
bution to the nal enthalpy, i.e., the largely positive interaction
energies that would vanish in the exponential of the Boltzmann
average. This rejection condition is expected to speed up the calcula-
tions, since the energy calculation is avoided for the rejected
sampling points. The energy calculation accounts for the largest
portion of the CPU time spent on the surface sampling. For the
structure KAXQIL,44 the Lennard-Jones potential calculation
represents up to 90% of the calculation time for 100 000 sampling
points per sphere (with the initial algorithm). The higher the factor
m, the more rejections there would be. But, if too many points are
rejected, the accuracy will drop. Here again, we used a statistical
analysis to determine the optimal value of m, making our sampling
faster without compromising the accuracy of the enthalpy calcu-
lation. The results are displayed in Fig. 6. Inspired by typical methods for accessible surface calcula-
tions, we implemented a hard sphere rejection condition based
on the distance to neighbors. If the adsorbate is too close to
another atom of the structure, the sampling point is rejected,
i.e., its energy is not calculated (or considered to be innite). We
based this distance threshold on the sij parameter of the
Lennard-Jones potential. To determine the optimal threshold,
we introduced a factor m with real values between 0 and 1 that
changes the size of the hard sphere rejection condition. If the
guest–host distance is lower than dm = m × s, then the point is
rejected. If m = 0, then there is no rejection condition. And if m =
1, we reject all points with a positive energy interaction with at
least one atom of the structure. This condition could be a bit The values of RMSE and time in Fig. This article is licensed under a Creative Commons Attribution 3.0 This is far less than the 150 s (Table
S2†) required for a Widom insertion to reach its plateau value, for
an RMSE of 0.10 kJ mol−1 with 12 000 cycles. Moreover, the Widom
insertion needs around 14 s to reach a similar RMSE of 1.0 kJ
mol−1, which is still slower than the surface sampling. We can
conclude that this initial implementation of the surface sampling
is faster than a standard Widom insertion, with a good accuracy. By choosing an optimal sampling sphere, we can more than
halve the error, while increasing the computation time by
around 20 percent, when comparing the case l = 1.6 with l =
1.1 (close to rmin). In most cases, it will be an acceptable trade-
off. However, in a case where the computation time is crucial,
like in a rapid screening, the optimal choice might not be to
increase the sampling sphere at l = 1.6 but to have it lower at l
= 1.4 or l = 1.2, and have an RMSE at around 0.5 kJ mol−1—still
quite acceptable. The new scale parameter introduced in this
section can therefore be tweaked to serve the users' purpose,
whether it is to focus on the accuracy or to optimize the
computation speed. If one wants to use it on a completely
different database under very different conditions, then one can
either choose a default value that works ne (e.g. l = 1.4) or one However, this initial implementation of the method is slower
than a Voronoi sampling that only needs to sample around 1600
points on average, instead of 13 000 sampled points on average
(if we multiply by the average number of unique atoms). The
sampling part would take approximately 0.15 s, and the Voronoi
node generation 0.28 s, so our surface sampling algorithm
remains 2 to 3 times slower (implemented in an identically
compiled language, in this case C++). In order to improve the
accuracy and performance, we have further tweaked the surface
sampling method, adjusting the size of the sampling sphere © 2023 The Author(s). Published by the Royal Society of Chemistry 1802 | Chem. Sci., 2023, 14, 1797–1807 Fig. 6
Influence of the rejection coefficient m on the average CPU
time required for a simulation of 100k sampling points and the RMSE
compared to the reference adsorption enthalpy. © 2023 The Author(s). Published by the Royal Society of Chemistry This article is licensed under a Creative Commons Attribution 3.0 The averaging is
performed only on the structures with a largest cavity diameter (LCD)
greater than 3.7 Å. Chemical Science
View Article Online View Article Online Fig. 4
Influence of the sampling sphere radius Rl on the average CPU
time required for a simulation of 100k sampling points and the RMSE,
compared to the reference adsorption enthalpy. The averaging is
performed only on the structures with a largest cavity diameter (LCD)
higher than 3.7 Å. Fig. 6
Influence of the rejection coefficient m on the average CPU
time required for a simulation of 100k sampling points and the RMSE
compared to the reference adsorption enthalpy. The averaging is
performed only on the structures with a largest cavity diameter (LCD)
greater than 3.7 Å. Edge Article
Chemical Science Edge Article This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 4
Influence of the sampling sphere radius Rl on the average CPU
time required for a simulation of 100k sampling points and the RMSE,
compared to the reference adsorption enthalpy. The averaging is
performed only on the structures with a largest cavity diameter (LCD)
higher than 3.7 Å. Fig. 6
Influence of the rejection coefficient m on the average CPU
time required for a simulation of 100k sampling points and the RMSE
compared to the reference adsorption enthalpy. The averaging is
performed only on the structures with a largest cavity diameter (LCD)
greater than 3.7 Å. This article is licensed under a Creative Commons Attribution 3.0 On very large databases, one
could consider that this increase in the required computational
time is not worth the accuracy improvement, and one could decide
to keep a smaller sampling sphere. If this method is transposed to
different molecular systems, its parameters should be tested on
the specic database and adsorbate of interest. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This article is licensed under a Creative Commons Attribution 3.0 Unported Fig. 7
Comparison of the RMSE to the reference Widom insertion
(100k cycles) and the average computation time for different types of
enthalpy calculation methods. The surface sampling calculations were
all performed with 2k sampling points on each sphere and the Widom
simulations were performed using 12k cycles. These values corre-
spond to the value at the convergence identified using Fig. 3. 3.4.2
Calculation of the Henry constant and surface area. The main goal of our sampling algorithm is to calculate
adsorption enthalpy at the zero-loading limit. But the method
can also calculate the Henry constant and surface area of the
materials at the same time, without signicant additional
computational cost. The Henry constant is a key metric for
assessing the affinity of an adsorbate to a nanoporous structure. The A/B gas selectivity at low pressure is dened as a ratio of the
Henry constants of components A and B. This important
property can be calculated using eqn (2) in a Widom insertion
calculation. Instead of using the interaction energies at the
Widom inserted points, we can now use the surface sampled
points to get an approximate value for the Henry constant. This article is licensed under a Creat higher values of m that trade a bit more accuracy in exchange of
further speed gains. For the simulations considered in Fig. 6, the use of a rejec-
tion condition m = 0.85 makes the simulation four times faster
than the standard algorithm. As we will see in the next section,
the combination of optimal values for the l and m parameters
generates an algorithm with very interesting performance
compared to Voronoi sampling or Widom insertion. Using the optimized set of parameters for surface sampling,
we assessed the performance of our algorithm on the values of
the Henry constant by comparing them to ground truth ob-
tained by 100 000 cycles of Widom insertion. This article is licensed under a Creative Commons Attribution 3.0 6 are averaged only on
the most interesting structures for xenon adsorption (LCD $ 3.7
Å). For m # 0.85, increasing the value of m improves the speed of
the calculation without changing the RMSE.‡ For high values of
m, the rejection condition is too strong and we reject points with
non-negligible contribution to the overall enthalpy. The RMSE
increases as a consequence. If we want to keep the accuracy
unchanged, the optimal value is therefore m x 0.85, because it
gives the lowest computation time with a similar RMSE. We
note that it would be possible, in specic cases, to explore Fig. 5
Simplified 2D representation of the principle of rejection conditions and the concept of sampling spheres inside the one-dimensional
channels of a nanoporous material. Fig. 5
Simplified 2D representation of the principle of rejection conditions and the concept of sampling spheres inside the one-dimensional
channels of a nanoporous material. Chem. Sci., 2023, 14, 1797–1807 | 1803 © 2023 The Author(s). Published by the Royal Society of Chemistry Edge Article
View Article Online Edge Article
View Article Online Chemical Science Fig. 7
Comparison of the RMSE to the reference Widom insertion
(100k cycles) and the average computation time for different types of
enthalpy calculation methods. The surface sampling calculations were
all performed with 2k sampling points on each sphere and the Widom
simulations were performed using 12k cycles. These values corre-
spond to the value at the convergence identified using Fig. 3. accurate and 26% faster than Voronoi sampling, and it is also
about 430 times faster than a Widom insertion with 12k cycles. Finally, we suggest that the values of the parameters optimized
in this work might need adjustment when applied to other
adsorption systems. The optimal m parameter depends on the size
of the adsorbent, and it should be tweaked differently when
considering another adsorbent. For instance, the set of structures
used for the optimization of m depends on the size of their cavities,
and the 3.7 Å threshold chosen here would need to be changed
according to the kinetic diameter of the adsorbate. Furthermore,
as aforementioned in the section on rejection conditions, it is
possible to trade-offa bit of accuracy for faster simulations espe-
cially in high-throughput screenings where speed is extremely
important. Similarly, in the case of xenon, the cost of increasing
the sphere size is around 10 to 20%. This article is licensed under a Creative Commons Attribution 3.0 Since the Henry
constant corresponds to the exponential of an adsorption free
energy and we are more interested in the precision of the free
energy, we are using a log-scale evaluation metric. For surface
sampling, the log-RMSE of KH is equal to 0.2, which means that
the order of magnitude of the values is well predicted (Table
S4†). If we consider the derived free energy DFads = −RT
log(rfRTKH), the RMSE is of the order of 1.1 kJ mol−1 reached in
about 1 s (Table S6†), whereas for Widom insertion, this level of
error is also reached in a similar amount of time and 0.1 kJ
mol−1 of RMSE is reached in about 86 s (Table S7†). For free
energy calculations, surface sampling is still 86 times faster to
converge. If we consider that the main target is the adsorption
enthalpy, the Henry constant can be calculated with little
additional computational cost and with reasonable accuracy: we
get two thermodynamic properties of interest for the price of
one. © 2023 The Author(s). Published by the Royal Society of Chemistry 1804 | Chem. Sci., 2023, 14, 1797–1807 This article is licensed under a Creative Commons Attribution 3.0 Unport p
y
Finally, although the algorithm in its present form can already
be applied in a wide range of applications, additional develop-
ment work could allow us to generalize it to polyatomic adsor-
bates. For instance, we would need to work on a denition of the
molecular radius for nonspherical adsorbates as well as all the
orientation conformations of the adsorbent. We could imagine
making the distance to the surface depend on the orientation of
the adsorbate or sample a band volume on the surface. Although
the best implementation of surface sampling for polyatomic
adsorbates remains an open question, in theory it should be
possible to apply it to more complex adsorbates than spherical
noble gas. This would add more complexity to the algorithm but
would not change the fundamental speed up due to surface
sampling, since these orientation moves are also performed in
other standard methodologies. To improve the accuracy even
more, we could test hybrid samplings with multiple sampling
spheres, or a combination of Voronoi nodes and sampling
spheres. Another idea could be to have fractions of spheres that
are oriented toward the center of pores given by the Voronoi
node. In theory, having a wider variety of sampling points can
only improve the sampling. There are therefore multiple possible
sampling techniques that could be built around the method
introduced herein. The code is made freely available on the
group's GitHub, where further development will be released. 4
Conclusions and perspectives In the present article, we described a novel algorithm for the
high-speed calculation of adsorption enthalpy in nanoporous
materials that takes a unique approach to reduce the sampling
necessary. This new algorithm is based on the core principle of
dimensional reduction, from a volume problem to a surface
one. The algorithm is proven to be signicantly faster than the
reference Widom insertion (random sampling of porous space). Moreover, the error associated is found to be in the order of 0.4
kJ mol−1, tested throughout the entire CoRE MOF 2019 data-
base, for xenon adsorption. Even when compared to existing
very fast sampling techniques such as Voronoi sampling, this
surface
sampling
technique
requires
similar
CPU
time,
combined with a better accuracy. Based on these results, this algorithm has important
potential for applications in the current computational analysis
workows of material databases, such as high-throughput
screening studies. For instance, this algorithm can be used to
get a fast approximation of the low-loading adsorption enthalpy
of a molecule inside nanoporous materials. This cheap evalu-
ation of enthalpy can be used to screen out the structures with
little affinity with the targeted adsorbate molecule. It can also be
used as a thermodynamic descriptor for selectivity prediction in
a machine learning model, as performed by Simon et al.14 The
computational speed-up brought about by this novel method-
ology can also enable the screening of materials databases at
a larger scale in the future. Data availability Data and code related to this study are available from our group
repository
at
https://github.com/fxcoudert/citable-data. The
RAESS code is available at https://github.com/coudertlab/RAESS. 3.4
Final surface sampling algorithm 3.4.1
Performance comparison. For the calculation of
adsorption enthalpy, our proposed surface sampling method is
a good compromise between the accuracy of Widom insertion (full
sampling of the porous space) and the speed of a less accurate
method such as Voronoi sampling. The performance of our algo-
rithm, including the two new features (sampling sphere scaling
and rejection criterion) is illustrated in Fig. 7, where we can see the
improvement brought about by each feature and how it compares
to reference simulations. All CPU times are calculated using the
smallest possible number of sampling points so that the respective
algorithms reach convergence. With the implementation of
a rejection condition, we nd that surface sampling is even quicker
than Voronoi sampling. Moreover, the increase in the size of the
sampling sphere makes the surface sampling much more accu-
rate, reaching an RMSE of 0.33 kJ mol−1 and an MAE of 0.21 kJ
mol−1. The ideal set of parameters, determined for porous mate-
rials from the CoRE MOF 2019 database, is (l = 1.6, m = 0.85) in
order to combine the lowest error and smallest computational
cost. By combining both of these new features into the algorithm,
we have a nal surface sampling method with an RMSE of 0.33 kJ
mol−1 and an average computation time of 0.34 s per structure. According to the data in Table S3,† it is about 6 times more The same goes for the determination of the surface area. We
can adapt our algorithm to count the number of points of the
sampling spheres that have a negative energy. These represent
the points where a guest molecule can favorably interact; © 2023 The Author(s). Published by the Royal Society of Chemistry 1804 | Chem. Sci., 2023, 14, 1797–1807 Chemical Science
View Article Online Chemical Science
View Article Online Chemical Science
View Article Online This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. where V is the volume of the cell; a is the number of atoms of the
cell; Naccessible(a) is the number of accessible points around the
atom a; Ntotal is the total number of sampling points; r(a) is the
radius of the sampling sphere around the atom a. When we set l
= 1, we are sampling spheres that have a radius of s and it is
equivalent to considering hard spheres all dened using s
(convention used by RASPA2 to calculate surface areas). If we
compare simulation with l = 1, we obtain surface areas that are
very close to the one obtained by RASPA2 (see Fig. S11 in the
ESI†). However, when we consider l = 1.6, we lose the accor-
dance previously obtained and the points are weakly correlated
at the log-scale (see Fig. S10 in the ESI†). The difference can be
explained by the fact that the sphere size is larger, but the
proportion of adsorbable points also changes. The relationship
between these two adsorption surface areas is not trivial at all. Since the calculation of surface areas is quite cheap, this
implementation would not be very useful, except for having
a rough idea of the surface area. where V is the volume of the cell; a is the number of atoms of the
cell; Naccessible(a) is the number of accessible points around the
atom a; Ntotal is the total number of sampling points; r(a) is the
radius of the sampling sphere around the atom a. When we set l
= 1, we are sampling spheres that have a radius of s and it is
equivalent to considering hard spheres all dened using s
(convention used by RASPA2 to calculate surface areas). If we
compare simulation with l = 1, we obtain surface areas that are
very close to the one obtained by RASPA2 (see Fig. S11 in the
ESI†). However, when we consider l = 1.6, we lose the accor-
dance previously obtained and the points are weakly correlated
at the log-scale (see Fig. S10 in the ESI†). The difference can be
explained by the fact that the sphere size is larger, but the
proportion of adsorbable points also changes. The relationship
between these two adsorption surface areas is not trivial at all. © 2023 The Author(s). Published by the Royal Society of Chemistry This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Since the calculation of surface areas is quite cheap, this
implementation would not be very useful, except for having
a rough idea of the surface area. Edge Article therefore when dividing it by the number of sampled points, we
obtain a proportion of the adsorbable area of the sphere. Summing this over all atoms, we obtain the total surface area. This implementation is summed up in eqn (3): a simple Lennard-Jones interaction potential, this sampling
technique could equally be used to speed up samplings of space
based on more expensive modeling strategies, including polar-
izable force elds or density functional theory (DFT) calculations. In the literature, the need for cheap ab initio grade thermody-
namic properties is usually fullled by using an importance
sampling method based on a classical force eld.45 In our
method, the description of surface sampling is independent of
any force eld, and the sampling spheres can be dened
according to kinetic radius, van der Waals radius or any other
physically relevant distance. Consequently, given a denition of
atomic radii, it is possible to dene a surface on which to carry
out other types of simulations such as neural network potential,
DFT or any other force elds. Although the accuracy or relevance
of such a sampling remains an open question, the approach will
undeniably speed up the simulations. This could even be applied
to calculate adsorption enthalpies while considering intrinsic
structure exibility,46 a task whose main drawback is the high
computation time required. Since surface sampling is hundreds
of time faster than standard methodologies, we could use
hundreds of snapshots in a exibility-aware calculation. SA ¼ 1
V
X
a˛cell
NaccessibleðaÞ
Ntotal
4prðaÞ2
(3) (3) Notes and references 18 S. Kar and C. Chakravarty, Computational evaluation of
Henry's constants and isosteric heats of sorption for
Lennard-Jones sorbates in Na-Y zeolite, Mol. Phys., 2001,
99, 1517–1521. ‡ In fact, what we observe is a deterioration of the accuracy for structures with
small pores because the probability of rejection in a conned space is really high
and all sampled points end up rejected. But these points are not considered, if we
apply the condition on the cavity size (LCD $ 3.7 Å). This article is licensed under a Creative Commons Attribution 3.0 19 B. Widom, Some Topics in the Theory of Fluids, J. Chem. Phys., 1963, 39, 2808–2812. 1 D. S. Sholl and R. P. Lively, Seven chemical separations to
change the world, Nature, 2016, 532, 435–437. 20 D. Dubbeldam, S. Calero, D. E. Ellis and R. Q. Snurr, RASPA:
molecular simulation soware for adsorption and diffusion
in exible nanoporous materials, Mol. Simul., 2016, 42, 81–101. 2 National Academies of Sciences, Engineering, and Medicine, A
Research Agenda for Transforming Separation Science, National
Academies Press, 2019. 21 T. F. Willems, C. H. Rycro, M. Kazi, J. C. Meza and
M. Haranczyk, Algorithms and tools for high-throughput
geometry-based analysis of crystalline porous materials,
Microporous Mesoporous Mater., 2012, 149, 134–141. 3 R. Morris and P. Wheatley, Gas Storage in Nanoporous
Materials, Angew. Chem., Int. Ed., 2008, 47, 4966–4981. 4 M. Ding, R. W. Flaig, H.-L. Jiang and O. M. Yaghi, Carbon
capture and conversion using metal–organic frameworks and
MOF-based materials, Chem. Soc. Rev., 2019, 48, 2783–2828. 22 Y. G. Chung, E. Haldoupis, B. J. Bucior, M. Haranczyk, S. Lee,
H. Zhang, K. D. Vogiatzis, M. Milisavljevic, S. Ling,
J. S. Camp, B. Slater, J. I. Siepmann, D. S. Sholl and
R. Q. Snurr, Advances, Updates, and Analytics for the
Computation-Ready,
Experimental
Metal–Organic
Framework Database: CoRE MOF 2019, J. Chem. Eng. Data,
2019, 64, 5985–5998. 5 , The Materials Genome Initiative. , 2022, Available online at
https://www.mgi.gov/. 6 T. Kalil and C. Wadia Materials Genome Initiative for Global
Competitiveness. 2011. 23 A. Togo and I. Tanaka Spglib: a soware library for crystal
symmetry search. 2018; https://arxiv.org/abs/1808.01590. 7 S. Chibani and F.-X. Coudert, Systematic exploration of the
mechanical properties of 13 621 inorganic compounds,
Chem. Sci., 2019, 10, 8589–8599. 24 Y. J. Colon, D. A. Gomez-Gualdron and R. Q. Acknowledgements 16 R. Gaillac, S. Chibani and F.-X. Coudert, Speeding Up
Discovery
of
Auxetic
Zeolite
Frameworks
by
Machine
Learning, Chem. Mater., 2020, 32, 2653–2663. This work was nancially supported by Orano. The authors
thank Philippe Guilbaud and Isabelle Hablot for discussions on
the topic of adsorption-based separation. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Commons Attribution 3.0 Unported Licence. 17 K. Jorner , Solvent accessible surface area. , 2021; Available
online at https://kjelljorner.github.io/morfeus/sasa.html. Conflicts of interest There are no conicts to declare. 15 C. E. Wilmer, M. Leaf, C. Y. Lee, O. K. Farha, B. G. Hauser,
J. T. Hupp and R. Q. Snurr, Large-scale screening of
hypothetical metal–organic frameworks, Nat. Chem., 2011,
4, 83–89. Author contributions Both authors designed the study, analysed the results, wrote
and revised the article. E. R. wrote the RAESS soware and ran
the molecular simulations. We note, moreover, that the speed of our method resides in
the sampling technique itself, rather than in the actual energy
calculation. While we have benchmarked it in this work for © 2023 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2023, 14, 1797–1807 | 1805 Edge Article
View Article Online View Article Online Chemical Science a Xenon/Krypton Mixture?, Chem. Mater., 2015, 27, 4459–
4475. a Xenon/Krypton Mixture?, Chem. Mater., 2015, 27, 4459–
4475. Notes and references Snurr,
Topologically Guided, Automated Construction of Metal–
Organic Frameworks and Their Evaluation for Energy-
Related Applications, Cryst. Growth Des., 2017, 17, 5801–
5810. 8 E. Ren and F.-X. Coudert, Thermodynamic exploration of
xenon/krypton separation based on a high-throughput
screening, Faraday Discuss., 2021, 231, 201–223. 9 E. Ren, P. Guilbaud and F.-X. Coudert, High-throughput
computational
screening
of
nanoporous
materials
in
targeted applications, Digital Discovery, 2022, 1, 355–374. 25 S. M. Moosavi, A. Chidambaram, L. Talirz, M. Haranczyk,
K. C. Stylianou and B. Smit, Capturing chemical intuition
in synthesis of metal-organic frameworks, Nat. Commun.,
2019, 10, 539. 10 J. Greeley, T. F. Jaramillo, J. Bonde, I. Chorkendorffand
J. K. Nørskov, Computational high-throughput screening of
electrocatalytic
materials
for
hydrogen
evolution,
Nat. Mater., 2006, 5, 909–913. 26 A. Shrake and J. Rupley, Environment and exposure to
solvent of protein atoms. Lysozyme and insulin, J. Mol. Biol., 1973, 79, 351–371. 11 A. K. Singh, K. Mathew, H. L. Zhuang and R. G. Hennig,
Computational
Screening
of
2D
Materials
for
Photocatalysis, J. Phys. Chem. Lett., 2015, 6, 1087–1098. 27 C. H. Rycro, VORO++: A three-dimensional Voronoi cell
library in C++, Chaos, 2009, 19, 041111. 12 S. Back, K. Tran and Z. W. Ulissi, Discovery of Acid-Stable
Oxygen
Evolution
Catalysts:
High-Throughput
Computational Screening of Equimolar Bimetallic Oxides,
ACS Appl. Mater. Interfaces, 2020, 12, 38256–38265. 28 A. K. Rappe, C. J. Casewit, K. Colwell, W. A. Goddard III and
W. M. Skiff, UFF, a full periodic table force eld for
molecular
mechanics
and
molecular
dynamics
simulations, J. Am. Chem. Soc., 1992, 114, 10024–10035. 29 P. Ryan, O. K. Farha, L. J. Broadbelt and R. Q. Snurr,
Computational screening of metal-organic frameworks for
xenon/krypton separation, AIChE J., 2010, 57, 1759–1766. 13 S. Han, Y. Huang, T. Watanabe, Y. Dai, K. S. Walton, S. Nair,
D. S. Sholl and J. C. Meredith, High-Throughput Screening of
Metal–Organic Frameworks for CO2Separation, ACS Comb. Sci., 2012, 14, 263–267. 30 J. O. Hirschfelder; C. F. Curtiss and R. B. Bird Molecular
theory of gases and liquids. Molecular theory of gases and
liquids 1964. 14 C. M. Simon, R. Mercado, S. K. Schnell, B. Smit and
M. Haranczyk, What Are the Best Materials To Separate © 2023 The Author(s). Published by the Royal Society of Chemistry 1806 | Chem. Sci., 2023, 14, 1797–1807 Chemical Science
View Article Online View Article Online Edge Article Chemical Science 31 H. A. Notes and references Lorentz, Ueber die Anwendung des Satzes vom Virial in
der kinetischen Theorie der Gase, Ann. Phys., 1881, 248, 127–
136. 39 J. R. Morris, D. M. Deaven and K. M. Ho, Genetic-algorithm
energy minimization for point charges on a sphere, Phys. Rev. B: Condens. Matter Mater. Phys., 1996, 53, R1740–R1743. 32 S. L. Mayo, B. D. Olafson and W. A. Goddard, DREIDING:
a generic force eld for molecular simulations, J. Phys. Chem., 1990, 94, 8897–8909. 40 A. Gonzalez, Measurement of Areas on a Sphere Using
Fibonacci and Latitude–Longitude Lattices, Math. Geosci.,
2009, 42, 49–64. 33 D. Frenkel and B. Smit Understanding molecular simulation:
from algorithms to applications, Elsevier, 2001, vol. 1. 41 R. L. June, A. T. Bell and D. N. Theodorou, Prediction of low
occupancy sorption of alkanes in silicalite, J. Phys. Chem.,
1990, 94, 1508–1516. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 34 F. Eisenhaber, P. Lijnzaad, P. Argos, C. Sander and
M. Scharf, The double cubic lattice method: Efficient
approaches to numerical integration of surface area and
volume
and
to
dot
surface
contouring
of
molecular
assemblies, J. Comput. Chem., 1995, 16, 273–284. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 42 L. Sarkisov, R. Bueno-Perez, M. Sutharson and D. Fairen-
Jimenez, Materials Informatics with PoreBlazer v4.0 and
the CSD MOF Database, Chem. Mater., 2020, 32, 9849–9867. This article is licensed under a Creative Commons Attribution 3.0 Unpor 43 M. Pinheiro, R. L. Martin, C. H. Rycro, A. Jones, E. Iglesia
and M. Haranczyk, Characterization and comparison of
pore landscapes in crystalline porous materials, J. Mol. Graphics Modell., 2013, 44, 208–219. 35 B. Edward and A. B. K. Saff, Distributing many points on
a sphere, Math. Intell, 1997, 19, 5–11. 36 G. Marsaglia, Choosing a Point from the Surface of a Sphere,
Ann. Math. Stat., 1972, 43, 645–646. 44 D. Banerjee, Z. Zhang, A. M. Plonka, J. Li and J. B. Parise, A
Calcium
Coordination
Framework
Having
Permanent
Porosity and High CO2/N2 Selectivity, Cryst. Growth Des.,
2012, 12, 2162–2165. 37 C. Simon Generating uniformly distributed numbers on
a
sphere. 2015,
Available
online
at
http://
corysimon.github.io/articles/uniformdistn-on-sphere/. 45 S. Vandenbrande, M. Waroquier, V. V. Speybroeck and
T. Verstraelen, Ab Initio Evaluation of Henry Coefficients
Using Importance Sampling, J. Chem. Theory Comput.,
2018, 14, 6359–6369. 38 J. J. Chem. Sci., 2023, 14, 1797–1807 | 1807 © 2023 The Author(s). Published by the Royal Society of Chemistry Notes and references Thomson, On the structure of the atom: an investigation
of the stability and periods of oscillation of a number of
corpuscles
arranged
at
equal
intervals
around
the
circumference of a circle; with application of the results to
the theory of atomic structure, Lond. Edinb. Dublin Philos. Mag. J. Sci., 1904, 7, 237–265. 46 M. Witman, S. Ling, S. Jawahery, P. G. Boyd, M. Haranczyk,
B. Slater and B. Smit, The Inuence of Intrinsic Framework
Flexibility on Adsorption in Nanoporous Materials, J. Am. Chem. Soc., 2017, 139, 5547–5557. Chem. Sci., 2023, 14, 1797–1807 | 1807 © 2023 The Author(s). Published by the Royal Society of Chemistry
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Challenges to the Israeli healthcare system: attracting medical students to primary care and to the periphery
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Israel journal of health policy research
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Abstract Background: The greatest challenges facing healthcare systems include ensuring a sufficient supply of primary
care physicians and physicians willing to work in rural or peripheral areas. Especially challenging is enticing
young physicians to practice primary care in rural/peripheral areas. Identifying medical students interested in
primary care and in residencies in Israel’s periphery should aid the healthcare leadership. It may be
particularly important to do so during the clinical years, as this is the stage at which many future physicians
begin to crystallize their specialty and location preferences. Methods: Questionnaires, distributed to 6 consecutive 5th-year classes of the Hebrew University – Hadassah
School of Medicine, from 2010 to 2016, elicited information on criteria for choosing a career specialty, criteria
for choosing a residency program and whether one-time monetary grants authorized in the 2011 physicians’
union contract would attract students to residencies in the periphery. Results: Completed questionnaires were returned by 511 of 740 (69%) students. Ninety-eight (19%) were
interested in a primary care residency, 184 (36%) were unsure and 229 (45%) were not interested. Students
interested in primary care were significantly less interested in specialties that perform procedures/surgeries
and in joining a medical school faculty, while being more inclined towards specialties dealing with social
problems, controllable lifestyles and working limited hours. The percentage of students interested in primary
care was stable during the study period. Forty-eight of the students indicated interest in residencies in the country’s periphery, and 42% of them were
also interested in primary care residencies. Overall, only 3.7% of students were interested in both a primary
care residency and a residency in the periphery. Thirty percent of the students indicated that the monetary incentives tempted them to consider a residency
in the periphery. Fifty-three percent of these students reported that they did not yet know the geographic
area where they wished to do their residency, as compared to only 22% among those not interested in
incentives. (Continued on next page) * Correspondence: charles@hadassah.org.il Correspondence: charles@hadassah.org.il
1Department of Anesthesiology and Critical Care Medicine,
Hadassah-Hebrew University Medical Center, Hebrew University – Hadassah
School of Medicine, Kiryat Hadassah, POB 12000, 91120 Jerusalem, Israel
Full list of author information is available at the end of the article © 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. Correspondence: charles@hadassah.org.il
1Department of Anesthesiology and Critical Care Medicine,
Hadassah-Hebrew University Medical Center, Hebrew University – Hadassah
School of Medicine, Kiryat Hadassah, POB 12000, 91120 Jerusalem, Israel
Full list of author information is available at the end of the article Weissman et al. Israel Journal of Health Policy Research (2018) 7:28
https://doi.org/10.1186/s13584-018-0218-z Weissman et al. Israel Journal of Health Policy Research (2018) 7:28
https://doi.org/10.1186/s13584-018-0218-z Open Access Challenges to the Israeli healthcare system:
attracting medical students to primary care
and to the periphery es Weissman1*, Rachel Yaffa Zisk-Rony2, Alexander Avidan1, Uriel Elchalal3 and Howard Tandete Background specialists and pediatricians, especially in the county’s
peripheral
areas. This
shortage
is
predicted
to
increase as the population grows, ages, and life spans
lengthen. Furthermore, the primary care physician
population is aging as many physicians who emigrated
from the former Soviet Union in the 1990’s are reach-
ing retirement age [8]. Therefore, it is necessary to at-
tract
more
medical
students
to
primary
care
residencies. To increase recruitment it is important
that the medical education and healthcare system
leadership better understand the characteristics of stu-
dents interested in pursuing primary care and how
they differ from those without such interests. The
leadership needs similar information on students in-
terested
in
residency
positions
in
the
county’s
periphery. The greatest challenges facing healthcare systems in-
clude providing sufficient numbers of primary care
and rural physicians [1]. Especially challenging is en-
ticing young physicians to establish primary care
practices in rural areas [1, 2]. Among the counties
where such challenges exist are the United States,
Australia and Canada [1, 3, 4]. Israel suffers from a
similar problem with shortages of family medicine
specialists in peripheral areas of the country. The lat-
ter include the northern and southern regions of the
country
which
encompass
the
majority
of
the
county’s rural districts. These areas chronically suffer
from physician shortages, greater infant mortality and
lower life expectancy than the rest of the country
[5]. In an attempt to remedy this maldistribution, the
physicians’ union contract of 2011 included both pay
increases for practicing in the periphery and one-
time monetary incentives for moving and committing
to work in the periphery [6, 7]. This programs also
provided incentives for medical students interested in
residencies in specialties suffering workforce short-
ages. The original program excluded family medicine
residents from both the incentives for work in the
periphery and the incentives to work in distressed
specialties. However, in 2015 the Israel Ministry of Health
began to provide financial incentives to family medicine
residents willing to train in peripheral areas. The present study compared the characteristics of
Israeli 5th-year (out of 6 years) medical students
interested in primary care residencies and residencies
in peripheral areas with those without such interests. The dataset used was collected over a 6-year period
from a single Israeli medical school and includes over
500 students. © 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. Page 2 of 17 Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 (Continued from previous page) Conclusions: This study provides the healthcare leadership with information on the characteristics of the
students at a centrally-located medical school who tend to be more interested in primary care and in
working in the periphery. Specifically, the study found that students interested in primary care desire a
positive life/work balance, something that Israeli non-hospital primary care practice provides. Students
considering residencies in the periphery were similarly inclined. Moreover, about a third of students had
positive thoughts about monetary incentives for residencies in peripheral hospitals. These students should be
identified early during their clinical experience so that attempts to recruit them to the periphery can
commence before their specialty and location preferences have fully crystallized. Parallel studies should be
performed at additional Israeli medical schools. Keywords: Medical students, Medical education, Residency, Medical specialty selection, Career choice Data analysis Data were entered into Microsoft Excel (Redmond, WA)
spreadsheets and analyses were performed with Systat
12 (San Jose CA). Background The two hypotheses tested were that
the student interest in primary care would be greater
among women medical students and that interest in
residencies
in
peripheral
areas
would
be
greater
among students who attended high school in the
northern and southern regions. We also explored
whether the one-time monetary grants approved in
the 2011 physicians’ union contract would encourage
students to consider a residency in a peripheral hospital. The ultimate goal was to provide the medical education
and healthcare leaderships with the attributes that typify
students interested in primary care and/or rural practice. Fifth-year students were studied since our previous study
revealed that most had already begun the process of
deciding on a specialty [9]. In order for the healthcare
leadership to influence specialty decisions, it is important
to be cognizant of the thought patterns of the students
early in their decision process. The
Israeli
healthcare
system
is
dependent
on
primary
care
physicians
to provide
comprehensive
out-patient care and to serve along with community spe-
cialists and internal Health Maintenance Organization
regulations and pre-authorization
systems as gate-
keepers
for
secondary
and
tertiary
care. These
primary care physicians, family medicine specialists,
pediatricians, internists and general practitioners, are
based
in
health
maintenance
organization
clinics. Despite family medicine and pediatrics being popular
specialties among Israeli medical students, there is
still
a
nationwide
shortage
of
family
medicine Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 Page 3 of 17 Page 3 of 17 Incentives The data set was divided into three groups as per the
responses to the multiple- choice query “As the result
of the union contract of 2011, residents in peripheral
hospitals receive a one-time monetary incentive and
higher salaries: (1) These incentives attract me to a
residency in the periphery (2) I already plan to do a
residency in the periphery (3) The incentives do not
attract me to a residency in the periphery”. The
differences between the characteristics of the three
groups were determined. After two small (15 students) preliminary studies
designed to identify problems and test the question-
naire’s
user-friendliness,
the
questionnaires
were
distributed to the 5th year classes of the Hebrew
University
–
Hadassah
School
of
Medicine
in
Jerusalem during the 2010–2011, 2011–2012, 2012–
2013, 2013–2014, 2014–2015 and 2015–2016 school-
years. A parallel article examining medical student
subgroups also utilized this dataset [10]. Based on prior research demonstrating significant
gender differences associated with specialty selection,
an a priori decision was made to separately analyze
and compare the male and female data [11]. Responses to multiple choice questions are pre-
sented as frequency distributions. When the Likert
Scale results were considered continuous variables,
statistical analyses were performed using all 5 points. When presented as categorical variables the Likert
Scale results were compressed into three categories,
(the two points representing negative tendencies and
the two points representing positive tendencies were
each combined). The percentages of total responses
for each of the three categories (positive tendency,
middle
point
and
negative
tendency)
were
then
computed. Rural (periphery) workforce p
p
y
Initial data analysis showed that a significant number
of students interested in primary care were inter-
ested in a residency in the county’s periphery. There-
fore,
a
post-hoc
examination
was
made
of
the
characteristics of students interested in a residency
in the country’s periphery. The dataset was divided
into two groups based on the answer to the 5-point
Likert scale question: “Are you interested in a resi-
dency in the country’s periphery?” Group A included
the two points representing positive tendencies while
Group B included the two points representing nega-
tive tendencies. Methods included
the
two
points
representing
positive
tendencies; Group 2 included the neutral point; and
Group
3
the
two
points
representing
negative
tendencies. This permitted us to compare students
interested and not interested in a primary care resi-
dency, while also examining those who were unsure. The results from each of the 6 school-years were
compared to determine whether there were differ-
ences between years. This study included students from 6 consecutive
5th-year classes of the Hebrew University – Hadassah
School of Medicine in Jerusalem (2010–2016). It
utilized a questionnaire to examine various aspects
of the medical specialty selection process. The ques-
tionnaire was based on the results of factor analysis
from a questionnaire used previously [9]. This per-
mitted us to reduce repeat Likert scale questions
thus providing space for new ones that investigated
additional issues. Among the new topics examined
were the influence of family and colleagues on specialty
and residency program decisions as well as the interests of
the students in academic pursuits. The questionnaire
included multiple choice questions, free-text queries
and 5-point Likert scales. In addition to demographic
information,
the
questionnaire
elicited
information
about: (1) Whether the student had already consid-
ered a specialty for their residency, which specialty or
specialties
they
were
considering
(free-text),
when
they had first considered a specialty and whether and
when (prior to beginning medical school or when
during the first 5 years of medical school) they had
changed their mind; (2) The criteria for choosing a
career specialty {20 items, 10 new, 5-point Likert
scale}; (3) The criteria for choosing a residency pro-
gram {20 items, 9 new, 5-point Likert scale}; and (4)
The importance of interest in a specific specialty
when choosing a residency {3 new items, multiple
choice}. (5) Whether the one-time monetary grants
authorized in the 2011 physicians’ union contract
would attract them to a residency in a peripheral
hospital (1 new item, final 4 classes). Rural (periphery) workforce y
Differences
between
students
expressing
and
not
interested in a residency in the country’s periphery are
found in Table 4 (Appendix B). Results of multivariate
and logistic regression analyses are in Table 3. Criteria
for
specialty
and
residency
program
selection were subjected to factor analysis (principal
components analysis) using varimax rotation with set
eigenvalues of ≥1.0. The data were also analyzed with
hierarchal cluster analysis. Interactions between the replies to the questions
“Are you interested in a residency in the country’s
periphery?” and “Are you interested in a primary care
residency?” revealed that 4% of all the students were
interested in both a primary care residency and a
residency in the periphery (Appendix C). The Institutional
Review
Board
of
the
Hadassah
Medical Organization approved this study. Completion
of the questionnaire by the student was considered tacit
consent. Discussion The present study identified several medical student
characteristics associated with interest in a primary care
residency and those interested in a residency in the per-
iphery among 5th year students at the Hadassah-Hebrew
University Medical School. Primary care
h Ninety-eight
(19%)
students
were
interested
in
pursuing a primary care residency, 184 (36%) were
unsure and 229 (45%) were not interested. Demo-
graphic information is found in Table 1. Interest in
primary care among 5th-year students was stable
over the 6-year study period ranging from 17%–21%
annually. Compared to students not interested in primary
care, those interested in primary care were signifi-
cantly less interested in a specialty with procedures/
surgeries and becoming “members of a medical school
faculty”, while being more inclined towards a specialty
dealing with social problems (Tables 2 and 3). When
choosing a residency program, students attracted to
primary
care
were
more
interested
than
their
colleagues in a residency in the country’s periphery. Alternately, 42% of the 48 students who indicated
interest
in
a
residency
program
in
the
country’s
periphery
were
also
interested
in
a
primary
care
residency (Table 2). When asked about specialties
they were considering, students interested in primary
care expressed significantly more interest in family
medicine and pediatrics than those not interested in Incentives Completed questionnaires were returned by 511 of 740
(69%) 5th-year medical students. Responses to the query about monetary incentives
for a residency in peripheral hospitals are in Tables 5
(Appendix
D). Thirty
percent
of
the
students
reported that the incentives interested them, while
another 6% had already decided to do a residency in
the
periphery. The
relationships
between
the
responses to this question and those to the query
“are you interested in a residency in the country’s
periphery?” revealed that 82% of the students who
replied they were not attracted by the incentives
indicated that they were not interested in a residency
in the periphery while those that reported that the
incentives interested them showed less aversion (20%
negative tendency and 54% positive tendency) to a
residency in the periphery. Primary care The dataset was divided into three groups based on
the answer to the 5-point Likert scale question: “Are
you interested in a primary care residency?” Group 1 Page 4 of 17 Page 4 of 17 Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 primary care. Among the latter, none were consider-
ing family medicine (Table 1). For continuous data, differences between the groups
were analyzed using analysis of variance with Tukey
post-hoc tests. Categorical data were analyzed using
χ2 or Fisher exact tests, as appropriate. A p value < 0. 05 was assumed to represent statistical significance. Univariate
linear
regression
analysis
was
used
to
examine the association between the answers to two
queries. Backward multivariate and logistic regression
analyses were performed with the dependent variable
being either interest in a residency in primary care or
a peripheral hospital. The independent variables were
the
demographic
parameters
and
specialty
and
resident selection criteria. As can be seen in Tables 1 and 2 (Appendix A),
there was similar interest in a primary care residency
between female (17%) and male (21%) students. Com-
parisons between men and women students interested
in primary care showed that women rated lifestyle is-
sues, such as family time, more highly than men and
were more interested in pediatrics (Tables 1 and 2,
Appendix A). Primary care There were many differences between 5th-year Israeli
medical students interested and not interested in a Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 Page 5 of 17 Table 1 Primary care - demographic and other information
Primary
care
Undecided
No primary
care
Primary care vs
no primary care
Primary care
Females
vs males
Females
Males
N
98
184
229
41
57
Female
42%
51%
49%
Male
58%
50%
51%
NS
Age (years)
18–20
0%
0%
0.4%
0%
0%
21–23
25%
14%
17%
27%
23%
24–26
19%
31%
29%
34%
9%
27–29
37%
42%
39%
27%
44%
30–32
14%
10%
12%
10%
18%
+ 32
5%
4%
3%
NS
2%
7%
p < 0.01
Marital status
Single
70%
63%
73%
66%
72%
Married
30%
36%
26. %
34%
26%
Divorced
1%
1%
0.4%
NS
0%
2%
NS
Thought of a specialty when started
Yes
85%
79%
82%
85%
84%
No
15%
21%
18%
NS
15%
16%
NS
When did you start thinking of a specialty? Pre - med school
25%
29%
26%
27%
23%
Year 1
4%
4%
4%
6%
2%
Year 2
4%
4%
2%
3%
4%
Year 3
4%
3%
5%
0%
6%
Year 4
56%
42%
48%
62%
51%
Year 5
9%
19%
16%
NS
3%
13%
NS
Have you changed your mind? Yes
61%
59%
63%
59%
62%
No
40%
41%
38%
NS
41%
38%
NS
When did you change your mind? Future residency location Primary care Year 1
0%
0%
2%
0%
0%
Year 2
0%
3%
1%
0%
0%
Year 3
2%
1%
2%
0%
4%
Year 4
41%
44%
42%
35%
46%
Year 5
57%
52%
54%
NS
65%
50%
NS
Specialties under consideration
Family medicine
16%
2%
0%
14%
17%
Pediatrics
41%
42%
24%
54%
30%
Internal med
26%
30%
32%
26%
26%
Ob/Gyn
19%
20%
22%
34%
7%
Emergency med
4%
0%
2%
0%
7%
Surgical*
20%
31%
31%
9%
30%
Other
22%
44%
39%
p < 0.01
20%
30%
p < 0.01
High school location
Israel
97%
97%
96%
95%
98%
Other
3%
3%
4%
NS
5%
2%
NS
High school location in israel
Jerusalem
32%
25%
23%
26%
36%
Central
29%
37%
44%
31%
29%
North
26%
28%
23%
33%
21%
South
13%
9%
10%
10%
14%
Other
0%
1%
1%
NS
0%
0%
NS
Future residency location
Israel
98%
98%
99%
97%
98%
Other
2%
2%
1%
NS
3%
2%
NS Table 1 Primary care - demographic and other information Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 Page 6 of 17 Table 1 Primary care - demographic and other information (Continued) Table 1 Primary care - demographic and other information (Continued)
Primary
care
Undecided
No primary
care
Primary care vs
no primary care
Primary care
Females
vs males
Females
Males
Perferred residency location in Israel
Jerusalem
28%
18%
23%
23%
31%
Central
22%
32%
34%
13%
29%
North
17%
11%
9%
23%
12%
South
1%
1%
2%
3%
0%
Don’t know
33%
39%
32%
p < 0.05
39%
29%
p < 0.01
*Surgical specialties male students. Students inclined toward primary care
were less interested in academic pursuits, such as
being academic faculty members. Reduced interest in
academic
activities
was
also
observed
among
Japanese
medical
students
with
preferences
for primary
care
residency. Students
inclined
toward
primary care were more interested in lifestyle: spend-
ing time with their families, working limited hours
and working only during the daytime. Percent of "agree" and "agree much" responses on 5-point Likert Scale Numbers in parenthesis are the results of factor analysis
aclusters per cluster analysis nalysis
he higher value in a statistically significant pair p
y
Bold result indicates the higher value in a statistically significant pair d "agree much" responses on 5-point Likert Scale Numbers in parenthesis are the results of factor analysis
nalysis Primary care This import-
ance of lifestyle was more pronounced in female than Table 2 Primary care - selection criteria
Primary
care
Undecided
No primary
care
Primary care vs
no primary
Primary care
Females vs males
Females
Males
N
98
184
229
41
57
98
Criteria for choosing a specialty
Time with family (1)
76%
73%
65%
p < 0.03
85%
68%
p < 0.05
Specialty with team work
57%
54%
45%
p < 0.04
56%
58%
NS
Influence of spouse
43%
43%
31%
p < 0.007
49%
39%
p < 0.05
Specialty that deals with social issues (3)
42%
35%
22%
p < 0.001
51%
35%
p < 0.009
Daytime work only (1)
26%
29%
16%
p < 0.03
34%
19%
p < 0.03
Work only in the community
9%
3%
4%
p < 0.001
12%
7%
NS
Procedures/surgery
43%
41%
57%
p < 0.01
34%
49%
p < 0.02
High salary
36%
48%
51%
p < 0.002
34%
38%
NS
Opportunity for research (2)
34%
37%
44%
p < 0.05
27%
39%
NS
Academic faculty member
19%
26%
30%
p < 0.05
24%
16%
NS
Criteria for choosing a residency
Much supervision by senior physicians
53%
45%
37%
p < 0.001
68%
42%
p < 0.04
Limited work hours
29%
30%
17%
p < 0.001
33%
27%
NS
Short residency (<4.5. years)
28%
15%
12%
p < 0.001
29%
26%
NS
Much clinic time (2)
23%
16%
5%
p < 0.001
20%
25%
NS
Hospital in the periphery (3)
19%
7%
6%
p < 0.001
20%
19%
NS
Leading department (1)a
67%
77%
79%
p < 0.03
71%
65%
NS
Large hospitial
52%
55%
62%
p < 0.04
59%
47%
NS
Family living location
78%
75%
64%
NS
88%
70%
p < 0.05
Making clinical decisions on your own
66%
50%
55%
NS
56%
74%
p < 0.05
Pre-determined work hours (2)
47%
48%
35%
NS
59%
39%
p < 0.05
Influence of family
42%
37%
31%
NS
54%
33%
p < 0.01
Many on-call shifts
10%
11%
12%
NS
5%
14%
p < 0.03
Percent of "agree" and "agree much" responses on 5-point Likert Scale Numbers in parenthesis are the results of factor analysis
aclusters per cluster analysis Table 2 Primary care - selection criteria Weissman et al. Primary care Israel Journal of Health Policy Research (2018) 7:28 Page 8 of 17 Table 3 Regression analysis (Continued)
Demographics/criteria
Multivariable Backward
regression
coefficient
Backward logistic regression
p
Odds ratio
95% confidence limits
P
Monetary incentives for residency in the periphery
r - 0.58
Opportunity for research
−0.126
0.047
0.580
0.360–0.933
0.025
Specialty advancing rapidly
−0.143
0.022
0.620
0.393–0.90
0.041
Specific location
−0.204
0.001
0.428
0.275–0.667
0.001
Primary care
0.115
0.023
Specialty that deals with social issues
0.126
0.008
1.530
1.044–2.242
0.029
Peripheral hospital
0.258
0.001
2.835
1.782–4.511
0.001 entering
general
internal
medicine
and
pediatric
practices [17]. The major reasons cited for the dearth
of
students
entering
primary
care
in
the
United
States are relatively low incomes in the face of high
student debt burdens, many administrative tasks and
time pressures [16]. Many medical schools have in-
stituted programs to attract more students to pri-
mary care, with a multi-year exposure to primary
care being more successful than adding a single pri-
mary care course to a conventional curriculum [18]. Other countries face similar problems. In Vietnam
less than a third of commune (collective farming
communities) health stations are staffed by a phys-
ician even though the number of medical school
graduates almost tripled between 2004 and 2011
[19]. The reasons include poor working conditions,
low income and lack of opportunities for career de-
velopment [19]. In a survey of 9499 South Korean
medical students only 2.2% expressed interest in
family medicine [20]. family medicine [12]. Lack of interest in academic
endeavors is problematic since it reduces the number
of family medicine faculty members able to serve as
medical student mentors. This lack of mentors might
decrease
the
ability
to
attract
students
to
the
specialty. p
y
It is important to note that the query on the
questionnaire was about the broader area of primary
care
and
not
specifically
about
family
medicine. Unlike a previous study where we found a female-
predominance
among
6th
year
Israeli
medical
students interested in family medicine, in the present
study
we
did
not
find
such
predominance
[13]. Furthermore,
the
proportion
of
women
medical
students interested and not interested in primary care
was comparable. Similarly, upon multiple regression
analysis, interest in primary care was not associated
with
being
female. We
thus
failed to
prove
our
hypothesis that interest in primary care would be
greater among women than men medical students. Primary care Israel Journal of Health Policy Research _#####################_ Page 7 of 17 Table 3 Regression analysis
Demographics/criteria
Multivariable Backward
regression
coefficient
Backward logistic regression
p
Odds ratio
95% confidence limits
P
Interest in a primary care residency
r = 0.43
Male gender
0.264
0.015
2.251
1.195–4.241
0.012
Criteria for choosing a specialty
Opportunity for research
−0.096
0.033
0.755
0.578–0.987
0.04
High salary
−0.207
0.002
0.580
0.376–0.893
0.013
Influency of spouse
0.122
0.018
Specialty that deals with social issues
0.146
0.004
1.442
1.079–1.927
0.013
Work only in the community
0.278
0.001
Wide range of medical problems
1.512
1.004–2.277
0.048
Criteria for choosing a residency program
r = 0.45
Leading department
−0.125
0.053
0.581
0.395–0.854
0.006
Influence of family
0.094
0.026
1.414
1.083–1.847
0.011
Short residency (< 4.5 years)
0.159
0.001
1.524
1.122–2.069
0.007
Peripheral hospital
0.188
0.001
Much supervision by senior physicians
0.202
0.001
1.836
1.340–2.515
0.001
Much clinic time
0.207
0.001
1.495
1.110–2.013
0.008
Interest in a residency in a peripheral hospital
r = 0.43
Older age
0.157
0.004
1.732
1.144–2.621
0.009
Have considered a specialty
−0.234
0.048
High school location (periphery)
0.138
0.002
1.546
1.089–2.193
0.015
Criteria for choosing a specialty
Wide range of medical problems
0.144
0.008
Time with family
0.145
0.009
1.686
1.001–2.839
0.049
Work only in the community
0.178
0.002
1.701
1.147–2.524
0.008
Influence of spouse
1.488
1.019–2.173
0.039
Private practice
−0.109
0.012
Specialty that deals with social issues
0.123
0.003
Narrow range of medical problems
0.298
0.001
2.156
1.377–3.376
0.001
Criteria for choosing a residency program
r = 0.52
Specific location in Israel
−0.106
0.031
Leading department
−0.280
0.001
0.480
0.091–0.791
0.004
Limited work hours
0.108
0.031
Family living location
0.115
0.046
Many on-call shifts
0.121
0.011
Teaching students
0.144
0.001
1.563
1.061–2.304
0.024
Physical challenge
0.157
0.001
Primary care
0.168
0.002
Much clinic time
0.198
0.003
1.893
1.288–2.783
0.001
Much supervision by senior physicians
0.045
1.010–2.455
0.045
Influence of family
1.430
1.030–1.986
0.022 Weissman et al. Primary care Therefore, in many countries with large rur
Table 4 Residency in a peripheral hospital
Periphery No
periphery
Periphery
vs no
Periphery
N
48
382
Gender
Female
45%
48%
Male
55%
52%
NS
Age (years)
18–20
0%
0.3%
21–23
17%
18%
24–26
19%
30%
27–29
42%
39%
30–32
17%
10%
+ 32
6%
3%
NS
Marital status
Single
63%
71%
Married
38%
29%
Divorced
0%
1%
NS
High school location
Israel
100%
97%
Other
0%
3%
NS
High school location in
Israel
Jerusalem
17%
27%
Central
31%
42%
North
38%
22%
South
15%
8%
Other
0%
1%
p < 0.05
Residency location
Israel
100%
98%
Other
0%
2%
NS
Future residency location
in Israel
Jerusalem
13%
26%
Central
16%
36%
North
40%
7%
South
13%
1%
Don’t know
18%
31%
p < 0.01
Specialties under
consideration
Family
medicne
0%
2%
Pediatrics
46%
30%
Internal
medicine
24%
32%
Ob/Gyn
15%
21%
Emergency
medicine
5%
1%
Surgical
specialties
32%
30%
Other
specialties
32%
35%
p < 0.05
Criteria for choosing a specialty
Time with family
85%
66%
p < 0.001
Controllable lifestyle
75%
65%
p < 0.04
Influency of spouse
56%
37%
p < 0.01
Specialty that deals
with social issues
46%
27%
p < 0.004
Table 4 Residency in a peripheral hospital (Continued)
Periphery No
periphery
Periphe
vs no
Periphe
Work only in the
community
19%
3%
p < 0.00
Narrow range of
medical problems
10%
2%
p < 0.00
Advancing rapidly
48%
62%
p < 0.05
Opportunity for
research
29%
41%
p < 0.03
Criteria for choosing a residency program
Family living location
81%
69%
p < 0.04
Teaching students
57%
40%
p < 0.05
Pre-determined work
hours
56%
41%
p < 0.05
Influence of family
52%
33%
p < 0.0
Much supervision
bysenior physicians
50%
42%
p < 0.03
Primary care
42%
15%
p < 0.00
Limited work hours
42%
21%
p < 0.03
Much clinic time
33%
8%
p < 0.00
Leading department
58%
79%
p < 0.00
Large hosptial
47%
60%
p < 0.00
Percent of “agree” and “agree much” responses on 5-point Likert Scale Table 4 Residency in a peripheral hospital (Continued)
Periphery No
periphery
Periphery
vs no
Periphery
Work only in the
community
19%
3%
p < 0.001
Narrow range of
medical problems
10%
2%
p < 0.001
Advancing rapidly
48%
62%
p < 0.05
Opportunity for
research
29%
41%
p < 0.03
Criteria for choosing a residency program
Family living location
81%
69%
p < 0.04
Teaching students
57%
40%
p < 0.05
Pre-determined work
hours
56%
41%
p < 0.05
Influence of family
52%
33%
p < 0.02
Much supervision
bysenior physicians
50%
42%
p < 0.03
Primary care
42%
15%
p < 0.0004
Limited work hours
42%
21%
p < 0.03
Much clinic time
33%
8%
p < 0.001
Leading department
58%
79%
p < 0.0001
Large hosptial
47%
60%
p < 0.003
Percent of “agree” and “agree much” responses on 5-point Likert Scale Table 4 Residency in a peripheral hospital (Continued) greater interest in short (in years) residency programs
with limited hours and with much time spent in
clinics. Primary care This variance with our previous studies is attributable
to
primary
care
incorporating
general
internal
medicine, general pediatrics and some aspects of ob-
stetrics/gynecology, in addition to family medicine. We previously found that In Israel, internal medicine
and
obstetrics/gynecology
residencies
attract
many
male students [13]. When asked which specialties
they were considering, students interested in primary
care listed pediatrics and internal medicine more fre-
quently than family medicine. Shortages of primary care physicians are generally
attributed
to
low
salaries,
lack
of
prestige
and
glamor; long hours with frequent on-call responsibil-
ities; and lack of a controllable lifestyle [21, 22]. The
situation in Israel differs from other countries in that
primary care physicians mainly work in health main-
tenance clinics, receive salaries comparable to other
physicians, have few on-call obligations and have set
hours [8]. This was reflected in our previous study
where Israeli 6th-year students rated family medicine
and pediatrics as specialties with controllable life-
styles and positive relationships between controllable
lifestyle and remuneration [13]. This was also found
in the present study, where compared to 5th-year
students not interested in primary care, those inter-
ested in primary care wanted a specialty with time
for family involving only daytime work and practice
in the community (i.e. outside the hospital). This
interest profile was similarly demonstrated by their In many countries, attracting medical students to
primary care careers is a daunting task [14]. The
reasons for this difficulty differ between countries
[15]. In the United States, the proportion of medical
students selecting primary care specialties dropped
from 73% in 1996 to 44% in 2008, although subse-
quently there has been some stabilization [16]. More-
over, more internal medicine and pediatric residents
are choosing to subspecialize, reducing the numbers Weissman et al. Primary care Israel Journal of Health Policy Research (2018) 7:28 Page 9 of 17 Table 4 Residency in a peripheral hospital
Periphery No
periphery
Periphery
vs no
Periphery
N
48
382
Gender
Female
45%
48%
Male
55%
52%
NS
Age (years)
18–20
0%
0.3%
21–23
17%
18%
24–26
19%
30%
27–29
42%
39%
30–32
17%
10%
+ 32
6%
3%
NS
Marital status
Single
63%
71%
Married
38%
29%
Divorced
0%
1%
NS
High school location
Israel
100%
97%
Other
0%
3%
NS
High school location in
Israel
Jerusalem
17%
27%
Central
31%
42%
North
38%
22%
South
15%
8%
Other
0%
1%
p < 0.05
Residency location
Israel
100%
98%
Other
0%
2%
NS
Future residency location
in Israel
Jerusalem
13%
26%
Central
16%
36%
North
40%
7%
South
13%
1%
Don’t know
18%
31%
p < 0.01
Specialties under
consideration
Family
medicne
0%
2%
Pediatrics
46%
30%
Internal
medicine
24%
32%
Ob/Gyn
15%
21%
Emergency
medicine
5%
1%
Surgical
specialties
32%
30%
Other
specialties
32%
35%
p < 0.05
Criteria for choosing a specialty
Time with family
85%
66%
p < 0.001
Controllable lifestyle
75%
65%
p < 0.04
Influency of spouse
56%
37%
p < 0.01
Specialty that deals
with social issues
46%
27%
p < 0.004 greater interest in short (in years) residency program
with limited hours and with much time spent
clinics. This grouping of interests indicates a desi
for positive life/work balance, something that Isra
non-hospital primary care practice provides. A rece
study of Israeli family medicine residents report
similar findings. Specifically, more than 85% of res
dents reported that factors that positively influen
their choice included the ability to combine wor
family, and free time; direct, meaningful contact wi
patients; the diversity of patients and medical cond
tions; and attractive working conditions [23]. Th
interest profile is similar to those reported from oth
countries among students interested in primary ca
and family medicine [12, 24]. However, primary ca
in isolated Israeli rural village has been reported
lead to unclear boundaries between private life an
physician roles leading to problems with life/wo
balance [25]. This may be among the reasons for th
shortage of primary care practitioners in the country
periphery. Rural (periphery) workforce
Worldwide, rural areas often suffer physician shor
ages. Primary care This grouping of interests indicates a desire
for positive life/work balance, something that Israeli
non-hospital primary care practice provides. A recent
study of Israeli family medicine residents reported
similar findings. Specifically, more than 85% of resi-
dents reported that factors that positively influence
their choice included the ability to combine work,
family, and free time; direct, meaningful contact with
patients; the diversity of patients and medical condi-
tions; and attractive working conditions [23]. This
interest profile is similar to those reported from other
countries among students interested in primary care
and family medicine [12, 24]. However, primary care
in isolated Israeli rural village has been reported to
lead to unclear boundaries between private life and
physician roles leading to problems with life/work
balance [25]. This may be among the reasons for the
shortage of primary care practitioners in the country’s
periphery. Rural (periphery) workforce Rural (periphery) workforce
Worldwide, rural areas often suffer physician short-
ages. Therefore, in many countries with large rural Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 Page 10 of 17 Table 5 Incentives - residency in periphery
1. Incentive
interests me
2. Plan peripheral
residency
3. Rural (periphery) workforce Incentives don’t
interest me
1 vs 3
1 vs 2
2 vs 3
Leading department (1)a
69%
45%
85%
p < 0.0002
p < 0.004
p < 0.0009
Specific location
51%
68%
70%
p < 0.0001
NS
NS
Large hospitial
50%
26%
68%
p < 0.005
p < 0.04
p < 0.0006
Opportunity for research
17%
5%
29%
p < 0.02
p < 0.01
p < 0.0005
Much supervision by senior physicians
44%
20%
46%
NS
NS
p < 0.04
Percent of “agree” and “agree much” responses on 5-point Likert Scale
Numbers in parenthesis are the results of factor analysis
aClusters per cluster analysis
Bold result indicares the higher value in a statistically significant pair Table 5 Incentives - residency in periphery (Continued) areas,
such
as
the
United
States,
Canada
and
Australia, emphasis has been placed on encouraging
more medical students to become rural primary care
physicians [26, 27]. To attract students to rural areas,
medical schools have programs that expose students
to rural practice and have increased the recruitment
of students from rural areas [28]. The current study
showed that of the more than 500 5th-year Israeli
students studied, 8.9% were considering residency in
the country’s periphery. This percentage is greater
than that reported in our previous study of 5th year
students (4.6%) and might be attributable to the re-
cent introduction of monetary incentives (one-time
grants and salary increases) for physicians choosing to
train and practice in the periphery. Shortages of rural
physicians frequently includes a lack of primary care
physicians; a situation also present in Israel. Among
students
interested
in
primary
care,
19%
would
choose
a
residency
in
the
periphery. Alternately,
among students considering a residency in the periph-
ery, 42% were interested in primary care. This attrac-
tion to primary care among students interested in
living
in
rural
areas
was
also
observed
among
Japanese medical students [29]. However, when we
examined our overall student sample, only 3.7% of
the
5th-year
students
reported
interest
in
both
primary care and peripheral residencies. the periphery”, 63% had gone to high school in the
northern or southern areas. Therefore, we confirmed
the hypothesis that interest in residencies in periph-
eral areas is greater among students who attended
high school in peripheral regions. Similar observa-
tions were made in Kenya, United States, Japan and
Australia where students of rural origin were more
interested in rural practice [2, 11, 28, 29]. Rural (periphery) workforce Incentives don’t
interest me
1 vs 3
1 vs 2
2 vs 3
N
106
20
223
Percent
30%
6%
64%
Gender
Female
48%
47%
50%
Male
52%
53%
50%
NS
NS
NS
Age (years)
18–20
0%
0%
0%
21–23
22%
20%
19%
24–26
27%
15%
31%
27–29
36%
50%
35%
30–32
10%
10%
11%
+ 32
5%
5%
3%
NS
NS
p < 0.02
Marital status
Single
68%
60%
72%
Married
30%
40%
27%
Divorced
2%
0%
1%
NS
p< 0.05
p < 0.02
High school location in Israel
Jerusalem
27%
5%
25%
Central
36%
32%
40%
South
10%
26%
10%
Other
1%
0%
1%
NS
p < 0.01
NS
Future residency location in Israel
Jerusalem
14%
10%
29%
Central
20%
0%
41%
North
14%
55%
8%
South
0%
20%
1%
Don’t know
53%
15%
22%
p< 0.03
p < 0.01
p< 0.01
Specialties under consideration
Pediatrics
39%
31%
31%
Internal medicine
21%
6%
35%
Ob/Gyn
29%
19%
15%
Emergency medicine
0%
13%
1%
Surgical specialties
32%
31%
29%
Other specialties
31%
31%
37%
p < 0.04
p < 0.01
p < 0.02
Criteria for choosing a specialty
Time with family (1)
78%
60%
63%
p < 0.004
p < 0.04
NS
Specialty that deals with social issues (3)
35%
35%
25%
p < 0.02
NS
NS
Advancing rapidly (2)
51%
45%
69%
p < 0.0007
NS
p < 0.04
Opportunity for research (2)
29%
5%
47%
p < 0.0001
p < 0.04
p < 0.0001
Controllable lifestyle (1)
74%
50%
66%
NS
NS
NS
Independent practice
54%
40%
51%
NS
p < 0.03
p < 0.03
High salary
51%
20%
45%
NS
p < 0.006
p < 0.01
Procedures/surgery
45%
32%
51%
NS
NS
p < 0.04
Private practice
37%
10%
404%
NS
p < 0.008
p < 0.009
Criteria for choosing a residency
Controllable lifestyle
73%
45%
58%
p < 0.04
p < 0.03
NS
Primary care
27%
32%
15%
p < 0.001
NS
p < 0.002
Hospital in the periphery (3)
8%
55%
4%
p < 0.001
p < 0.0007
p < 0.001
Intellectual challenge (1)a
8%
60%
87%
p < 0.004
NS
p < 0.007 Table 5 Incentives - residency in periphery Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 Page 11 of 17 Table 5 Incentives - residency in periphery (Continued)
1. Incentive
interests me
2. Plan peripheral
residency
3. Rural (periphery) workforce These
results have potential healthcare policy implications. Firstly,
they
can
contribute
to
decisions
about
whether to admit more medical school applicants
residing in the periphery in an attempt to reduce the
shortage of physicians practicing there. Secondly, the
results
can
also
contribute
to
decisions
about
whether to include rural medicine rotations during
the clinical years to provide all students with expos-
ure to such medical practice [30]. The
characteristics
of
students
considering
a
residency
in
the
periphery
were
similar
to
those
interested in a primary care residency. Although this
may be partially attributable to the many students
interested in a primary care residency, it also likely
reflects that those interested in residencies in the per-
iphery are more interested in life-work balance and
are less interested in a residency in a large hospital
and in a leading department. Regression
analysis
showed
that
attending
high
school in Israel’s south and north was associated with
interest in residency in the same regions. Notably,
significantly more students interested in residencies
in the periphery reported that the locale of their
family was an important criterion for choosing a
residency
program. Furthermore,
among
students
who responded to the question concerning the effects
of one-time monetary incentives to do a residency in
the periphery, “I already plan to do a residency in Strengths and limitations The strength of this study is that the large number of
students studied allowed us to examine subgroups,
such as those interested in primary care. A further
strength was its multi-year design showing that the
proportion of students interested in primary care and
peripheral hospitals remained steady throughout the
study. The
major
limitation
is
that
the
study
was
performed in a single institution that is located in
the center of the country and is focused on academic
medicine. Hence the findings are not automatically
generalizable to the entire Israeli medical student
population, and parallel studies should be carried out
at additional Israeli medical schools.1 Conclusions
h
h The characteristics of students showing interest in
primary care and practice in peripheral areas, that
were delineated by this study, should aid department
heads and residency program directors in identifying
potential residents. Moreover, the present study re-
vealed that for Israeli medical students the 5th-year is
an important juncture in their choice of a medical
specialty. Eighty percent had already considered vari-
ous specialties. Although, about a quarter had begun
their considerations before beginning medical school,
the majority had begun during their 4th and 5th-
years. Furthermore, 60% of those who had begun the
thought process had already changed their minds. Therefore, the 4th and 5th years of medical school
appear to be an opportune time to market the various
specialties to medical students and might also be the
time to begin informing them about residency pro-
grams. Since the students’ decisions as to specialty
and residency program decisions have major influ-
ences on the composition and geographic distribution
of the future physician workforce, it is for the health-
care leadership to take the initiative and provide the
students with direction, counseling and information
to help them with their choices. Incentives Alternately, they were less interested in re-
search opportunities, a rapidly advancing specialty
and a residency in a leading department in a large
hospital. For medical educators and healthcare leaders
these results point to a group with distinct character-
istics who might be encouraged to join residency pro-
grams in the periphery. The challenge is to better
characterize this student group, identify them early
during
their
specialty/residency
program
decision
process and provide positive information and counsel-
ing about residency programs and lifestyle in the per-
iphery. Thirty percent of the residents working in
Israel’s periphery reported that the incentives had in-
fluenced them greatly, even though initially they had
intended to work there [23]. Previous studies have
shown that Israeli primary care practitioners in the
periphery are more satisfied and had a broader scope
of practice than urban practitioners [29, 31, 32]. While the aim is to market residency programs in
the periphery to the students, the results of this
study show that a third of the students reported that
the influence of family was an important part of
their
residency
selection
process. Therefore,
consideration should be given to including spouses
in recruitment efforts. from
such
schools
[23]. Yet,
unlike
the
foreign
graduates who only enter the Israeli healthcare sys-
tem as interns, having had exposure to the various
specialties in foreign healthcare systems during med-
ical school, the Israeli medical students are part of
the Israeli healthcare system while they are medical
students providing the Israeli healthcare leadership
the opportunity to directly expose them to Israeli
primary and rural practices early in their clinical
experience. A further limitation is that there might be selection
criteria that were not included in the questionnaire. However, both the factor and cluster analyses showed
few factors and clusters indicating that a wide-variety
of topics were queried. Incentives The last four years of the study provided an oppor-
tunity to explore the student’s thoughts about the
monetary incentives for rural residency included in
the 2011 union contract [7]. Although only 6% were
already planning to do a residency in the periphery,
30% indicated that the incentives induced them to
consider a residency there. Fifty-three percent of the
latter students reported that they did not yet know Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 Page 12 of 17 Page 12 of 17 where they wished to do their residency as opposed
to 22% among those not interested in the incentives. Moreover, there were more differences between the
two groups. Students interested in the incentives were
more interested in primary care residencies, special-
ties dealing with social issues, specialties providing
family time and residencies affording controllable life-
styles. Alternately, they were less interested in re-
search opportunities, a rapidly advancing specialty
and a residency in a leading department in a large
hospital. For medical educators and healthcare leaders
these results point to a group with distinct character-
istics who might be encouraged to join residency pro-
grams in the periphery. The challenge is to better
characterize this student group, identify them early
during
their
specialty/residency
program
decision
process and provide positive information and counsel-
ing about residency programs and lifestyle in the per-
iphery. Thirty percent of the residents working in
Israel’s periphery reported that the incentives had in-
fluenced them greatly, even though initially they had
intended to work there [23]. Previous studies have
shown that Israeli primary care practitioners in the
periphery are more satisfied and had a broader scope
of practice than urban practitioners [29, 31, 32]. While the aim is to market residency programs in
the periphery to the students, the results of this
study show that a third of the students reported that
the influence of family was an important part of
their
residency
selection
process. Therefore,
consideration should be given to including spouses
in recruitment efforts. where they wished to do their residency as opposed
to 22% among those not interested in the incentives. Moreover, there were more differences between the
two groups. Students interested in the incentives were
more interested in primary care residencies, special-
ties dealing with social issues, specialties providing
family time and residencies affording controllable life-
styles. Endnote 1Our hypothesis is that the findings for the other
medical schools will be similar to what we found for
Hebrew University-Hadassah. This is because a) Des-
pite its location in the center of the country, the
Hebrew University- Hadassah Medical School draws
students from across the country; b) Earlier studies of
family medicine specialty preferences found similar-
ities between this medical school and the total for all
Israeli medical schools [23]. Another limitation is that the study only included
Israeli medical students when half the medical in-
ternship workforce is comprised of Israeli who grad-
uated from foreign medical schools and immigrants
and 58% of the family medicine residents graduated Weissman et al. Endnote Israel Journal of Health Policy Research (2018) 7:28 Page 13 of 17 Table 6 Primary care - selection criteria
Primary
care
Undecided
No primary
care
Primary care vs
no primary
Primary care
Females
vs males
Females
Males
N
98
184
229
41
Criteria for choosing a specialty
Time with family (1)
75.51%
72.68%
65.07%
p < 0.03
85.37%
68.42%
p < 0.05
Specialty with team work
57.14%
53.80%
44.98%
p < 0.04
56.10%
57.89%
NS
Influence of spouse
42.55%
42.62%
31.14%
p < 0.007
48.65%
38.60%
p < 0.05
Specialty that deals with social issues (3)
41.84%
34.62%
21.93%
p < 0.001
51.22%
35.09%
p < 0.009
Daytime work only (1)
25.51%
28.73%
16.23%
p < 0.03
34.15%
19.30%
p < 0.03
Work only in the community
9.18%
2.73%
3.51%
p < 0.001
12.20%
7.02%
NS
Procedures/surgery
42.86%
40.98%
56.58%
p < 0.01
34.15%
49.12%
p < 0.02
High salary
36.08%
48.09%
51.09%
p < 0.002
34.15%
37.50%
NS
Opportunity for research (2)
33.67%
37.16%
43.67%
p < 0.05
26.83%
38.60%
NS
Academic faculty member
19.39%
25.68%
30.26%
p < 0.05
24.39%
15.79%
NS
Bedside specialty
93.88%
92.93%
92.07%
NS
92.68%
94.74%
NS
Wide range of medical problems
79.38%
71.04%
68.12%
NS
75.61%
82.14%
NS
Controllable lifestyle (1)
69.39%
73.91%
64.19%
NS
78.05%
63.16%
NS
Advancing rapidly (2)
56.12%
61.41%
58.77%
NS
51.22%
59.65%
NS
Independent practice
51.02%
60.11%
52.40%
NS
56.10%
47.37%
NS
Private practice
39.80%
36.96%
42.48%
NS
41.46%
38.60%
NS
Influence of family
12.37%
9.78%
8.81%
NS
15.00%
10.53%
NS
Specialty that my coleagues choosea
2.06%
1.64%
0.00%
NS
0.00%
3.57%
NS
Influence of classmatesa
2.06%
1.63%
0.44%
NS
0.00%
3.57%
NS
Narrow range of medical problemsa
2.04%
3.26%
3.49%
NS
0.00%
3.51%
NS
Criteria for choosing a residency
Much supervision by senior physicians
53.06%
45.11%
36.68%
p < 0.001
68.29%
42.11%
p < 0.04
Limited work hours
29.17%
29.89%
16.67%
p < 0.001
32.50%
26.79%
NS
Short residency (< 4.5. Endnote Years)
27.55%
15.30%
11.79%
p < 0.001
29.27%
26.32%
NS
Much clinic time (2)
22.45%
16.30%
4.82%
p < 0.001
19.51%
24.56%
NS
Hospital in the periphery (3)
19.39%
6.52%
6.11%
p < 0.001
19.51%
19.30%
NS
Leading department (1)a
67.35%
77.17%
79.04%
p < 0.03
70.73%
64.91%
NS
Large hospitial
52.04%
54.64%
61.84%
p < 0.04
58.54%
47.37%
NS
Intellectual challenge (1)a
83.67%
82.07%
83.41%
NS
87.80%
80.70%
NS
Family living location
77.55%
74.86%
63.76%
NS
87.80%
70.18%
p < 0.05
Controllable lifestyle
69.39%
70.49%
60.70%
NS
73.17%
66.67%
NS
Making clinical decisions on your own
66.33%
49.46%
55.02%
NS
56.10%
73.68%
p < 0.05
Specific location in Israel
65.31%
64.48%
63.88%
NS
68.29%
63.16%
NS
Much “action”
52.04%
33.15%
46.49%
NS
51.22%
52.63%
NS
Pre-determined work hours (2)
46.94%
48.37%
35.37%
NS
58.54%
38.60%
p < 0.05
Physical challenge
47.96%
36.96%
46.93%
NS
41.46%
52.63%
NS
Teaching students
43.88%
43.48%
43.42%
NS
39.02%
47.37%
NS
Influence of family
41.84%
37.16%
31.00%
NS
53.66%
33.33%
p < 0.01
Opportunity for research
20.41%
25.00%
27.95%
NS
19.51%
21.05%
NS
Many on-call shifts
10.20%
11.41%
11.79%
NS
4.88%
14.04%
p < 0.03
Percent of “agree” and “agree much” responses on 5-point Likert Scale
Numbers in parenthesis are the results of factor analysis Weissman et al. Endnote Israel Journal of Health Policy Research (2018) 7:28 Page 14 of 17 Appendix B
Table 7 Residency in a peripheral hospital
Periphery
No periphery
Periphery vs no periph
N
48
382
Criteria for choosing a specialty
Time with family
85.42%
66.23%
p < 0.001
Controllable lifestyle
75.00%
65.01%
p < 0.04
Influency of spouse
56.25%
37.40%
p < 0.01
Specialty that deals with social issues
45.83%
27.03%
p < 0.004
Work only in the community
18.75%
2.62%
p < 0.001
Narrow range of medical problems
10.42%
1.83%
p < 0.001
Advancing rapidly
47.92%
62.30%
p < 0.05
Opportunity for research
29.17%
40.94%
p < 0.03
Bedside specialty
91.67%
93.10%
NS
Wide rangeof medical problems
62.50%
72.44%
NS
Independent practice
60.42%
55.24%
NS
Specialty with teamwork
54.17%
47.52%
NS
Procedures/surgery
50.00%
47.24%
NS
High salary
50.00%
47.64%
NS
Private practice
47.92%
41.05%
NS
Daytime work only
27.08%
21.78%
NS
Influence of family
14.89%
10.76%
NS
Academic faculty member
18.75%
28.61%
NS
Influence of classmate
4.17%
1.31%
NS
Specialty that my coleagues choose
2.08%
0.79%
NS
Criteria for choosing a residency
Family living location
81.25%
68.59%
p < 0.04
Teaching students
56.25%
40.05%
p < 0.05
Pre-determined work hours
56.25%
40.73%
p < 0.05
Influence of family
52.08%
32.98%
p < 0.02
Much supervision by senior physicians
50.00%
42.15%
p < 0.03
Primary care
42.22%
14.72%
p < 0.0004
Limited work hours
41.67%
21.26%
p < 0.03
Much clinic time
33.33%
8.38%
p < 0.001
Leading department
58.33%
78.59%
p < 0.0001
Large hosptial
46.81%
60.05%
p < 0.003
Intellectual challenge
77.08%
82.77%
NS
Controllable lifestyle
77.08%
61.56%
NS
Specific location
75.00%
65.79%
NS
Making clinical decisions on your own
62.50%
53.26%
NS
Much “action”
43.75%
40.31%
NS
Physical challenge
41.67%
41.10%
NS
Short residency
22.92%
15.18%
NS
Opportunity for research
20.83%
25.85%
NS
Many on-call shifts
16 67%
9 14%
NS Page 15 of 17 Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. 9. Weissman C, Zisk-Rony RY, Schroeder JE, Weiss YG, Avidan A, Elchalal U,
Tandeter H. Medical specialty considerations by medical students early in
their clinical experience. Isr J Health Policy Res. 2012;1:13. References The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request. 1. Allen SM, Ballweg RA, Cosgrove EM, Engle KA, Robinson LR, Rosenblatt RA,
Skillman SM, Wenrich MD. Challenges and opportunities in building a
sustainable rural primary care workforce in alignment with the Affordable
Care Act: the WWAMI program as a case study. Acad Med. 2013;88:1862–9. 1. Allen SM, Ballweg RA, Cosgrove EM, Engle KA, Robinson LR, Rosenblatt RA,
Skillman SM, Wenrich MD. Challenges and opportunities in building a
sustainable rural primary care workforce in alignment with the Affordable
Care Act: the WWAMI program as a case study. Acad Med. 2013;88:1862–9. 1. Allen SM, Ballweg RA, Cosgrove EM, Engle KA, Robinson LR, Rosenblatt RA,
Skillman SM, Wenrich MD. Challenges and opportunities in building a
sustainable rural primary care workforce in alignment with the Affordable
Care Act: the WWAMI program as a case study. Acad Med. 2013;88:1862–9. Competing interests Competing interests
The authors declare that they have no competing interests. p
g
The authors declare that they have no competing interests. 8. OECD Reviews of Health Care Quality: Israel 2012: Raising Standards OCED
Library http://www.oecd-ilibrary.org/social-issues-migration-health/oecd-
reviews-of-health-care-quality-israel-2012_9789264029941-en
Accessed 26 Feb 2017. 8. OECD Reviews of Health Care Quality: Israel 2012: Raising Standards OCED
Library http://www.oecd-ilibrary.org/social-issues-migration-health/oecd-
reviews-of-health-care-quality-israel-2012_9789264029941-en
Accessed 26 Feb 2017. Authors’ contributions CW – Designed study, collected and analyzed data, wrote and edited
manuscript. RYZ – Equal contribution with first author – designed study, edited
questionnaire, analyzed data, edited, commented and revised manuscript. AA –
Reviewed and edited questionnaire, reviewed data, edited and commented on
manuscript. UA – Reviewed and edited questionnaire, edited and commented
on manuscript. HT – Designed study, revised questionnaire, reviewed
manuscript. All the authors read and approved the final manuscript. 2. Duffrin C, Diaz S, Cashion M, Watson R, Cummings D, Jackson N. Factors
associated with placement of rural primary care physicians in North
Carolina. South Med J. 2014;107:728–33. 3. Sempowski IP. Effectiveness of financial incentives in exchange for rural and
underserviced area return-of-service commitments: systematic review of the
literature. Can J Rural Med. 2004;9:82–8. 4. Del Mar C. New investments in primary care in Australia. BMC Health Serv
Res. 2011;11:39. Appendix C Appendix C
Table 8 In “residency in the periphery” and “primary care residency”
Residency in the periphery
Not intersted
Neutral
Interested
Total
Primary
Not interested
37.4%
4.7%
2.7%
44.8%
Care
Neutral
22.7%
11.0%
2.3%
36.0%
Residency
Interested
10.4%
5.1%
3.7%
19.2%
Total
70.5%
20.7%
8.8%
Appendix D
Table 9 Primary care - selection criteria
1
Incentive
interests me
2
Plan peripheral
residency
3
Incentives don’t
interest me
1 vs 3
1 vs 2
2 vs 3
N
106
20
223
Criteria for choosing a specialty
Time with family (1)
78.30%
60.00%
63.39%
p < 0.004
p < 0.04
NS
Specialty that deals with social issues (3)
35.24%
35.00%
25.11%
p < 0.02
NS
NS
Advancing rapidly (2)
51.43%
45.00%
68.75%
p < 0.0007
NS
p < 0.04
Opportunity for research (2)
29.25%
5.00%
46.64%
p < 0.0001
p < 0.04
p < 0.0001
Bedside specialty
95.28%
95.00%
92.79%
NS
NS
NS
Wide range of medical problems
73.58%
63.16%
73.66%
NS
NS
NS
Controllable lifestyle (1)
73.58%
50.00%
66.07%
NS
NS
NS
Independent practice practice
53.77%
40.00%
51.28%
NS
p < 0.03
p < 0.03
High salary
50.48%
20.00%
45.09%
NS
p < 0.006
p < 0.01
Specialty with team work
48.11%
55.00%
50.89%
NS
NS
NS
Procedures/surgery
45.28%
31.58%
50.67%
NS
NS
p < 0.04
Influence of spouse
41.35%
45.00%
34.84%
NS
NS
NS
Private practice
36.79%
10.00%
39.64%
NS
p < 0.008
p < 0.009
Daytime work only (1)
23.30%
21.05%
19.20%
NS
NS
NS
Academic faculty member
22.86%
10.53%
31.25%
NS
NS
NS
Influence of family
9.43%
10.00%
11.21%
NS
NS
NS
Work only in the community
4.72%
10.00%
3.14%
NS
NS
NS
Narrow range of medical problemsa
4.72%
5.00%
2.68%
NS
NS
NS
Specialty that my coleagues choosea
1.90%
5.00%
0.90%
NS
NS
NS
Influence of classmatesa
0.95%
0.00%
1.34%
NS
NS
NS
Criteria for choosing a residency
Controllable lifestyle
73.33%
45.00%
58.12%
p < 0.04
p < 0.03
NS
Primary care
27.18%
31.58%
15.32%
p < 0.001
NS
p < 0.002
Hospital in the periphery (3)
7.55%
55.00%
3.85%
p < 0.001
p < 0.0007
p < 0.001
Intellectual challenge (1)a
80.19%
60.00%
87.05%
p < 0.004
NS
p < 0.007
Leading department (1)a
68.87%
45.00%
84.82%
p < 0.0002
p < 0.004
p < 0.0009
Specific location
50.94%
68.42%
69.82%
p < 0.0001
NS
NS
Large hospitial
50.00%
26.32%
67.71%
p < 0.005
p < 0.04
p < 0.0006
Opportunity for research
16.98%
5.00%
29.02%
p < 0.02
p < 0.01
p < 0.0005
Family living location
68.87%
70.00%
69.06%
NS
NS
NS
Making clinical decisions on your own
49.06%
50.00%
58.04%
NS
NS
NS Weissman et al. Ethics approval and consent to participate 5. Israel Ministry of Health: Disparity in health services and ways of coping
with it. 2015, Jerusalem; www.health.gov.il/publicationsfiles/inequality-2015. pdf (Accessed 12 Mar 2017). The Institutional Review Board of the Hadassah Medical Organization
approved this study. Completion of the questionnaire by the student was
considered tacit consent. The Institutional Review Board of the Hadassah Medical Organization
approved this study. Completion of the questionnaire by the student was
considered tacit consent. 6. Weil LG, Bin Nun G, Mckee M. Recent physician strike in Israel: a health
system under stress? Israel J Health Policy Res. 2013;2:33. 7. Simonstein F. Priorities in the Israeli health care system. Med Health Care
Philos. 2013;16:341–7. Acknowledgements The authors thank the many students who participated in this study. The authors thank the many students who participated in this study. Received: 15 November 2017 Accepted: 25 April 2018
/ Appendix C Israel Journal of Health Policy Research (2018) 7:28 Page 16 of 17 Table 9 Primary care - selection criteria (Continued)
1
Incentive
interests me
2
Plan peripheral
residency
3
Incentives don’t
interest me
1 vs 3
1 vs 2
2 vs 3
Much “action”
45.28%
40.00%
43.75%
NS
NS
NS
Much supervision by senior physicians
43.81%
20.00%
46.43%
NS
NS
p < 0.04
Limited work hours
43.40%
30.00%
40.18%
NS
NS
NS
Physical challenge
42.45%
30.00%
41.70%
NS
NS
NS
Teaching students
38.10%
50.00%
48.21%
NS
NS
NS
Influence of family
32.38%
30.00%
34.38%
NS
NS
NSb
Pre-determined work hours (2)
25.71%
25.00%
20.72%
NS
NS
NS
Short residency
15.09%
10.00%
16.52%
NS
NS
NS
Much clinic time (2)
13.33%
20.00%
9.38%
NS
NS
NS
Many on-call shifts
12.26%
10.00%
10.71%
NS
NS
NS
Percent of “agree” and “agree much” responses on 5-point Likert Scale
Numbers in parenthesis are the results of factor analysis
aClusters per cluster analysis
Bold results represent the higher value of a statistically significant pair Table 9 Primary care - selection criteria (Continued) Jerusalem, Israel. 4Department of Family Medicine, Ben Gurion University
Joyce and Irving Goldman School of Medicine, Be’er Sheva, Israel. Acknowledgements Received: 15 November 2017 Accepted: 25 April 2018
/ Received: 15 November 2017 Accepted: 25 April 2018
/ Funding
Israel National Institute for Health Policy Research. Funding
Israel National Institute for Health Policy Research. Author details
1 10. Avidan A, Weissman C, Elchalal U, Tandeter H, Zisk-Rony RY. Medical
specialty selection criteria of Israeli medical students early in their clinical
experience: subgroups. Isr J Health Policy Res. 2018;7:20. 1Department of Anesthesiology and Critical Care Medicine,
Hadassah-Hebrew University Medical Center, Hebrew University – Hadassah
School of Medicine, Kiryat Hadassah, POB 12000, 91120 Jerusalem, Israel. 2Hadassah Henrietta Szold School of Nursing, Hebrew University, Jerusalem,
Israel. 3Department of Obstetrics and Gynecology, Hadassah-Hebrew
University Medical Center, Hebrew University Hadassah School of Medicine, 1Department of Anesthesiology and Critical Care Medicine,
Hadassah-Hebrew University Medical Center, Hebrew University – Hadassah
School of Medicine, Kiryat Hadassah, POB 12000, 91120 Jerusalem, Israel. 2Hadassah Henrietta Szold School of Nursing, Hebrew University, Jerusalem,
Israel. 3Department of Obstetrics and Gynecology, Hadassah-Hebrew
University Medical Center, Hebrew University Hadassah School of Medicine, 11. Dossajee H, Obonyo N, Ahmed SM. Career preferences of final year medical
students as a medical school in Kenya – a cross sectional study. BMC Medical Education. 2016;16:5. 11. Dossajee H, Obonyo N, Ahmed SM. Career preferences of final year medical
students as a medical school in Kenya – a cross sectional study. BMC Medical Education. 2016;16:5. Page 17 of 17 Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 12. Kawamoto R, Ninomiya D, Kasai Y, Kusunoki T, Ohtsuka N, Kumagi T, Abe M. Factors associated with the choice of general medicine as a career among
Japanese medical students. Med Educ Online. 2016;21:29448. 13. Weissman C, Tandeter H, Zisk-Rony RY, Weiss YG, Elchalal U, Avidan A. Schroeder JE Israeli medical student's perceptions of six key medical
specialties. Israel J Health Policy Res. 2013;2:19. 14. Petterson SM, Liaw WR, Tran C, Bazemore AW. Estimating the residency
expansion required to avoid projected primary care physician shortages by
2035. Ann Fam Med. 2015;13:107–14. 15. Kringos D, Boerma W, Bourgueil Y, Cartier T, Dedeu T, Hasvold T,
Hutchinson A, Lember M, Oleszczyk M, Rotar Pavlic D, Svab I, Tedeschi P,
Wilm S, Wilson A, Windak A, Van der Zee J, Groenewegen P. The strength of
primary care in Europe: an international comparative study. Br J Gen Pract. 2013;63:e742–50. 16. Teng VC, Lin SY. Renewing US medical students’ interest in primary care:
bridging the role model gap. Postgrad Med. 2014;90:1–2. 17. Messinger CJ, Hafler J, Khan AM, Long T. Recent trends in primary care
interest and career choices among medical students at an academic
medical institution. Teach Learn Med. 2017;29:42–51. Author details
1 18. Pfarrwaller E, Sommer J, Chung C, Maisooeuve H, Nendaz M, Perron NJ,
Haller DM. Impact of interventions to increase the proportion of medical
students choosing a primary career: a systemic review. J Gen Intern Med. 2015;30:1349–58. 19. Giang KB, Minh HV, Hien NV, Ngoc NM, Hinh ND. Knowledge of primary
health care and career choice at primary health care settings among final
year medical students – challenges to human resources for health in
Vietnam. Global Pub Heal. 2017;10(sup 1):S120–30. 20. Kim YY, Kim UN, Kim YS, Lee JS. Factors associated with the specialty choice
of Korean medical students: a cross-sectional survey. Hum Resource Health. 2016;14:45. 21. Gold JA, Barg FK, Margo K. Undergraduate students’ perspectives on
primary care. J Prim Care Comm Health. 2014;5:279–83. 22. Clinite KL, DeZee KJ, Durning SJ, Kogan JR, Blevins T, Chou CL, Diemer G,
Dunne DW, Fagan MJ, Hartung PJ, Kazantsev SM, Mechaber HF, Paauw DS,
Wong JG, Reddy ST. Lifestyle factors and primary care specialty selection:
comparing 2012-2013 graduating and matriculating medical students’
thoughts on specialty lifestyle. Acad Med. 2014;89:1483–9. 23. Ashkenazi Y, Gordon M, Yankellevich A, Rosen B. Attracting medical
residents to the periphery and to medical specialties in crisis following the
2011 collective agreement. Jerusalem: Myers-JDC-Brookdale Institute
Smokler Center for Health Policy Research; 2017. 24. Kiobassa K, Miksch A, Hermann K, Loh A, Szecsenyi J, Joos S, et al. Becoming
a general practitioner – which factors have more impact on career choice
of medical students? BMC Fam Pract. 2011;12:25. 25. Monnickendam SM, Borkan JM, Matalon A, Zalewski S. Trials and tribulations
of country doctors: a qualitative study of doctor-patient relationships in
rural Israel. Isr J Med Sci. 1996;32:239–47. 26. Royston PJ, Mathieson K, Leafman J, Ojan-Sheehan O. Medical student
characteristics predictive of intent for rural practice. Rural Remote Health. 2012;12:2107. 27. Russell DJ, Wakerman J, Humphreys JS. What is a reasonable length of
employment for health workers in Australian rural and remote primary
healthcare services? Aust Health Rev. 2013;37:256–61. 28. Puddey IB, Mercer A, Playford DE, Riley GJ. Medical student selection criteria
and socio-demographic factors as predictors of ultimately working rurally
after graduation. BMC Med Educ. 2017;17:1. 29. Kawamoto R, Uemoto A, Ninomiya D, Hasegawa Y, Ohtsuka N, Kusunoki T,
Kumagi T, Abe M. Characteristics of Japanese medical students associated
with their intention for rural practice. Rural Remote Health. 2015;15:3112. 30. Williamson MI, Wilson R, McKechnie R, Ross J. Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 Author details
1 Does the positive influence of
an undergraduate rural placement persist into postgraduate years? Rural
Remote Health. 2012;12:2011. 31. Fennig S, Yuval D, Greenstein M, Rabin S, Weingarten M. Job satisfaction
among certified and non-certified general practitioners. Isr Med Assoc J. 2000;2:823–7. 32. Biderman A, Shvartzman P, Anson O, Almagor G, Grol R. Responsibility
taking and role definition in family practice: effect of training and practice
setting. Isr J Med Sci. 1996;32:545–50.
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Single-Dose Microparticle Delivery of a Malaria Transmission- Blocking Vaccine Elicits a Long-Lasting Functional Antibody Response
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Current molecular medicine
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R.R. Dinglasan*,1, J.S. Armistead1,#, J.F. Nyland2,#, X. Jiang3,4 and H.Q. Mao3,4 1W. Harry Feinstone Department of Molecular Microbiology & Immunology, Johns Hopkins Bloomberg School
of Public Health, 615 N. Wolfe Street, Baltimore, MD 21205, USA 2Department of Pathology, Microbiology & Immunology, University of South Carolina School of Medicine,
6439 Garner's Ferry Road, Columbia, SC 29209, USA 3Department of Materials Science and Engineering, Johns Hopkins University, 3400 North Charles Street,
Baltimore, MD 21218, USA 3Department of Materials Science and Engineering, Johns Hopkins University, 3400 North Charles Street,
Baltimore, MD 21218, USA
4 4Translational Tissue Engineering Center, Whitaker Biomedical Engineering Institute, Johns Hopkins School
of Medicine, 400 North Broadway, Baltimore, MD 21287, USA 4Translational Tissue Engineering Center, Whitaker Biomedical Engineering Institute, Johns Hopkins School
of Medicine, 400 North Broadway, Baltimore, MD 21287, USA Abstract: Malaria sexual stage and mosquito transmission-blocking vaccines (SSM-TBV) have recently gained
prominence as a necessary tool for malaria eradication. SSM-TBVs are unique in that, with the exception of
parasite gametocyte antigens, they primarily target parasite or mosquito midgut surface antigens expressed
only inside the mosquito. As such, the primary perceived limitation of SSM-TBVs is that the absence of natural
boosting following immunization will limit its efficacy, since the antigens are never presented to the human
immune system. An ideal, safe SSM-TBV formulation must overcome this limitation. We provide a focused
evaluation of relevant nano-/microparticle technologies that can be applied toward the development of leading
SSM-TBV candidates, and data from a proof-of-concept study demonstrating that a single inoculation and
controlled release of antigen in mice, can elicit long-lasting protective antibody titers. We conclude by
identifying the remaining critical gaps in knowledge and opportunities for moving SSM-TBVs to the field. Keywords: Antigen, controlled release, immunity, malaria, midgut, mosquito, nanotechnology, natural boosting,
sexual stages, transmission-blocking vaccine. Send Orders of Reprints at reprints@benthamscience.net Send Orders of Reprints at reprints@benthamscience.net Current Molecular Medicine 2013, 13, 479-487 479 © 2013 Bentham Science Publishers *Address correspondence to this author at the W. Harry Feinstone
Department of Molecular Microbiology & Immunology, Johns Hopkins
Bloomberg School of Public Health, 615 N. Wolfe Street, Rm. E5646,
Baltimore, MD 21205, USA; Tel: +1-410-614-4839;
Fax: +1-410-955-0105; E-mail: rdinglas@jhsph.edu #These authors contributed equally to this manuscript. Single-Dose Microparticle Delivery of a Malaria Transmission-
Blocking Vaccine Elicits a Long-Lasting Functional Antibody
Response R. Dinglasan*,1, J.S. Armistead1,#, J.F. Nyland2,#, X. Jiang3,4 and H.Q. Mao3 INTRODUCTION vivax
Efficacy
>85% efficacy and reduction of the Case Reproductive Rate (R0) below 1
Duration
Effective for at least 2 years
Safety
Vaccine has a safety and efficacy profile comparable to Hepatitis B vaccine
Target Population
All age groups
Administration Route
Administered orally, or by intramuscular or subcutaneous injection or other innovative device
Immunization Schedule
A single dosage schedule that can be administered by mass administration or clinic-based programs. Booster
dose after 2 years may be required
Minimal schedule is three doses, administered over 6 months
Stability & Storage
Minimum shelf life of 36 months and can be stored at ambient temperature and withstand freeze thawing
Minimum is stability at 37°C for 30 minutes and 2 years at 2-8°C
Co-administration
No interference or interactions with other vaccines expected to be concurrently administered Target Product Profile of a SSM-TBV “natural boosting” issue (Table 1). Furthermore, we
have also used APN1 as a model antigen to directly
address the above issue using nano- and microparticle
technologies. [14, 15], thus it is possible that gametocyte exposure in
the NHPs following challenge was responsible for
boosting. This study further supported the notion that
boosting would increase the efficacy and utility of SSM-
TBVs but raised the question of the need for highly
potent adjuvants such as FCA, which is considered a
serious obstacle in human vaccine development. An ideal SSM-TBV formulation with a highly
immunogenic antigen must therefore have the following
characteristics: (i) it should be safe; (ii) it should not
require
a
cold-chain;
(iii)
it
should
easily
be
administered; and (iv) a single immunization should
confer
long-lasting
protection. A
biodegradable
microparticle (BMP) system, which provides sustained
release of antigen and adjuvant properties, is capable
of meeting these challenges. Several recent studies
have demonstrated the utility of this general vaccine
approach in vertebrate models [36-40]. Microparticle
size is an important determinant for cell uptake [41, 42]
and may also influence the antigen release rate [43]. In
line with this, recent studies have shown that smaller
particle delivery systems are effective in eliciting a
robust immune response to the target immunogen [44-
47]. The bioabsorption rate of BMPs and antigen
release rate can be engineered to provide boosting
from weeks to several months. INTRODUCTION (gamete, zygote, or ookinete) or Anopheles mosquito
midgut surface antigens that are only expressed in the
mosquito. As such, one of the potential limitations of
the TBV approach is that since the antigens are never
naturally presented to the human immune system, the
absence of natural boosting following immunization will
limit their efficacy [8-13]. A complete P. knowlesi model
in non-human primates (NHP) has been used to test
the “natural boosting” hypothesis for Plasmodium
gamete antigens [13]. It was found that following a two-
dose immunization regimen using 105-107 P. knowlesi
microgametes and macrogametes in a Freund’s
complete adjuvant (FCA), the majority of the monkeys
maintained a high level of functional transmission-
blocking antibody titer for more than 1 year. Furthermore, annual challenge infections over a six
year period were found to be sufficient for boosting and
transmission-blocking
immunity
persisted
in
the
majority of splenectomized NHPs. Importantly, as
expected, they observed that transmission-blocking
activity waned over time in the absence of boosting and
that the challenge infection resulted in an increase in
gamete-specific antibody levels. Although the likely
gamete antigens had not yet been fully characterized at
the time of this study, it was already known that
gametocytes and gametes shared surface antigens The malaria eradication research agenda has re-
emphasized the need for effective sexual stage and
mosquito transmission-blocking vaccines (SSM-TBV)
[1], which prevents malaria parasite development in its
mosquito vector and the subsequent cascade of
secondary infections [2-5]. SSM-TBVs, in general, work
through the action of inhibitory antibodies [5-7]. Thus,
the minimum objective of immunization is to induce
high titer antibodies sustainable for at least one
transmission season (~3-6 months), but preferably for
2
years. Achieving
this
minimum
goal
would
theoretically drive the case reproductive rate, (R0) <1. A
summary of the target product profile (TPP) for SSM-
TBVs is shown in Table 1. With the exception of
Plasmodium falciparum or P. vivax gametocyte surface
antigens that are expressed in the human, SSM-TBVs
are considered unique in that they target parasite #These authors contributed equally to this manuscript. /13 $58.00+.00 © 2013 Bentham Science Publishers 0 Current Molecular Medicine, 2013, Vol. 13, No. 4 480 Dinglasan et al. Table 1. The Proposed Target Product Profile (TPP) for a Malaria Sexual Stage and Mosquito Transmission-Blocking
Vaccine (SSM-TBV) [61]
Target Product Profile of a SSM-TBV
Indication
Prevention of transmission of P. falciparum or P. INTRODUCTION Particles carrying single
or multiple antigens can arguably mimic viral antigen
presentation thus rapidly inducing a potent and long-
lasting cellular and humoral response either by direct
immune stimulation of antigen presenting cells (APCs)
or/and by delivering antigen to the lymph node [30, 37,
48]. In fact, virosomes follow this approach and have
shown to be effective carriers for proteins and subunit
vaccines against a variety of pathogens, including
malaria [49], but to date, this approach has not been
used to deliver SSM-TBV antigens. With these goals in
mind, we conducted proof-of-concept studies to test the
hypothesis that safe biodegradable microparticles can
mimic natural boosting through sustained release of
antigen and, in doing so, elicit significant transmission-
blocking antibodies against Plasmodium. The four leading SSM-TBVs (Table 2) include two
gametocyte surface antigens, Pfs230 [16-20] and
Pfs48/45 [21], the ookinete surface protein Pfs25 [22]
and the Anopheles gambiae alanyl aminopeptidase N
(APN1), which is an abundant, midgut-specific apical
microvilli surface glycoprotein that has been shown to
mediate ookinete invasion and oocyst development [7,
23]. Of these, only Pfs25 and APN1 are expressed
explicitly inside the mosquito midgut. Note that the goal
of this report is not to evaluate the complete repertoire
of proven and possible SSM-TBV candidates, and the
reader is directed to several excellent reviews for
additional information [3, 4, 24-29]. Among the four
leading candidates, only Pfs25 has completed Phase I
clinical trials, albeit with equivocal results [29]. Efforts
are underway to produce the full-length Pfs/Pvs230
[30-32] and Pfs48/45 antigens [33-35], which have
proven to be a difficult undertaking using different
expression
platforms
due
to
their
size
and/or
conformation, as well as the high A+T content of
plasmodial genes; and these issues have a direct
impact on vaccine process development. The APN1
antigen, on the other hand, does not require the full-
length antigen, is highly immunogenic [7] and is
entering process development, with an optimistic
initiation of Phase I clinical trials within the next 3-4
years. Since Pfs25 and APN1-based vaccines are the
least likely to benefit from boosting following natural
infection, we focused on these two antigens in this
article to examine their current state of development,
as well as similarities and differences in the context of
several identified target product profiles and the Current Molecular Medicine, 2013, Vol. 13, No. 4 481 Current Molecular Medicine, 2013, Vol. 13, No. 4 481 Single-Dose Microparticle Delivery Table 2. Preparation
of
Biodegradable
Microparticles
(BMPs) with Different Size Range and Different
Antigen Loading Levels Recombinant APN1 was produced in E. coli as
previously described [23]. Polylactofate (PLE) was
used to prepare BMPs. PLE is a poly(lactide-co-
glycolide) derivative with good biocompatibility and
better control of biodegradation rate and physical
properties [50, 51] (Fig. 1A). BMPs were prepared by a
modified
double
emulsion
method
[50],
and
characterized by scanning electron microscopy. The
release kinetics of APN1 from BMPs was characterized
by monitoring the concentration of APN1 using ELISA. To modulate APN1 release, we used bovine serum
albumin (BSA) as a filler protein. MATERIALS AND METHODS MATERIALS AND METHODS Immunizations BALB/c female mice were immunized with either (A)
recombinant APN1 in PBS in suspension with alum, or
(B) recombinant APN1 in PBS emulsified with
incomplete Freund’s adjuvant (IFA), or (C) BMP-
encapsulated recombinant APN1 delivered with alum,
or (D) BMP encapsulated APN1 with IFA or (E) empty
BMP with alum or (F) empty BMP with IFA. For all
treatment groups, mice received 2 g antigen/mouse/
dose. At day 0, mice received a subcutaneous (s.c.)
injection of the appropriate inoculum in a volume of 100
l per mouse. At 2, 4 and 6 weeks post priming, mice
in the Control cohorts (treatments A and B, above)
were boosted intraperitoneally (i.p.) with the same dose
of the inoculum per mouse, whereas the BMP cohorts
were boosted only with PBS. At these time points, each
mouse was bled to collect sera for anti-APN1 antibody
titer determination via ELISA (Fig. 1C). INTRODUCTION Update of the Current Status and Characteristics of the Leading SSM-TBV Candidates
Target Antigen
Current Status
Attributes
P230
Recombinant antigen expression
through a variety of systems including
plant, cell free wheat germ systems. Present in the gametocyte and can confer natural boosting [10, 19]
Immunogenicity is poor and requires a strong adjuvant [19, 62, 63]
Molecule is large, resulting in difficulty in expression and maintenance of
conformational epitopes [63]
P48/45
Recombinant antigen expression using
E. coli (codon harmonized)
Conformational epitopes necessitates an appropriate expression system
[33]
Immunogenic protein in animals (alum) and is further enhanced by using a
strong adjuvant [33]
P25
Phase I clinical trials
+ Conjugated to recombinant
Pseudomonas aeruginosa ExoProtein A
[62]
Phase I Clinical trial of ExoProtein A
product is ongoing
Immunogenic varies depending on route [63] but is generally considered
poorly immunogenic by itself and may require a strong adjuvant [29, 64-66]
or conjugation to a molecular adjuvant or protein carrier [67]
Reactogenic formulations prevented continuation of the first Phase I clinical
trial [65]
Successfully produced the small immunogen in yeast and plants [68]
APN1
Entering Process Development
Immunogenic in mice [7] and non-human primates (Dinglasan, unpublished)
using alum as adjuvant
Does not require an adjuvant for complete seroconversion in mice [7] Update of the Current Status and Characteristics of the Leading SSM-TBV Candidates ELISA and Cytokine Assay ELISAs were performed as previously described,
using recombinant APN1 as coating antigen [7]. For
cytokine
assays,
the
spleen
was
removed
and
homogenized at 10% wt/vol in 2% fetal bovine
serum/minimal essential medium, and supernatants
stored at -80°C until used. Cytokines were measured in
tissue homogenates using bead-based multiplex cytokine
kits (Bio-Plex, Bio-Rad), according to manufacturer’s
instructions. The limits of detection were as follows:
interleukin (IL)-1, 1.32 pg/ml, IL-1, 1.70 pg/ml; IL-2,
1.98 pg/ml; IL-3, 1.32 pg/ml; IL-4, 2.43 pg/ml; IL-5, 1.69
pg/ml; IL-5, 1.69 pg/ml; IL-6, 1.02 pg/ml; IL-9, 1.36 pg/ml;
IL-10, 1.04 pg/ml; IL-12/23 p40, 1.15 pg/ml; IL-12 p70,
1.20 pg/ml; IL-13, 1.57 pg/ml; IL-17a, 1.44 pg/ml;
interferon (IFN)-, 1.30 pg/ml; eotaxin, 1.70 pg/ml;
granulocyte-colony
stimulating
factor,
1.69
pg/ml;
granulocyte-macrophage-colony stimulating factor, 1.58
pg/ml; monocyte chemo-attractant protein, 1.71 pg/ml;
macrophage inflammatory protein (MIP)-1, 1.57 pg/ml;
MIP-1, 1.20 pg/ml; RANTES, 0.95 pg/ml; tumor necrosis
factor (TNF)-, 1.73 pg/ml. Cytokine measurements
below the limit of detection as determined by the standard
curve for each individual cytokine were assigned a value
of the limit of detection/2 for statistical analysis and
plotting. Statistical significance was determined by One-
way ANOVA with Bonferroni Post Test, = 0.05. Preparation
of
Biodegradable
Microparticles
(BMPs) with Different Size Range and Different
Antigen Loading Levels Preparation
of
Biodegradable
Microparticles
(BMPs) with Different Size Range and Different
Antigen Loading Levels Transmission-Blocking Assays *
.
/*
+#, #
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+#! %
%! *
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/* Fig. (1). Polylactofate biodegradable microparticles for single inoculation delivery of a malaria transmission-blocking
vaccine antigen. (A) Structure of polylactofate (PLE). (B) Scanning electron micrographs of three batches of BMPs with 0.52%,
3.53% and 6.77% protein loading, respectively (Scale bars = 10 m). (C) Effect of protein loading level on the cumulative
release profile of encapsulated proteins from BMPs. (D) Immunization dosing regimens for BMP and control groups, and
functional analysis by direct feeding assay (DFA). Transmission-Blocking Assays The Direct Feeding Assays (DFA) were conducted
as previously described [7] at 2 months and at 6
months post-priming immunization (Fig. 1D). Since
Plasmodium
oocyst
numbers
are
generally
overdispersed in our system, statistical significance
was assessed using the non-parametric Mann Whitney
U Test, = 0.05. 482 Current Molecular Medicine, 2013, Vol. 13, No. 4
Dinglasan et al. Fig. (1). Polylactofate biodegradable microparticles for single inoculation delivery of a malaria transmission-blocking
vaccine antigen. (A) Structure of polylactofate (PLE). (B) Scanning electron micrographs of three batches of BMPs with 0.52%,
3.53% and 6.77% protein loading, respectively (Scale bars = 10 m). (C) Effect of protein loading level on the cumulative
release profile of encapsulated proteins from BMPs. (D) Immunization dosing regimens for BMP and control groups, and
functional analysis by direct feeding assay (DFA).
!" #
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#
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1 Fig. (1). Polylactofate biodegradable microparticles for single inoculation delivery of a malaria transmission-blocking
vaccine antigen. (A) Structure of polylactofate (PLE). (B) Scanning electron micrographs of three batches of BMPs with 0.52%,
3.53% and 6.77% protein loading, respectively (Scale bars = 10 m). (C) Effect of protein loading level on the cumulative
release profile of encapsulated proteins from BMPs. (D) Immunization dosing regimens for BMP and control groups, and
functional analysis by direct feeding assay (DFA).
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4 3 Fig. (2). Characterization of the immune response and activity of antibodies elicited following immunization with APN1. (A) APN1-specific antibody titers (at bleeds 1-3) for mice that received only a single inoculation of BMP encapsulated APN1 with
alum or IFA. RESULTS gambiae (Keele) mosquitoes for groups in (C) at two months
nization. For A-D: Median oocyst numbers are represented by the horizontal line; control infections were f
hed, unimmunized mouse; and the P-value was determined by Mann Whitney U Test and asterisks (*) indica
cance at = 0.05. (E-G) APN1-BMP induces pro-T-cell and B-cell cytokines. Twenty-three cytokines m
genized spleen samples from mice that received either BMP (empty) or APN1-encapsulated BMPs. Data e
of tissue basis (corrected for spleen weight). RESULTS single inoculation of APN1-containing BMPs plus IFA
or Alum alone (Fig. 2A) mounted a relatively poor
antibody response in comparison to a prime and 3-
boost regimen of APN1 plus IFA/Alum (Fig. 2C). Surprisingly, the immunoglobulin subtypes (IgG1,
IgG2a, and IgG2b) generated in the group that
received a single immunization of APN1-BMPs/alum
were similar to that elicited by the APN1-alum (data not
shown). We generated PLE BMPs (Fig. 1B) and optimized
the protocol for controlling the protein antigen loading
levels. We then used loading level as a parameter to
adjust the release rate of the antigen. Using bovine
serum albumin (BSA) as a model antigen, we have
shown that the amount of antigen released from the
BMPs can be controlled by loading level as shown in
Fig. (1C). For example, BMPs with 3.53% protein
loading level released protein antigen at a rate of ~ 104
ng/day per mg of BMPs, after an initial burst release of
9.3% of the total protein loaded. These release rates
amounted to a release of approximately 15% of total
protein within the first 22 days. For this pilot study, we
used BMPs with 3.53% of protein loading level. We
compared the humoral response of mice using the
schedule outlined in Fig. (1D). Mice immunized with a To determine the short-term and long-term efficacy
of transmission-blocking serum antibodies against P. berghei we performed direct feeding assays (DFAs)
two weeks following the final boost in the control group
at 2 months (60 days) and at 6 months (180 days)
(Figs. 1D, 2B, D). We compared parasite development
in mosquitoes that were fed on four groups: (i) control
cohort (primed with APN1/alum followed by three Current Molecular Medicine, 2013, Vol. 13, No. 4 483 Single-Dose Microparticle Delivery 2). Characterization of the immune response and activity of antibodies elicited following immunization
PN1-specific antibody titers (at bleeds 1-3) for mice that received only a single inoculation of BMP encapsulate
or IFA. (B) Direct Feeding Assay to assess short-term transmission-blocking potential of mouse APN1 anti
modium berghei (ANKA 2.34) in Anopheles gambiae (KEELE) mosquitoes for groups in (A) at two months
nization (see Fig. 1D). (C) APN1-specific antibody titers (at bleeds 1-3, at two week intervals) for mice that rec
either alum or IFA as adjuvant. (D) Direct Feeding Assay to assess short-term transmission-blocking potent
antisera against P. berghei (ANKA 2.34) in An. RESULTS (B) Direct Feeding Assay to assess short-term transmission-blocking potential of mouse APN1 antisera against
Plasmodium berghei (ANKA 2.34) in Anopheles gambiae (KEELE) mosquitoes for groups in (A) at two months post-priming
immunization (see Fig. 1D). (C) APN1-specific antibody titers (at bleeds 1-3, at two week intervals) for mice that received APN1
with either alum or IFA as adjuvant. (D) Direct Feeding Assay to assess short-term transmission-blocking potential of mouse
APN1 antisera against P. berghei (ANKA 2.34) in An. gambiae (Keele) mosquitoes for groups in (C) at two months post-priming
immunization. For A-D: Median oocyst numbers are represented by the horizontal line; control infections were from an age-
matched, unimmunized mouse; and the P-value was determined by Mann Whitney U Test and asterisks (*) indicate statistical
significance at = 0.05. (E-G) APN1-BMP induces pro-T-cell and B-cell cytokines. Twenty-three cytokines measured in
homogenized spleen samples from mice that received either BMP (empty) or APN1-encapsulated BMPs. Data expressed on
pg/g of tissue basis (corrected for spleen weight). The two significantly different cytokines (E) IL-2 and (F) IL-5 and one cytokine,
TNF-, which was not significantly different (G), are shown. Data presented as box and whiskers plots with outliers identified as
dots. Median is the horizontal line within the box. Statistical significance was determined by one way ANOVA with Bonferroni
Post Test, = 0.05. 484 Current Molecular Medicine, 2013, Vol. 13, No. 4 Dinglasan et al. Table 3
Direct Feeding Assays (DFA) to Assess Long-Term Transmission-Blocking Potential of Mouse APN1 Antisera
Against Plasmodium berghei (ANKA 2.34) in Anopheles gambiae (Keele) Mosquitoes. DFAs were Performed
at 6 Months Post-Priming Immunization (see Fig. 1D) Table 3
Direct Feeding Assays (DFA) to Assess Long-Term Transmission-Blocking Potential of Mouse APN1 Antisera
Against Plasmodium berghei (ANKA 2.34) in Anopheles gambiae (Keele) Mosquitoes. DFAs were Performed
at 6 Months Post-Priming Immunization (see Fig. 1D)
Group (Mouse #)
N
Median Oocyst # (Range)
% Inhibition
Prevalence
P-Value
Long-Term
APN1-Alum Control (M3)
23
82 (1-181)
100%
APN1-BMP-Alum (M4)
22
8.5 (0-84)
90
82%
<0.0001
APN1-BMP-Alum (M5)
n.d.
APN1-BMP-IFA (M4)
32
16 (0-124)
81
59%
<0.0001
APN1-BMP-IFA (M5)
22
0 (0-1)
100
9%
<0.0001
Groups: APN1-Alum Control = recombinant APN1 + alum, using a prime + 3 boost immunization regimen (age-matched with BMP groups); APN1-BMP =APN1-BMP
+ alum (single inoculation); APN1-BMPIFA=APN1-BMP +Incomplete Freund's adjuvant (single inoculation). RESULTS n.d., not determined since the mouse did not survive the
mosquito feeding. P-value determined by Mann Whitney U Test, = 0.05. Groups: APN1-Alum Control = recombinant APN1 + alum, using a prime + 3 boost immunization regimen (age-matched with BMP groups); APN1-BMP =APN1-BMP
+ alum (single inoculation); APN1-BMPIFA=APN1-BMP +Incomplete Freund's adjuvant (single inoculation). n.d., not determined since the mouse did not survive the
mosquito feeding. P-value determined by Mann Whitney U Test, = 0.05. boosts); (ii) treatment group receiving a single
inoculation of APN1-BMPplus alum, (iii) treatment
group receiving a single inoculation of APN1-BMP plus
IFA, and (iv) control (naïve/unimmunized or empty
BMP immunized) infected mice. At 60 days, both the
APN1-alum and APN1-IFA immunized controls elicited
functional transmission-blocking antibodies against P. berghei (Fig. 2B). Despite the lower antibody titer
observed
previously,
APN1-BMP-immunized
mice
generated a significant level of functional antibody titers
that can effectively inhibit oocyst development in An. gambiae (Fig. 2C, D). We observed that at 6 months
post-priming
immunization,
serum
from
mice
immunized with APN1/alum, following a standard
immunization regimen, no longer contained any
transmission-blocking antibodies [refer to median
oocyst number/prevalence for APN1-Alum Control
(M3)]. In contrast, individual mice that received either
APN1-BMPs/alum or APN1-BMPs/IFA still retained
functional transmission-blocking antibody (Table 3). Cytokine levels analyzed by multiplex assay also
demonstrated that APN1-BMPs significantly induced
splenic pro-T-cell and B-cell cytokines such as IL-2 and
IL-5. These data suggest a cell-specific immune effect
rather than a general inflammatory process in response
to BMP dosing, thereby validating the specificity of the
immune response to the vaccine formulation (Fig. 2D-
F). gambiae mosquitoes; and (3) that with a potent
adjuvant such as incomplete Freund’s adjuvant,
immunization
with
BMPs
elicits
and
maintains
transmission-blocking titers in mice for 6 months. Furthermore, the protracted release kinetics of model
antigen over 16 days in vitro by our PLE BMP
demonstrates a more controlled profile as compared to
gel core liposome or conventional liposome particles
which have been shown to exhibit a 50% cumulative
percentage release of antigen at 10-15 days and 5
days, respectively [44]. These data suggest that larger
microparticles allow for enhanced control over the
release
profile. Recently,
it
was
shown
that
incorporation of TLR9 agonists in 1-m gel core
liposomes can significantly enhance the immune
response to the poorly immunogenic Pfs25 SSM-TBV
antigen [44]. Thus, the use of molecular adjuvants as
filler molecules may also be considered in future
formulations. RESULTS Taken together with our proof-of-concept
data, we anticipate that co-encapsulation of adjuvant
and administration of different BMPs with different
release profiles (e.g. burst and fast release serve as
priming and sustained/delayed release as boosting
dose) will significantly enhance the overall immune
responses. DISCUSSION Although nano- and microparticle technology has
been already shown to potentiate the immune
response to pathogen-derived antigens [52, 53],
including malaria [44, 45, 49, 54], its use in TBV
delivery while previously postulated [9], remained
relatively untested [44]. Our small scale study adds to
the growing body of data, and moreover, successfully
demonstrates that (1) APN1-BMPs with alum adjuvant
elicit antigen-specific antibody titers after single dose
immunization and induce the production of cell-
activation rather than broad-spectrum pro-inflammatory
cytokines; (2) the functional transmission-blocking
activity of APN1 antisera against P. berghei from mice
immunized with a single dose of APN1-BMP in An. CONCLUSION AND FUTURE PERSPECTIVES Vaccines are traditionally developed with the
prospect of eventual parenteral administration, and the
TPP for SSM-TBVs suggests that this is the primary
consideration for the development of the leading
candidates (Table 1). Given the uniqueness of the
SSM-TBV approach it is argued that non-classical
concepts for vaccine delivery may be more suitable. In
this section we highlight some concerns surrounding
the use of NPs and BMPs when considering vaccine
delivery not only through parenteral, oral or mucosal
routes, but specifically via cutaneous immunization. REFERENCES [1]
Alonso PL, Brown G, Arevalo-Herrera M, et al. A research
agenda to underpin malaria eradication. PLoS Med 2011;
8(1): e1000406. ( )
[2]
Carter R. Transmission blocking malaria vaccines. Vaccine
2001; 19(17-19): 2309-2314. (
)
[3]
Dinglasan RR, Jacobs-Lorena M. Flipping the paradigm on
malaria transmission-blocking vaccines. Trends Parasitol
2008; 24(8): 364-370. [4]
Lavazec C, Bourgouin C. Mosquito-based transmission
blocking vaccines for interrupting Plasmodium development. Microbes Infect 2008; 10(8): 845-849. ( )
Sinden RE. A biologist's perspective on malaria vaccine
development. Hum Vaccin 2010; 6(1): 3-11. While there are clear opportunities for the utility of
BNPs and BMPs in the development of the next
generation of SSM-TBVs, the current working model by
many
vaccine
developers
remains
generally
conservative. This is rightly so, since malaria vaccines
must be low cost to allow for general distribution. The
huge number of vaccine doses to cover the more than
one third of the world’s population is likely to be borne
by public-private partnerships and other novel funding
models. However, there is hope for this approach since
the prevailing strategy has been more recently revisited
by the PATH Malaria Vaccine Initiative [59]. One of the
biggest benefits of the BNP/BMP approach, namely the
potential to mimic natural boosting, is quite attractive,
especially in light of the prediction that titers of antibody
(produced either naturally or following vaccination)
against sexual stage and mosquito antigens will likely
wane over time [60]. Furthermore, there is optimism Ranawaka GR, Fleck SL, Blanco AR, Sinden RE. Characterization of the modes of action of anti-Pbs21 malaria
transmission-blocking
immunity:
ookinete
to
oocyst
differentiation in vivo. Parasitology 1994; 109 (Pt 4): 403-411. Ranawaka GR, Fleck SL, Blanco AR, Sinden RE. Characterization of the modes of action of anti-Pbs21 malaria
transmission-blocking
immunity:
ookinete
to
oocyst
differentiation in vivo. Parasitology 1994; 109 (Pt 4): 403-411. Mathias DK, Plieskatt JL, Armistead JS, et al. Expression,
immunogenicity, histopathology, and potency of a mosquito-
based malaria transmission-blocking recombinant vaccine. Infect Immun 2012; 80(4): 1606-1614. Mathias DK, Plieskatt JL, Armistead JS, et al. Expression,
immunogenicity, histopathology, and potency of a mosquito-
based malaria transmission-blocking recombinant vaccine. Infect Immun 2012; 80(4): 1606-1614. ( )
Kaslow DC. Immunogenicity of Plasmodium falciparum
sexual stage antigens: implications for the design of a
transmission blocking vaccine. Immunol Lett 1990; 25(1-3):
83-86. [9] Kaslow DC. Transmission-blocking immunity against malaria
and other vector-borne diseases. Curr Opin Immunol 1993;
5(4): 557-565. Kaslow DC. Does Route of Delivery Matter? It has been shown that size also has a direct
influence on the effectiveness of delivery when the
route of administration is considered. Intradermal or
subcutaneous
inoculation
of
BNPs
and
BMPs
bypasses the issue of tissue barriers and proteolytic
environments, in the case of oral administration. However, the clear potential of this technology lies in
the idea of needle-free vaccination. The use of BNP
and BMPs as carriers for transcutaneous or cutaneous
immunization has been extensively studied [57, 58] and
it is well recognized that the main barrier for trans- or
percutaneous delivery of antigen payload to the rich
population of APCs in the epidermis and dermis is the
stratum corneum lipid bilayer overlaying the epidermis
[57]. Passive
diffusion
of
antigen
carried
via
nanocarriers through intercellular or follicular routes to
access to the APCs in the epidermis and preferably the
dermis has been demonstrated, strongly implying that
presentation is size dependent [58]. Does Size Matter? In the context
of SSM-TBVs, it remains to be seen if different carrier
modes can further potentiate the humoral response to
confer long-term protection. that by leveraging the potential advantages conferred
by particle-based approaches, the community will
ultimately see the incorporation of vaccine antigens
targeting different life stages of the parasite in a single
particle formulation. ABBREVIATIONS ABBREVIATIONS
BMP
= Biodegradable microparticle
NP
= Nanoparticle
TBV
= Transmission-blocking vaccine
SSM-TBV = Sexual stage and mosquito TBV
APN1
= Alanyl aminopeptidase N 1
PLGA
= Poly-lactic-co-glycolic acid
FCA
= Freund’s complete adjuvant
IFA
= Incomplete Freund’s adjuvant. ACKNOWLEDGEMENTS This work was supported by the Bloomberg Family
Foundation and the Johns Hopkins Malaria Research
Institute Pilot Grant Award Program. The authors thank
Hilary Hurd and Paul Eggleston for the Anopheles
gambiae KEELE strain. CONFLICT OF INTEREST The authors confirm that this article content has no
conflicts of interest. Does Size Matter? It has been shown that 40 nm polystyrene
nanoparticles (NP) that are surface-coated with antigen
can be targeted to the lymph nodes to generate a
robust immune response [46-48, 55]. NPs have also Current Molecular Medicine, 2013, Vol. 13, No. 4 Single-Dose Microparticle Delivery 485 been shown to increase the breadth and avidity of the
humoral response to a Plasmodium vivax blood stage
antigen [37, 45] arising in part through a synergistic
effect of surface displayed and encapsulated antigen in
a single formulation. However, it is likely that the nature
of the particle, the characteristics of the antigen,
including intrinsic immunogenicity and molecular size,
presence of conformational antibody epitopes, as well
as the type of immune response that should be
engendered will have a direct influence on the selection
of biodegradable nanoparticles (BNP) vs BMP as
carrier (reviewed in [56, 57]). It was found that larger
particles engender a Type 2 response while smaller,
virus-sized particles induced a largely cell-mediated
Type 1 response [46]. An interesting approach would
be to use different BNP and BMP carriers, leveraging
the advantages of antigen targeting and antigen depot
effect endowed by each type of particles to reach a
specific immune response endpoint [36]. In the context
of SSM-TBVs, it remains to be seen if different carrier
modes can further potentiate the humoral response to
confer long-term protection. been shown to increase the breadth and avidity of the
humoral response to a Plasmodium vivax blood stage
antigen [37, 45] arising in part through a synergistic
effect of surface displayed and encapsulated antigen in
a single formulation. However, it is likely that the nature
of the particle, the characteristics of the antigen,
including intrinsic immunogenicity and molecular size,
presence of conformational antibody epitopes, as well
as the type of immune response that should be
engendered will have a direct influence on the selection
of biodegradable nanoparticles (BNP) vs BMP as
carrier (reviewed in [56, 57]). It was found that larger
particles engender a Type 2 response while smaller,
virus-sized particles induced a largely cell-mediated
Type 1 response [46]. An interesting approach would
be to use different BNP and BMP carriers, leveraging
the advantages of antigen targeting and antigen depot
effect endowed by each type of particles to reach a
specific immune response endpoint [36]. 486 Current Molecular Medicine, 2013, Vol. 13, No. 4 486 Current Molecular Medicine, 2013, Vol. 13, No. 4 Dinglasan et al. blocking activity. Clin Vaccine Immunol 2011; 18(8): 1343-
1350. [12]
Halloran ME, Struchiner CJ, Spielman A. Modeling malaria
vaccines. II: Population effects of stage-specific malaria
vaccines dependent on natural boosting. Math Biosci 1989;
94(1): 115-149. [32] Farrance CE, Rhee A, Jones RM, et al. A plant-produced
Pfs230
vaccine
candidate
blocks
transmission
of
Plasmodium falciparum. Clin Vaccine Immunol 2011; 18(8):
1351-1357. ( )
[13]
Gwadz RW, Koontz LC. Plasmodium knowlesi: persistence
of transmission blocking immunity in monkeys immunized
with gamete antigens. Infect Immun 1984; 44(1): 137-140. [33] Chowdhury DR, Angov E, Kariuki T, Kumar N. A potent
malaria transmission blocking vaccine based on codon
harmonized full length Pfs48/45 expressed in Escherichia
coli. PLoS One 2009; 4(7): e6352. [14] [14]
Rener J, Carter R, Rosenberg Y, Miller LH. Anti-gamete
monoclonal antibodies synergistically block transmission of
malaria by preventing fertilization in the mosquito. Proc Natl
Acad Sci USA 1980; 77(11): 6797-6799. [34] [34]
Outchkourov NS, Roeffen W, Kaan A, et al. Correctly folded
Pfs48/45 protein of Plasmodium falciparum elicits malaria
transmission-blocking immunity in mice. Proc Natl Acad Sci
USA 2008; 105(11): 4301-4305. [15] (
)
[15]
Kaushal DC, Carter R, Rener J, Grotendorst CA, Miller LH,
Howard
RJ. Monoclonal
antibodies
against
surface
determinants on gametes of Plasmodium gallinaceum block
transmission of malaria parasites to mosquitoes. J Immunol
1983; 131(5): 2557-2562. [35] 5]
Jones CS, Luong T, Hannon M, et al. Heterologous
expression of the C-terminal antigenic domain of the malaria
vaccine
candidate
Pfs48/45
in
the
green
algae
Chlamydomonas reinhardtii. Appl Microbiol Biotechnol 2012;
in press. [16]
Quakyi IA, Carter R, Rener J, Kumar N, Good MF, Miller LH. The 230-kDa gamete surface protein of Plasmodium
falciparum is also a target for transmission-blocking
antibodies. J Immunol 1987; 139(12): 4213-4217. [36] [36]
Fredriksen BN, Grip J. PLGA/PLA micro- and nanoparticle
formulations serve as antigen depots and induce elevated
humoral responses after immunization of Atlantic salmon
(Salmo salar L.). Vaccine 2012; 30(3): 656-667. [17]
Healer J, McGuinness D, Hopcroft P, Haley S, Carter R,
Riley
E. Complement-mediated
lysis
of
Plasmodium
falciparum gametes by malaria-immune human sera is
associated with antibodies to the gamete surface antigen
Pfs230. Infect Immun 1997; 65(8): 3017-3023. [37] [37]
Moon JJ, Suh H, Li AV, Ockenhouse CF, Yadava A, Irvine
DJ. Enhancing humoral responses to a malaria antigen with
nanoparticle vaccines that expand Tfh cells and promote
germinal center induction. 486 Current Molecular Medicine, 2013, Vol. 13, No. 4 Proc Natl Acad Sci USA 2012;
109(4): 1080-1085. [18]
Williamson KC, Keister DB, Muratova O, Kaslow DC. Recombinant Pfs230, a Plasmodium falciparum gametocyte
protein, induces antisera that reduce the infectivity of
Plasmodium
falciparum
to
mosquitoes. Mol
Biochem
Parasitol 1995; 75(1): 33-42. [38] ( )
[38]
Uppada SB, Bhat AA, Sah A, Donthamshetty RN. Enhanced
humoral and mucosal immune responses after intranasal
immunization with chimeric multiple antigen peptide of LcrV
antigen epitopes of Yersinia pestis coupled to palmitate in
mice. Vaccine 2011; 29(50): 9352-9360. [19] [19]
Graves PM, Carter R, Burkot TR, Rener J, Kaushal DC,
Williams JL. Effects of transmission-blocking monoclonal
antibodies on different isolates of Plasmodium falciparum. Infect Immun 1985; 48(3): 611-616. [39] [39]
Fredriksen BN, Saevareid K, McAuley L, Lane ME, Bogwald
J, Dalmo RA. Early immune responses in Atlantic salmon
(Salmo salar L.) after immunization with PLGA nanoparticles
loaded with a model antigen and beta-glucan. Vaccine 2011;
29(46): 8338-8349. ( )
[20]
Williamson KC. Pfs230: from malaria transmission-blocking
vaccine candidate toward function. Parasite Immunol 2003;
25(7): 351-359. [21]
Kocken CH, Jansen J, Kaan AM, et al. Cloning and
expression of the gene coding for the transmission blocking
target antigen Pfs48/45 of Plasmodium falciparum. Mol
Biochem Parasitol 1993; 61(1): 59-68. [40] [40]
dos Santos SA, Zarate-Blades CR, de Sa Galetti FC, et al. A
subunit vaccine based on biodegradable microspheres
carrying rHsp65 protein and KLK protects BALB/c mice
against tuberculosis infection. Hum Vaccin 2010; 6(12):
1047-1053. [22]
Stowers AW, Keister DB, Muratova O, Kaslow DC. A region
of Plasmodium falciparum antigen Pfs25 that is the target of
highly potent transmission-blocking antibodies. Infect Immun
2000; 68(10): 5530-5538. [41] [41]
Xiang SD, Scholzen A, Minigo G, et al. Pathogen recognition
and development of particulate vaccines: does size matter? Methods 2006; 40(1): 1-9. [23]
Dinglasan RR, Kalume DE, Kanzok SM, et al. Disruption of
Plasmodium falciparum development by antibodies against a
conserved mosquito midgut antigen. Proc Natl Acad Sci USA
2007; 104(33): 13461-13466. [42] ( )
[42]
Cruz LJ, Tacken PJ, Fokkink R, et al. Targeted PLGA nano-
but not microparticles specifically deliver antigen to human
dendritic cells via DC-SIGN in vitro. J Control Release 2010;
144(2): 118-126. (
)
[24]
Kaslow DC. Transmission-blocking vaccines. Chem Immunol
2002; 80: 287-307. [43] ( )
[43]
De Koker S, Lambrecht BN, Willart MA, et al. Designing
polymeric particles for antigen delivery. Chem Soc Rev 2011;
40(1): 320-339. REFERENCES Transmission-blocking vaccines: uses and
current status of development. Int J Parasitol 1997; 27(2):
183-189. [11] Mendis KN, David PH, Carter R. Human immune responses
against sexual stages of malaria parasites: considerations for
malaria vaccines. Int J Parasitol 1990; 20(4): 497-502. [37] [47] 486 Current Molecular Medicine, 2013, Vol. 13, No. 4 [25]
Sinden RE, Carter R, Drakeley C, Leroy D. The biology of
sexual development of Plasmodium: the design and
implementation of transmission-blocking strategies. Malar J
2012; 11: 70. [44] [44]
Tiwari S, Goyal AK, Mishra N, et al. Development and
characterization of novel carrier gel core liposomes based
transmission blocking malaria vaccine. J Control Release
2009; 140(2): 157-165. [26]
Coutinho-Abreu
IV,
Ramalho-Ortigao
M. Transmission
blocking vaccines to control insect-borne diseases: a review. Mem Inst Oswaldo Cruz 2010; 105(1): 1-12. [45] [45]
Moon JJ, Suh H, Polhemus ME, Ockenhouse CF, Yadava A,
Irvine
DJ. Antigen-displaying
lipid-enveloped
PLGA
nanoparticles as delivery agents for a Plasmodium vivax
malaria vaccine. PLoS One 2012; 7(2): e31472. [27]
Arevalo-Herrera M, Solarte Y, Marin C, et al. Malaria
transmission blocking immunity and sexual stage vaccines
for interrupting malaria transmission in Latin America. Mem
Inst Oswaldo Cruz 2011; 106 (Suppl 1): 202-211. [46] ( )
[46]
Fifis T, Gamvrellis A, Crimeen-Irwin B, et al. Size-dependent
immunogenicity: therapeutic and protective properties of
nano-vaccines against tumors. J Immunol 2004; 173(5):
3148-3154. [28]
Bousema T, Drakeley C. Epidemiology and infectivity of
Plasmodium falciparum and Plasmodium vivax gametocytes
in relation to malaria control and elimination. Clin Microbiol
Rev 2011; 24(2): 377-410. [47] [47]
Hardy CL, LeMasurier JS, Belz GT, et al. Inert 50-nm
polystyrene nanoparticles that modify pulmonary dendritic
cell function and inhibit allergic airway inflammation. J
Immunol 2012; 188(3): 1431-1441. [29]
Pradel G. Proteins of the malaria parasite sexual stages:
expression, function and potential for transmission blocking
strategies. Parasitology 2007; 134 (Pt 14): 1911-1929. [48] ( )
[48]
Fifis T, Mottram P, Bogdanoska V, Hanley J, Plebanski M. Short peptide sequences containing MHC class I and/or
class II epitopes linked to nano-beads induce strong
immunity and inhibition of growth of antigen-specific tumour
challenge in mice. Vaccine 2004; 23(2): 258-266. [30]
Tachibana M, Sato C, Otsuki H, et al. Plasmodium vivax
gametocyte protein Pvs230 is a transmission-blocking
vaccine candidate. Vaccine 2012; 30(10): 1807-1812. [31]
Tachibana M, Wu Y, Iriko H, et al. N-terminal prodomain of
Pfs230 synthesized using a cell-free system is sufficient to
induce
complement-dependent
malaria
transmission- Single-Dose Microparticle Delivery Current Molecular Medicine, 2013, Vol. 13, No. 4 487 [60] [60]
Bousema JT, Drakeley CJ, Sauerwein RW. Sexual-stage
antibody responses to P. falciparum in endemic populations. Curr Mol Med 2006; 6(2): 223-229. [49]
Moreno
R,
Jiang
L,
Moehle
K,
et
al. 486 Current Molecular Medicine, 2013, Vol. 13, No. 4 Exploiting
conformationally
constrained
peptidomimetics
and
an
efficient human-compatible delivery system in synthetic
vaccine design. Chembiochem 2001; 2(11): 838-843. [61] [61]
The malERA Consultative Group on Vaccines. A Research
Agenda for Malaria Eradication: Vaccines. PLoS Med 2011;
8(1): e1000398. [50]
Mao HQ, Zhao
Z, Dang W, et al. Biodegradable
poly(phosphoester)s. In: Encyclopedia of Controlled Drug
Delivery. Mathiowitz E, editor. New York, NY: Johns Wiley &
Sons, Inc., 1999: 45-60. [62] [62]
Bustamante PJ, Woodruff DC, Oh J, Keister DB, Muratova
O, Williamson KC. Differential ability of specific regions of
Plasmodium falciparum sexual-stage antigen, Pfs230, to
induce malaria transmission-blocking immunity. Parasite
Immunol 2000; 22(8): 373-380. [51]
McNeela EA, Lavelle EC. Recent advances in microparticle
and nanoparticle delivery vehicles for mucosal vaccination. Curr Top Microbiol Immunol 2012; 354: 75-99. [63] p
[52]
Zhao Z, Wang J, Mao HQ, Leong KW. Polyphosphoesters in
drug and gene delivery. Adv Drug Deliv Rev 2003; 55(4):
483-499. [63]
Vincent AA, Fanning S, Caira FC, Williamson KC. Immunogenicity of malaria transmission-blocking vaccine
candidate, y230.CA14 following crosslinking in the presence
of tetanus toxoid. Parasite Immunol 1999; 21(11): 573-581. [53]
Tyagi RK, Garg NK, Sahu T. Vaccination Strategies against
Malaria: novel carrier(s) more than a tour de force. J Control
Release 2012; 162(1): 242-254. [64] [64]
Qian F, Wu Y, Muratova O, et al. Conjugating recombinant
proteins to Pseudomonas aeruginosa ExoProtein A: a
strategy for enhancing immunogenicity of malaria vaccine
candidates. Vaccine 2007; 25(20): 3923-3933. [54]
Kaba SA, Brando C, Guo Q, et al. A nonadjuvanted
polypeptide
nanoparticle
vaccine
confers
long-lasting
protection against rodent malaria. J Immunol 2009; 183(11):
7268-7277. [65] Wu Y, Ellis RD, Shaffer D, et al. Phase 1 trial of malaria
transmission blocking vaccine candidates Pfs25 and Pvs25
formulated with montanide ISA 51. PLoS One 2008; 3(7):
e2636. [55]
Jilek S, Merkle HP, Walter E. DNA-loaded biodegradable
microparticles as vaccine delivery systems and their
interaction with dendritic cells. Adv Drug Deliv Rev 2005;
57(3): 377-390. [66] [66]
Kubler-Kielb J, Majadly F, Wu Y, et al. Long-lasting and
transmission-blocking activity of antibodies to Plasmodium
falciparum elicited in mice by protein conjugates of Pfs25. Proc Natl Acad Sci USA 2007; 104(1): 293-298. [56]
Jennings GT, Bachmann MF. Designing recombinant
vaccines with viral properties: a rational approach to more
effective vaccines. Curr Mol Med 2007; 7(2): 143-155. [67] [67]
Wu Y, Przysiecki C, Flanagan E, et al. Received: December 13, 2012 Revised: January 08, 2013
Accepted: January 12, 2013 [61] [66] 486 Current Molecular Medicine, 2013, Vol. 13, No. 4 Sustained high-titer
antibody responses induced by conjugating a malarial
vaccine candidate to outer-membrane protein complex. Proc
Natl Acad Sci USA 2006; 103(48): 18243-18248. ( )
[57]
Hansen S, Lehr CM. Nanoparticles for transcutaneous
vaccination. Microb Biotechnol 2012; 5(2): 156-167. [58]
Combadiere B, Mahe B. Particle-based vaccines for
transcutaneous vaccination. Comp Immunol Microbiol Infect
Dis 2008; 31(2-3): 293-315. [68] 68]
Farrance CE, Chichester JA, Musiychuk K, et al. Antibodies
to plant-produced Plasmodium falciparum sexual stage
protein Pfs25 exhibit transmission blocking activity. Hum
Vaccin 2011; 7 Suppl: 191-198. (
)
[59]
PATH-Malaria Vaccine Initiative. http://www.malariavaccine. org/rd-collaborations.php. Access November 2012. Received: December 13, 2012 Revised: January 08, 2013
Accepted: January 12, 2013
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Regulatory Role of Retinoic Acid in Male Pregnancy of the Seahorse
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Regulatory Role of Retinoic Acid
in Male Pregnancy of the Seahorse Chunyan Li,1,2 Yongxin Li,2,3 Geng Qin,1 Zelin Chen,1 Meng Qu,1 Bo Zhang,1 Xue Han,1 Xin Wang,1 Pei-yuan Qian,2,4,* and Qiang Lin1,2,*
1CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Innovation of South China Sea Ecology and Environmental
Engineering, Chinese Academy of Sciences, 510301 Guangzhou, China
2Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), 511458 Guangzhou, China
3Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China
4Department of Ocean Science and Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Hong Kong University of Science
and Technology, Kowloon, Hong Kong, China
*Correspondence: linqiang@scsio.ac.cn (Q.L.); boqianpy@ust.hk (P.-y.Q.)
Received: June 23, 2020; Accepted: October 12, 2020; Published: November 25, 2020; https://doi.org/10.1016/j.xinn.2020.100052
ª 2020 The Author(s). Regulatory Role of Retinoic Acid
in Male Pregnancy of the Seahorse
Chunyan Li,1,2 Yongxin Li,2,3 Geng Qin,1 Zelin Chen,1 Meng Qu,1 Bo Zhang,1 Xue Han,1 Xin Wang,1 Pei-yuan Qian,2,4,* and Qiang Lin1,2,*
1CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Innovation of South China Sea Ecology and Environmental
Engineering, Chinese Academy of Sciences, 510301 Guangzhou, China
2Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), 511458 Guangzhou, China
3Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China
4Department of Ocean Science and Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Hong Kong University of Science
and Technology, Kowloon, Hong Kong, China
*Correspondence: linqiang@scsio.ac.cn (Q.L.); boqianpy@ust.hk (P.-y.Q.)
Received: June 23, 2020; Accepted: October 12, 2020; Published: November 25, 2020; https://doi.org/10.1016/j.xinn.2020.100052
ª 2020 The Author(s).
Report Regulatory Role of Retinoic Acid
in Male Pregnancy of the Seahorse
Chunyan Li,1,2 Yongxin Li,2,3 Geng Qin,1 Zelin Chen,1 Meng Qu,1 Bo Zhang,1 Xue Han,1 Xin Wang,1 Pei-yuan Qian,2,4,* and Qiang Lin1,2,*
1CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Innovation of South China Sea Ecology and Environmental
Engineering, Chinese Academy of Sciences, 510301 Guangzhou, China
2Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), 511458 Guangzhou, China
3Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China
4Department of Ocean Science and Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Hong Kong University of Science
and Technology, Kowloon, Hong Kong, China
*Correspondence: linqiang@scsio.ac.cn (Q.L.); boqianpy@ust.hk (P.-y.Q.)
Received: June 23, 2020; Accepted: October 12, 2020; Published: November 25, 2020; https://doi.org/10.1016/j.xinn.2020.100052
ª 2020 The Author(s). Report The Innovation Regulatory Role of Retinoic Acid
in Male Pregnancy of the Seahorse Chunyan Li,1,2 Yongxin Li,2,3 Geng Qin,1 Zelin Chen,1 Meng Qu,1 Bo Zhang,1 Xue Han,1 Xin Wang,1 Pei-yuan Qian,2,4,* and Qiang Lin1,2,*
1CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Innovation of South China Sea Ecology and Environmental
Engineering, Chinese Academy of Sciences, 510301 Guangzhou, China
2Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), 511458 Guangzhou, China
3Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China
4Department of Ocean Science and Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Hong Kong University of Science
and Technology, Kowloon, Hong Kong, China Chunyan Li,1,2 Yongxin Li,2,3 Geng Qin,1 Zelin Chen,1 Meng Qu,1 Bo Zhang,1 Xue Han,1 Xin Wang,1 Pei-yuan Qian,2,4,* and Qiang Lin1,2,*
1CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Innovation of South China Sea Ecology and Environmental
Engineering, Chinese Academy of Sciences, 510301 Guangzhou, China
2Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), 511458 Guangzhou, China
3Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China Bio-Resources and Ecology, South China Sea Institute of Oceanology, Innovation of South China Sea Ecology and Environmenta
ces, 510301 Guangzhou, China 2Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), 511458 Guangzhou, China
3Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China
4Department of Ocean Science and Hong Kong Branch of Southern Marine Science and Engineering Guangdong L 2Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), 511458 Guangzhou, China
3Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China 2Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), 511458 Guangzhou, China
3Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China
4Department of Ocean Science and Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Hong Kong University of Science
and Technology, Kowloon, Hong Kong, China 3Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China
4Department of Ocean Science and Hong Kong Branch of Southern Marine Science and Engineering Guangdong Labo
and Technology, Kowloon, Hong Kong, China y
y
g
g
g
g
g
g
4Department of Ocean Science and Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Hong Kong University of Science
and Technology, Kowloon, Hong Kong, China g Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Hong Kong University of Science
Chi 4Department of Ocean Science and Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laborator
and Technology, Kowloon, Hong Kong, China Received: June 23, 2020; Accepted: October 12, 2020; Published: November 25, 2020; https://doi.org/10.1016/j.xinn.2020.100052
ª 2020 The Author(s). PUBLIC SUMMARY - Male seahorses epitomize the unique characteristic of male pregnancy - Retinoic acid (RA) plays key regulatory roles in brood pouch formation and
pregnancy - RA could influence the brood pouch formation by regulating steroid hormone
metabolism - RA may contribute to the successful gestation by regulating antioxidant defense ll ll www.cell.com/the-innovation The Innovation Changes in Genes and Metabolites during Brood Pouch Formation and
Pregnancy The results of two comparisons, that is, for pouch formation (unformed
[UF] versus newly formed [NF]) and pregnancy (pregnant [PG] versus [NF]),
of the lined seahorses (H. erectus) at UF, NF, and PG stages are shown in Fig-
ure 1A. In total, we quantified and annotated 21,232 transcripts and 210 me-
tabolites (Tables S1–S4 and S13). Principal-component analysis (PCA) of
both transcripts and metabolites showed large variations among seahorses
at different stages of brood pouch development (Figures 1B and 1C). A total
of 141 differentially expressed genes (DEGs) and 73 significantly different
metabolites (SDMs) were identified in pouch formation analysis (Figure 1D;
Tables S5 and S6), while 2,533 DEGs and 121 SDMs were identified in preg-
nancy comparison (Figure 1E; Tables S14 and S15). INTRODUCTION Syngnathid fish possess the unique reproductive strategy of male preg-
nancy, in which the male possesses an embryo-incubating structure called
a brood pouch.1,2 Both the structure and position of the brood pouch vary
substantially among syngnathids and pouch ultrastructure and functions
differ even among closely related species.3 Among syngnathid fish, sea-
horses (Hippocampus) not only have the most complex pouch structure
but also experience the most significant physiological changes during em-
bryo incubation.4 A brood pouch is not present in juvenile seahorses but
rather appears with growth in male seahorses.5 Male seahorses first form
a baggy structure from the primordium, followed by differentiation and estab-
lishment of brood pouch-specific tissues, which ultimately form a pouch with
well-developed blood vessels capable of incubating embryos.2 Seahorses
have placenta-like tissues that facilitate pregnancy, which are similar to the
placental structure in mammals.4 Moreover, the significant morphological
and functional changes of the brood pouch that occur during gestation are
equivalent to those in the uterus of the mammals.6 For example, it has
been suggested that heavily vascularized tissues in the brood pouch are
related to gas exchange between the embryos and the father,7 while abun-
dant C-type lectins present during early incubation mayhave an important im-
mune protective role for developing embryos.8 Although a few studies have
sought to identify specific genes and pathways associated with pouch func- y
(
)
The expression levels of most DEGs were higher in seahorses in the NF
stage and lower in the PG stage (Figure 2A). Two pathways (retinol meta-
bolism and ABC transporters) were significantly enriched for both pouch for-
mation and pregnancy (p < 0.01, Figure 2B; Tables S7–S10). Meanwhile,
SDMs exhibited diverse changes (Figure 2C). Specifically, aminoacyl-tRNA
biosynthesis, nitrogen metabolism, as well as phenylalanine, tyrosine, and
tryptophan biosynthesis were the three significantly enriched SDM pathways
identified for both pouch formation and pregnancy (p < 0.05, Figure 2D; Ta-
bles S16 and S17). Of the 32 co-enriched pathways for transcriptome and
metabolome analyses, 6 pathways, including aminoacyl-tRNA biosynthesis,
were co-enriched for both pouch formation and pregnancy (Figure 2E; Tables
S18 and S19). In particular, five SDMs and ten DEGs were mapped in the ste-
roid hormone metabolism pathway (Figure 2F; Table S20). Regulatory Role of Retinoic Acid
in Male Pregnancy of the Seahorse This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). tion,9,10 a comprehensive analysis of the regulatory gene and metabolite net-
works responsible for control of brood pouch formation and pregnancy has
not yet been conducted. Seahorses epitomize the exuberance of evolution. They have the unique
characteristic of male pregnancy, which includes the carrying of many
embryos in a brood pouch that incubates and nourishes the embryos,
similar to the mammalian placenta. However, the regulatory networks
underlying brood pouch formation and pregnancy remain largely un-
known. In this study, comparative transcriptomic and metabolomic
profiling on the lined seahorse Hippocampus erectus, with unformed,
newly formed, and pregnant brood pouches identified a total of 141
and 2,533 differentially expressed genes together with 73 and 121
significantly differential metabolites related to brood pouch formation
and pregnancy, respectively. Specifically, integrative omics analysis re-
vealed that retinoic acid (RA) synthesis and signaling pathway played
essential roles in the formation of the brood pouch and pregnancy. RA
might function upstream of testosterone and progesterone, thereby
directly influencing brood pouch formation by regulating the expression
of fshr and cyp7a1. Our results also revealed that RA regulates antioxi-
dant defenses, particularly during male pregnancy. Alternatively, preg-
nancy caused a consistent decrease in RA, canthaxanthin, astaxanthin,
and glutathione synthetase, and an increase in susceptibility to oxidative
stress, which may balance brood pouch development and reproduction
in seahorses and pave the way to successful gestation. Retinoic acid (RA) has multiple functions in a wide range of biological pro-
cesses, particularly in early organogenesis induction and patterning,11–14 as
well as in innate and adaptive immunity.15 RA receptors are involved in the
regulation of androgen biosynthesis,16,17 which is necessary for the produc-
tion of seahorse pouches.4 Due to the pleiotropic roles of RA in the immune
system,15 any deviation from the required concentration of RA can result in
oxidative stress, while impacting normal growth and differentiation.18 Howev-
er, the effects of RA signaling in syngnathids remain unclear despite the
diverse brooding types and structures present in this family. In this study,
we aimed to investigate the molecular mechanism underlying brood pouch
development by combining both transcriptomic and metabolomic analyses
of the lined seahorse (Hippocampus erectus) at different pouch develop-
mental stages with specific focus on the regulatory role of RA in brood pouch
formation and pregnancy. Figure 1. Transcriptome and Metabolome Mapping of
Transcripts and Metabolites over Different Brood Pouch
Developmental Stages in Seahorses (A) Illustration of un-
formed (UF), newly formed (NF), and pregnant (PG) brood
pouches of seahorses. Two comparisons for the brood
pouch formation and pregnancy are labeled. Principal-
component analysis (PCA) for RNA-seq (B) and metab-
olome (C) data. Each point represents one biological repli-
cate and points with different colors represent seahorses at
different
pouch
developmental
stages.
Volcano
plots
showing the relative abundance of transcripts (D) and me-
tabolites (E) in comparison for pouch formation and preg-
nancy. Important transcripts and metabolites are marked. Potential Multiple Functions of RA in the Development of the Retinol metabolism was significantly enriched in DEGs identified during
both pouch formation and pregnancy. In fact, it was also the most signifi-
cantly enriched pathway during pouch formation (Figure 2B; Tables S9 and
S10). Expression level of six genes related to RA synthesis and signaling
consistently decreased during pregnancy (Figure 3A; Table S11). In addition,
54 RA target genes were differentially expressed over different stages of
pouch formation and pregnancy (Table S11). Expression of most RA target
genes consistently decreased during pregnancy, while expression of several
genes were increased in UF seahorses (e.g., cholesterol 7-alpha-monooxyge-
nase [cyp7a1]) and PG (e.g., cytochrome b [558] subunit beta [cybb]) brood
pouches (Table S11). These RA target genes participate in diverse intercon-
nected functions, including steroid hormone metabolism (e.g., cyp7a1 and
follicle-stimulating hormone receptor [fshr]19), tissue remodeling (e.g.,
collagen alpha-1 (IV) chain [col4a1],20 collagen alpha-2 (IV) chain [col4a2],21
and keratin 18 [krt18]22), organ development (e.g., fshr23 and paired-like ho-
meodomain 2 [pitx2]24), and immune defense (e.g., cybb25 and transient re- INTRODUCTION Upstream metab-
olites of the pathway (prolactin [PRL] and progesterone [PR]) were abundant
in seahorses in the UF stage, while downstream metabolites of the pathways ll The Innovation 1, 100052, November 25, 2020 1 The Innovation Report A A A
B
D
E
C
Figure 1. Transcriptome and Metabolome Mapping of
Transcripts and Metabolites over Different Brood Pouch
Developmental Stages in Seahorses (A) Illustration of un-
formed (UF), newly formed (NF), and pregnant (PG) brood
pouches of seahorses. Two comparisons for the brood
pouch formation and pregnancy are labeled. Principal-
component analysis (PCA) for RNA-seq (B) and metab-
olome (C) data. Each point represents one biological repli-
cate and points with different colors represent seahorses at
different
pouch
developmental
stages. Volcano
plots
showing the relative abundance of transcripts (D) and me-
tabolites (E) in comparison for pouch formation and preg-
nancy. Important transcripts and metabolites are marked. (testosterone [TE], 5-beta-androstane-3,17-dione and 5-alpha-pregnane-3,20-
dione) were abundant in seahorses in the PG stage (Figure 2E). Unlike meta-
bolic regulation, most DEGs participating in steroid hormone metabolism
showed the highest expression levels in seahorses in the NF stage. ceptor potential c
Intriguingly, five g
RA target genes,
pregnancy (Figu
B
D
E
C B
C B C D E D E D D (testosterone [TE], 5-beta-androstane-3,17-dione and 5-alpha-pregnane-3,20-
dione) were abundant in seahorses in the PG stage (Figure 2E). Unlike meta-
bolic regulation, most DEGs participating in steroid hormone metabolism
showed the highest expression levels in seahorses in the NF stage. (testosterone [TE], 5-beta-androstane-3,17-dione and 5-alpha-pregnane-3,20-
dione) were abundant in seahorses in the PG stage (Figure 2E). Unlike meta-
bolic regulation, most DEGs participating in steroid hormone metabolism
showed the highest expression levels in seahorses in the NF stage. ceptor potential cation channel subfamily M member 2 [trpm2]26) (Figure 3B). Intriguingly, five genes involved in RA synthesis and signaling, as well as 18
RA target genes, were also differentially expressed in H. abdominalis during
pregnancy (Figure 3A; Table S12). Four RA target genes (fshr, col4a2,
col4a1, and krt18) were differentially expressed during pregnancy in both
H. erectus and H. abdominalis (Tables S11 and S12). In addition, data from
a previous publication suggest that genes involved in RA signaling and syn-
thesis tended to have higher expression levels in the pituitary and gonad,
whereas RA target genes tended to have higher expression in the brood
pouch (Figure S2).11 Antioxidant Defenses Regulated by RA during Brood Pouch Formation
and Pregnancy
27 Canthaxanthin, acting as an antioxidant,27 showed the highest fold change
(7-fold) in seahorses in the NF stage compared with the UF stage, whereas
ophthalmic acid, an oxidant,28 showed the greatest (5.9-fold) change in sea-
horses in the UF stage compared with the NF stage (Figure 4A). Compared
with seahorses in the NF stage, pregnant seahorses had lower levels of an-
tioxidants (canthaxanthin, 19.1-fold; astaxanthin, 50.3-fold) (Figure 4A). Expression of glutathione S-transferase (GST) also significantly decreased
in pregnant seahorses (Table S6). Ophthalmic acid and canthaxanthin showed reverse correlations with the
same DEGs identified during pouch formation (Figure 4B; Table S21). During The Innovation 1, 100052, November 25, 2020 The Innovation 1, 100052, November 25, 2020 www.cell.com/the-innovation 2 The Innovation Report Report A
B
C
D
E
F
re 2. Expression Pattern and Enrichment Analysis of DEGs and SDMs over Different Pouch Developmental Stages (A–D) Heatmaps of DEGs (A) and S
ficantly enriched pathways for DEGs (p < 0.01) (B) and SDMs (p < 0.05) (D). Pink and green bar charts in (B) represent pathways enriched for brood pouch
pregnancy processes, respectively; while the blue and orange bar charts in (D) indicate pathways enriched for SDMs identified for pouch formation and p
esses, respectively. (E) Co-enriched pathways for both DEGs and SDMs. Co-enriched pathways for both brood pouch formation and pregnancy are la
nging patterns of DEGs and SDMs involved in steroid hormone metabolism. The left graph shows a simplified pathway of steroid hormone metabolism and
h h
h
i
DEG
d SDM
ti i
ti
i
t
id h
t b li B A C D D C E F E Figure 2. Expression Pattern and Enrichment Analysis of DEGs and SDMs over Different Pouch Developmental Stages (A–D) Heatmaps of DEGs (A) and SDMs (C). Significantly enriched pathways for DEGs (p < 0.01) (B) and SDMs (p < 0.05) (D). Pink and green bar charts in (B) represent pathways enriched for brood pouch formation
and pregnancy processes, respectively; while the blue and orange bar charts in (D) indicate pathways enriched for SDMs identified for pouch formation and pregnancy
processes, respectively. (E) Co-enriched pathways for both DEGs and SDMs. Co-enriched pathways for both brood pouch formation and pregnancy are labeled. (F)
Changing patterns of DEGs and SDMs involved in steroid hormone metabolism. Antioxidant Defenses Regulated by RA during Brood Pouch Formation
and Pregnancy
27 The left graph shows a simplified pathway of steroid hormone metabolism and the right
graph shows changes in DEGs and SDMs participating in steroid hormone metabolism. ll The Innovation 1, 100052, November 25, 2020 3 A
B
Report The Innovation A
B A
B
Figure 3. Role of RA in Seahorse Brood Pouch Formation
and Pregnancy Detailed gene expression data are shown
in Tables S11 and S12. (A) Role of RA synthesis and signaling during pregnancy of
two seahorse species, H. erectus and H. abdominalis. RA
synthesis
and
signaling
pathway
is
simplified
from
Duester.11 Upward and downward arrows indicate upregu-
lation and downregulation of genes and metabolites during
pouch pregnancy, respectively. Each arrow represents one
gene. (B) Summarized functions of RA target genes in seahorse
brood pouch formation and pregnancy. RA might function
upstream of testosterone and progesterone by regulating
the expression of fshr and cyp7a1. Oval circles indicate RA
target genes that were significantly differentially expressed
during pouch formation or pregnancy and three rectangles
indicate diverse functions that may be regulated by RA. A Figure 3. Role of RA in Seahorse Brood Pouch Formation
and Pregnancy Detailed gene expression data are shown
in Tables S11 and S12. A
B (A) Role of RA synthesis and signaling during pregnancy of
two seahorse species, H. erectus and H. abdominalis. RA
synthesis
and
signaling
pathway
is
simplified
from
Duester.11 Upward and downward arrows indicate upregu-
lation and downregulation of genes and metabolites during
pouch pregnancy, respectively. Each arrow represents one
gene. g
(B) Summarized functions of RA target genes in seahorse
brood pouch formation and pregnancy. RA might function
upstream of testosterone and progesterone by regulating
the expression of fshr and cyp7a1. Oval circles indicate RA
target genes that were significantly differentially expressed
during pouch formation or pregnancy and three rectangles
indicate diverse functions that may be regulated by RA. Antioxidant Defenses Regulated by RA during Brood Pouch Formation
and Pregnancy
27 B B B reported,13,30,31 and patterned expression of RA-related genes may play spe-
cific roles (e.g., tissue remodeling, organ development, and immune defense)
in seahorse brood pouch development.9 For example, PITX2, a transcription
factor known for its role in left-right symmetry,24,32 may control seahorse
brood pouch symmetry by regulating the Wnt signaling and collagen-related
genes.33 Meanwhile, CYBB, a super-oxide-generating enzyme conserved in
most eukaryotic groups, is involved in antioxidant immune defense as it is
activated in response to pathogens.25 Diverse and pleiotropic functions of
genes in the brood pouch may be responsible for their diverse expression
pattern (Table S11). pregnancy, both canthaxanthin and astaxanthin were core metabolites of the
regulatory network and showed a positive correlation with many genes,
particularly those participating in transportation, including ATP-binding
cassette superfamily G member 2 (abcg2) (Figure4C; Table S22). Meanwhile,
canthaxanthin showed a positive correlation with RA receptor responder pro-
tein 3 (rarres3), and astaxanthin showed a positive correlation with UDP glu-
curonosyltransferase 2 family, polypeptide A2 (ugt2a2) and beta,beta-caro-
tene 15,150-dioxygenase (bco1) during pregnancy (Figure 4C). Rarres3,
ugt2a2, and bco1 were all involved in RA metabolism and signaling (Tables
S10 and S11). Contradictory results have been reported regarding the relationship be-
tween the RA signaling pathway and steroid hormone metabolism. On the
one hand, conditional deletion of aldehyde dehydrogenase 1 family member
A2 (aldh1a2), the enzyme that metabolizes the vitamin A-intermediate retinal-
dehyde into RA, results in embryonic pituitary dysmorphology and alters hor-
mone expression.34 On the other hand, TE has been reported as the key up-
stream signal that controls RA biogenesis by promoting the expression of
genes encoding RA synthesizing enzymes.35 Our results indicate that RA
may function upstream of steroid hormone metabolism in seahorses as
demonstrated by the consistent decrease in levels of upstream molecules
of steroid hormone metabolism with RA during pregnancy (Figures 2F and
3A). Therefore, PRL, PR, and TE play important roles in the maintenance of
brood pouch structure and function of brood pouch.4,36 Whether RAregulates
pouch development by steroid hormones requires further investigation. DISCUSSION (B and C) Significant correlations between significantly different antioxidants and DEGs (correlation coefficient >0.9 and p < 0.01). Fonts of antioxidants and genes involved in transporting or related to RA are in bold. Metabolites and genes are indicated with blue and green background colors,
respectively. Blue and green lines indicate significant positive and negative correlations, respectively. (B) Correlations between canthaxanthin and ophthalmic acid with
other DEGs identified for brood pouch formation. (C) Correlations between canthaxanthin (left) and astaxanthin (right) with other DEGs identified during pregnancy. ca4 grik4 Figure 4. Interactional Network of Antioxidant Defense in Seahorse Brood Pouch Development (A) Boxplots showing the changing tendency of one oxidant and two
antioxidants over different pouch developmental stages in seahorses. Each point represents one replicate (n = 12). Center line, median; box limits, upper and lower quartiles;
whiskers, 1.53 the interquartile range. (B and C) Significant correlations between significantly different antioxidants and DEGs (correlation coefficient >0.9 and p < 0.01). Fonts of antioxidants and genes involved in transporting or related to RA are in bold. Metabolites and genes are indicated with blue and green background colors,
respectively. Blue and green lines indicate significant positive and negative correlations, respectively. (B) Correlations between canthaxanthin and ophthalmic acid with
other DEGs identified for brood pouch formation. (C) Correlations between canthaxanthin (left) and astaxanthin (right) with other DEGs identified during pregnancy. ity, and the metabolic rate of pregnant seahorses increases from 10% to 52%
over pre-gravid levels.42 The increasing metabolic rate could induce oxidative
stress during pregnancy by consuming antioxidants and enzymes (Fig-
ure 4A), which may weaken the immune defense43 and facilitate successful
gestation. The trade-off between immunological tolerance and embryo rejec-
tion accompanies the evolution of unique male pregnancy.44 Accordingly, the
increased susceptibility to oxidative stress in seahorses during pregnancy
might result from the necessity to strike a balance between reproduction
and survival that contributes to seahorse reproduction.45,46 intermediate filament family of cytoskeletal proteins;22 all three of which may
be involved in brood pouch tissue remodeling. Our results may indicate the
various roles of RA in seahorse brood pouch formation and pregnancy by
regulating downstream genes with diverse functions. DISCUSSION In this study, we quantified transcripts and metabolites over different sea-
horse brood pouch developmental stages and suggested multiple roles of RA
in pouch formation and pregnancy by regulating steroid hormone meta-
bolism and antioxidant defense. In seahorses, interruption of PRL synthesis
leads to disruption of brooding tissues and spontaneous abortions during
pregnancy as PRL production is essential for PR and TE secretion.4 However,
no study has examined changes in sex steroid hormones over different
stages of brood pouch development or how they affect pouch development
in seahorses. Here, we detected high levels of PRL and PR in seahorses
before pregnancy and high levels of TE during pregnancy (Figure 2F). Further-
more, we indicated the different roles of upstream and downstream metab-
olites of steroid hormone metabolism in seahorse brood pouch formation
and pregnancy (Figure 2F). In addition, we observed nonsynchronous regula-
tion at the transcriptional and metabolic levels (Figure 2F). Owing to their
diverse roles, the concentrations of sex steroids fluctuate during the breeding
cycle of the lined seahorses.29 Future research on specific sex hormones
may help to better illustrate the complex roles of steroid hormone meta-
bolism in brood pouch development. pouch development by steroid hormones requires further investigation. DEGs that participate in RA signaling and pathway, or that are regulated by
RA, were also identified in H. abdominalis, suggesting a conserved role of RA
in Hippocampus brood pouch development (Figure 3A; Tables S11 and S12). Of the four RA target genes identified in comparison of the pregnancy pro-
cess in both H. erectus and H. abdominalis, fshr was reportedly expressed
during the luteal phase in the secretory endometrium of the mammalian
uterus23 and may be involved in the formation of placenta-like tissue during
pregnancy.37 FSHR may also be involved in pouch formation by affecting TE
production.19 Meanwhile, COL4A1 and COL4A2 are the major structural com-
ponents of basement membranes20,21 and KRT18 is a member of the In addition to the roles of steroid hormones, our results may indicate that
RA regulates 54 downstream target genes with diverse functions, indicating
its potential roles in seahorse brood pouch development (Figure 3). DISCUSSION In fact,
involvement of RA in teleost development and regeneration has been widely The Innovation 1, 100052, November 25, 2020 www.cell.com/the-innovation www.cell.com/the-innovation 4 The Innovation Report nme2
kirrel3
hgf
plxna1
igfbp1
dlgap4
wnt9a
clock
zmynd8
pdp1
epn3
il17re
nalcn
gpm6a
pex11b
sv2a
dnm1
map7d2
six1b
syt1
methocarbamol
ndrg1
methionine sulfoxide
nmes1
cacna1i
adenosine 5'-monophosphate
astaxanthin
shank1
cdo1
cecr1a
bco2
hykk
cep170b
grik2
pdcd4
aatk
gdi1
12-hydroperoxy-9z,13e,15-octadecatrienoic acid
sema6b
scg5
camsap2
cngb1
adprhl1
ppfia2
dhcr24
fosl1
ugt2a2
cdh24
adgrb2
pcsk2
syp
ncan
dnaaf5
tmem260
epb41l3
tnip1
mapt
vdac2
oprm1
abcg2
cebpb
gabrb2
pcdhgc5
col25a1
grik4
sbk2
rin2
osbpl7
kbp
acot2
olfr139
erich3
scn8a
pcdhac2
grm3
gria3
cpne2
ampd2
rap1gap
mdga2
nav1
gmeb1
col28a1
pcbp4
pfkm
uridine diphosphate-n-acetylglucosamine
efna2
43347
kcnj11
tnmd
taurocholic acid
maltotetraose
fry
slc27a2
cacna2d1
shank3
srl
tnrc6c
fam184b
grin2a
qpct
sgcb
hsd17b12a
chrng
wfikkn2
egfl6
ciart
tubb2a
pcdhga11
kcnab1
kcnma1
myo16
rab3c
cga
ptprn
ank3
sepiapterin
d-norvaline
spon1
5-deoxy-5'-methylthioadenosine
cxxc4
adora1
ldlr
chd4
slc22a23
ttbk1
zfhx3
sh2d3c
rtn1
slc5a3
hapln1
elp6
gfi1b
gyc88e
acadl
grin2b
trpm3
rfx1
pomcb
guca2a
gpt2l
zearalenone
camkv
arhgef28
igsf9
argininosuccinic acid
map2
sgip1
cdk5r1
gss
lmtk3
gnao1
nrxn2
tnk2
lrp1b
kiaa1549
slc4a3
astn1
kcnj12
napb
slc1a2
l-cysteine-glutathione gisulfide
celsr3
unc80
atp1a1
sez6
ank1
1-methylguanosine
gad2
map1a
sphkap
tubb4b
tenm4
csrp3
pcdh15
prickle2
caskin1
grin1
trpc5
akap6
map6
nup210
syn2
cpne5
srcin1
stox2
erc2
zfhx4
dlgap2
pg(16:0/0:0)[u]
serpine1
lect1
scn2a
ano10
gltscr1l
prex2
lta4h
tkt
cacna2d2
rarres3
chd3
ptch1
abcc4
ppm1b
mgat5b
klhl41b
wdr31
klhl33
acsf2
slc2a9
adnp2
il22ra2
canthaxanthine
8-hydroxy-2-deoxyguanosine
slc22a3
amacr
kcng2
siah1
ca4
hs3st3b1
thap4
mansc1
arhgef10
0e+00
2e−04
4e−04
6e−04
Stage
opthalmic_acid
0e+00
5e−05
1e−04
Stage
canthaxanthine
0e+00
2e−05
4e−05
Stage
astaxanthin
lect1
scn2a
ano10
gltscr1l
prex2
lta4h
tkt
cacna2d2
rarres3
chd3
ptch1
abcc4
ppm1b
mgat5b
klhl41b
wdr31
klhl33
acsf2
slc2a9
adnp2
il22ra2
canthaxanthine
8-hydroxy-2-deoxyguanosine
slc22a3
amacr
kcng2
siah1
ca4
hs3st3b1
thap4
mansc1
arhgef10
astaxanthin
shank1
cdo1
cecr1a
bco2
hykk
cep170b
grik2
pdcd4
aatk
gdi1
12-hydroperoxy-9z,13e,15-octadecatrienoic acid
sema6b
scg5
camsap2
cngb1
adprhl1
ppfia2
dhcr24
fosl1
ugt2a2
abcg2
cebpb
gabrb2
pcdhgc5
col25a1
grik4
sbk2
rin2
osbpl7
kbp
B
A
C
Figure 4. Interactional Network of Antioxidant Defense in Seahorse Brood Pouch Development (A) Boxplots showing the changing tendency of one oxidant and two
antioxidants over different pouch developmental stages in seahorses. Each point represents one replicate (n = 12). Center line, median; box limits, upper and lower quartiles;
whiskers, 1.53 the interquartile range. DISCUSSION In addition, different tis-
sue-specific expression pattern of genes participating in RA signaling and
synthesis (Figure 3A) and RA target genes (Figure 3B) may indicate that
changes in RA level was resulted from the differential expression of genes
in the pituitary and gonad, which further regulated pouch development by
regulating the expression of RA target genes in the brood pouch (Figure
S2). Meanwhile, in mammals, RA is required to promote differentiation of
the mesenchyme into the future uterus and vagina by acting on the Mullerian
duct, and plays a vital role in morphological and functional differentiation of
female reproductive organs.38,39 Therefore, future studies comparing the
specific morphological and functional changes in the reproductive organs
of female mammals and male seahorses during pregnancy post-RA chal-
lenge may better illustrate the role of RA in male and female pregnancy. In addition, the protective role of transporters against oxidative stress may
be responsible for their positive correlation with antioxidants (Figure 4C). For
example, ABCG2, an ABC transporter that discharges a variety of toxic sub-
stances, protects cells from reactive oxygen species-mediated cell dam-
age.47 We also detected a positive correlation between astaxanthin and
genes participating in RA signaling (Figure 4C). The consistent decreasing
trend in RA expression and antioxidants, as well as their correlation during
pregnancy, may suggest that antioxidant defense during brood pouch devel-
opment is partly under the control of RA. Low RA levels in the cell milieu are
relevant to the expression of several antioxidants and antioxidant enzymes,
including GST;18 in mammals, a higher oxidative state caused by RA treat-
ment promotes secretion of vascular endothelial growth factor, which may
be necessary for successful pregnancy.48 Therefore, decreasing RA and an-
tioxidants in the seahorse during pregnancy indicates that an RA-related anti-
oxidant defense mechanism plays a key role in successful reproduction dur-
ing seahorse pregnancy, which is similar to that in mammals. Correlations This study serves as the first application of metabolome in seahorse for
identifying important pathways and metabolites possibly related to brood
pouch formation and pregnancy. As the co-enriched pathways of DEGs
and SDMs identified in both pouch formation and pregnancy, aminoacyl-
tRNA biosynthesis may participate in the formation of placenta-like tissues
during male pregnancy by facilitating the growth of collagenous fibers.40
Furthermore, antioxidant defense regulation, which is necessary to maintain
a stable low concentration of reactive oxygen species,41 plays a key role in
male pregnancy (Figure 4). DISCUSSION (B and C) Significant correlations between significantly different antioxidants and DEGs (correlation coefficient >0.9 and p < 0.01). Fonts of antioxidants and genes involved in transporting or related to RA are in bold. Metabolites and genes are indicated with blue and green background colors,
respectively. Blue and green lines indicate significant positive and negative correlations, respectively. (B) Correlations between canthaxanthin and ophthalmic acid with
other DEGs identified for brood pouch formation. (C) Correlations between canthaxanthin (left) and astaxanthin (right) with other DEGs identified during pregnancy. DISCUSSION 0e+00
2e−04
4e−04
6e−04
Stage
opthalmic_acid
0e+00
5e−05
1e−04
Stage
canthaxanthine
A
C 0e+00
2e−05
4e−05
Stage
astaxanthin
B 0e+00
2e−04
4e−04
6e−04
Stage
opthalmic_acid
0e+00
5e−05
1e−04
Stage
canthaxanthine
0e+00
2e−05
4e−05
Stage
astaxanthin
B
A
C B A A C nme2
kirrel3
hgf
plxna1
igfbp1
dlgap4
wnt9a
clock
zmynd8
pdp1
epn3
il17re
nalcn
gpm6a
pex11b
sv2a
dnm1
map7d2
six1b
syt1
methocarbamol
ndrg1
methionine sulfoxide
nmes1
cacna1i
adenosine 5'-monophosphate
astaxanthin
shank1
cdo1
cecr1a
bco2
hykk
cep170b
grik2
pdcd4
aatk
gdi1
12-hydroperoxy-9z,13e,15-octadecatrienoic acid
sema6b
scg5
camsap2
cngb1
adprhl1
ppfia2
dhcr24
fosl1
ugt2a2
cdh24
adgrb2
pcsk2
syp
ncan
dnaaf5
tmem260
epb41l3
tnip1
mapt
vdac2
oprm1
abcg2
cebpb
gabrb2
pcdhgc5
col25a1
grik4
sbk2
rin2
osbpl7
kbp
acot2
olfr139
erich3
scn8a
pcdhac2
grm3
gria3
cpne2
ampd2
rap1gap
mdga2
nav1
gmeb1
col28a1
pcbp4
pfkm
uridine diphosphate-n-acetylglucosamine
efna2
43347
kcnj11
tnmd
taurocholic acid
maltotetraose
fry
slc27a2
cacna2d1
shank3
srl
tnrc6c
fam184b
grin2a
qpct
sgcb
hsd17b12a
chrng
wfikkn2
egfl6
ciart
tubb2a
pcdhga11
kcnab1
kcnma1
myo16
rab3c
cga
ptprn
ank3
sepiapterin
d-norvaline
spon1
5-deoxy-5'-methylthioadenosine
cxxc4
adora1
ldlr
chd4
slc22a23
ttbk1
zfhx3
sh2d3c
rtn1
slc5a3
hapln1
elp6
gfi1b
gyc88e
acadl
grin2b
trpm3
rfx1
pomcb
guca2a
gpt2l
zearalenone
camkv
arhgef28
igsf9
argininosuccinic acid
map2
sgip1
cdk5r1
gss
lmtk3
gnao1
nrxn2
tnk2
lrp1b
kiaa1549
slc4a3
astn1
kcnj12
napb
slc1a2
l-cysteine-glutathione gisulfide
celsr3
unc80
atp1a1
sez6
ank1
1-methylguanosine
gad2
map1a
sphkap
tubb4b
tenm4
csrp3
pcdh15
prickle2
caskin1
grin1
trpc5
akap6
map6
nup210
syn2
cpne5
srcin1
stox2
erc2
zfhx4
dlgap2
pg(16:0/0:0)[u]
serpine1
lect1
scn2a
ano10
gltscr1l
prex2
lta4h
tkt
cacna2d2
rarres3
chd3
ptch1
abcc4
ppm1b
mgat5b
klhl41b
wdr31
klhl33
acsf2
slc2a9
adnp2
il22ra2
canthaxanthine
8-hydroxy-2-deoxyguanosine
slc22a3
amacr
kcng2
siah1
ca4
hs3st3b1
thap4
mansc1
arhgef10
Stage
Stage
Stage
lect1
scn2a
ano10
gltscr1l
prex2
lta4h
tkt
cacna2d2
rarres3
chd3
ptch1
abcc4
ppm1b
mgat5b
klhl41b
wdr31
klhl33
acsf2
slc2a9
adnp2
il22ra2
canthaxanthine
8-hydroxy-2-deoxyguanosine
slc22a3
amacr
kcng2
siah1
ca4
hs3st3b1
thap4
mansc1
arhgef10
astaxanthin
shank1
cdo1
cecr1a
bco2
hykk
cep170b
grik2
pdcd4
aatk
gdi1
12-hydroperoxy-9z,13e,15-octadecatrienoic acid
sema6b
scg5
camsap2
cngb1
adprhl1
ppfia2
dhcr24
fosl1
ugt2a2
abcg2
cebpb
gabrb2
pcdhgc5
col25a1
grik4
sbk2
rin2
osbpl7
kbp
C
Figure 4. Interactional Network of Antioxidant Defense in Seahorse Brood Pouch Development (A) Boxplots showing the changing tendency of one oxidant and two
antioxidants over different pouch developmental stages in seahorses. Each point represents one replicate (n = 12). Center line, median; box limits, upper and lower quartiles;
whiskers, 1.53 the interquartile range. Detection of DEGs Related to RA in Seahorse We first recorded RA-related genes identified in previous studies, including genes
participating in the RA synthesis and signaling pathway,11 as well as those with expres-
sion regulated by RA (RA target genes).56 We then searched for RA-related genes from
the DEGs identified by comparing both the pouch formation and pregnancy process of
H. erectus. To better illustrate the role of RA in Hippocampus, we also searched RA-
related genes from among the DEGs identified in the pregnancy process of the pot-
bellied seahorse Hippocampus abdominalis.9 We then used in-house R scripts to
identify RA-related DEGs associated with the pregnancy process of both species
and investigated their expression tendency. To reduce errors caused by inconsistency
in gene names from different studies, only gene symbols generated by the Ensembl
dataset were used when gene lists identified from different studies were compared. Ethical Approval All the experiments were carried out with an approval from the Experimental Animal
Ethics Committee of the South China Sea Institute of Oceanology, Chinese Academy
of Sciences, China. DISCUSSION Reproduction isan energetically demanding activ- ll The Innovation 1, 100052, November 25, 2020 5 The Innovation The Innovation Report olome analysis and 3 out of the 12 samples were randomly selected and used for tran-
scriptome analysis9,49 (Figure S1). between antioxidants and genes with different functions could indicate the
synergistic network of molecules involved in brood pouch development
and require further research. Certain limitations were noted in this study. First, is the potential bias asso-
ciated with the sampling design for transcriptome analysis. This may result in
allometric changes in organ properties due to the samples being collected
from animals in different stages. However, quantifying and extracting RNA
from half a testis or the pituitary gland from one seahorse is challenging
due to their light weight. Although this bias likely had minimal effects on com-
parison of the pouch pregnancy process, since both NF and PG seahorses
contain mature tissues, it is important to consider these issues when inter-
preting transcriptome data for the pouch formation process. In addition,
similar to what has been reported in previous seahorse studies,9,49 we
used three replicates per group for transcriptomic analysis and chose edgeR
to identify DEGs due to its power in dealing with a low number of replicates.50
Despite the reasonable DEG dataset we obtained, Schurch et al.50 stated that
only a tiny fraction of DEGs can be detected with three replicates per group. Hence, future transcriptome studies with a higher number of replicates will
enrich the gene dataset responsible for pouch development. Furthermore,
although we carefully interpreted the correlation networks, we did not focus
on the specific correlations between a single transcript and metabolite due to
the low sampling size (n = 6) used for Pearson correlation analysis. Hence,
although an increased sample size may serve to generate more convincing
correlation results, we believe that the reasonable positive correlations pre-
sented here between antioxidants and RA-related genes and transporters
during pregnancy will not be significantly impacted. Experimental Materials A total of 100 lined seahorses were obtained from the Shenzhen Seahorse Breeding
Center (Shenzhen, China) in August 2017. These seahorses were categorized into
three developmental stages according to brood pouch morphology: (1) unformed
(UF) stage comprising 50 seahorses (5.0–5.7 cm in length and 0.39–0.54 g in weight)
with unformed brood pouches; (2) newly formed (NF) stage, which included 25 male
seahorses (6.2–7.4 cm in length and 0.78–1.43 g in weight) with newly formed brood
pouches; and (3) pregnant (PG) stage, including 25 male seahorses (9.0–9.3 cm in
length and 2.42–3.55 g in weight) during pregnancy. Detection of RA, PRL, PR, and TE As a complement to the non-target metabolome, using enzyme-linked immu-
nosorbent assay (ELISA) we detected levels of PRL, PR, and TE due to their re-
ported roles in seahorse brood pouch development.4 RA was also detected using
the same method. There were six replicates per developmental stage. Muscle tis-
sues were used for RA, PRL, PR, and TE detection. Muscles in each sample were
maintained at 2C–8C after melting and were homogenized by grinding in PBS
solution (pH 7.4). These samples were centrifuged at 3,000 rpm for 20 min and
the supernatant was collected and reserved for detection. RA (cat. no. RJ-
27871), PRL (cat. no. RJ-21563), TE (cat. no. RJ-21574), and PR (cat. no. RJ-
21719) were all detected according to the ELISA manufacturer's instructions (Re-
njie, Shanghai, China). In brief, the purified antibody was used to coat microtiter
plate wells and produce a solid-phase antibody. The muscle supernatant was
then added to the wells and combined with a horseradish peroxidase (HRP)-
labeled
antibody
to
form
an
antibody-antigen-enzyme-antibody
complex. 3,30,5,50-Tetramethylbenzidine (TMB) was added for coloration after washing
thoroughly; TMB turned blue after HRP catalysis and yellow after the addition
of sulfuric acid solution. Absorbance (optical density) was measured at
450 nm using a microplate reader (Synergy H1, BioTek, USA), and the concentra-
tion of the target protein in the sample was determined using the standard curve
method. RNA Sequencing and Detection and Analysis of DEGs q
g
y
RNA (three replicates per stage) was extracted using TRIzol (Invitrogen, USA) and
RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies,
Santa Clara, CA, USA). Each replicate was used to generate an independent library. The RNA sequencing paired-end (PE) libraries were prepared using a NEBNext Ultra
RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA), following the manufacturer's
protocols. Each library was sequenced on an Illumina HiSeq platform (Illumina, San
Diego, CA, USA) with 150 bp PE reads. A total of approximately 489 million clean PE
reads were generated. After reads filtering and removal of rRNA-mapped reads,51
high-quality reads were mapped to the reference genome of the lined seahorse
(NCBI accession number: PRJNA347499)52 and gene abundance was quantified using
the FPKM (fragments per million mapped reads) method.53 Based on the expression
pattern of all genes, PCA was performed using R package gmodels (http://www. r-project.org/) for sample relationship analysis. DEGs were detected with the edgeR
package using the following criteria: >2-fold change >2 and false discovery rate
<0.05.50,54 Using the annotations of all identified transcripts as a reference, gene
ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment
analysis were conducted using DAVID v.6.8 (https://david.ncifcrf.gov/) and KOBAS
software,55 respectively. GO terms or KEGG pathways with a calculated p value <
0.01 were considered significantly enriched. Conclusion We showed the general trends in transcript and metabolite changes over
different stages of seahorse brood pouch development and highlighted the
potential roles of RA, and its target genes, in brood pouch formation and
pregnancy. This first application of metabolome analysis in the lined sea-
horse uncovered an increased susceptibility to oxidative stress in seahorses
during pregnancy, which may have resulted from decreased RA expres-
sions. We also identified certain molecules with important functions in sea-
horse brood pouch formation and pregnancy, which will promote future
specific functional brood pouch studies post-RA and stress challenge in
syngnathids. Metabolite Extraction, Detection, and Analysis
( All remaining tissues from the 36 seahorses (12 replicates per stage) were freeze-
dried separately (Boyikang, Beijing, China) and made into powder using a tissue crush-
ing apparatus (Guangzhou Good, Guangzhou, China). After mixing evenly, approxi-
mately 20 mg of tissue per sample was collected for UHPLC-QE Orbitrap/MS analysis
according to the standard protocol (Biotree, Shanghai, China). The resulting data ma-
trix, including the peak number, sample name, and normalized peak area was imported
to the SIMCA 14.1 software package (v.14.1, Umea, Sweden) for PCA. Metabolites
with variable importance for the projection values > 1 and p value < 0.05 were identi-
fied as SDMs. Commercial databases, including KEGG (http://www.genome.jp/kegg/)
and MetaboAnalyst (http://www.metaboanalyst.ca/) were utilized to search for
metabolite pathways. Pathways with a calculated p value < 0.05 were considered
significantly enriched. REFERENCES (2005). The male seahorse synthesizes and se-
cretes a novel C-type lectin into the brood pouch during early pregnancy. FEBS J. 272,
1221–1235. 36. Scobell, S.K., and MacKenzie, D.S. (2011). Reproductive endocrinology of syngnathi-
dae. J. Fish Biol. 78, 1662–1680. 9. Whittington, C.M., Griffith, O.W., Qi, W., et al. (2015). Seahorse brood pouch transcrip-
tome reveals common genes associated with vertebrate pregnancy. Mol. Biol. Evol. 32, 3114–3131. 37. Harlin-Cognato, A., Hoffman, E., and Jones, A. (2006). Gene cooption without duplica-
tion during the evolution of a male-pregnancy gene in pipefish. Proc. Natl. Acad. Sci. U
S A 103, 19407–19412. 10. Lin, Q., Fan, S., Zhang, Y., et al. (2016). The seahorse genome and the evolution of its
specialized morphology. Nature 540, 395–399. 38. Nakajima, T., Sato, T., Iguchi, T., et al. (2019). Retinoic acid signaling determines the
fate of the uterus from the mouse M€ullerian duct. Reprod. Toxicol. 86, 56–61. 11. Duester, G. (2008). Retinoic acid synthesis and signaling during early organogenesis. Cell 134, 921–931. 39. Nakajima, T., Iguchi, T., and Sato, T. (2016). Retinoic acid signaling determines the fate
of uterine stroma in the mouse M€ullerian duct. Proc. Natl. Acad. Sci. U S A 113,
14354–14359. 12. Gibert, Y., Gajewski, A., Meyer, A., et al. (2006). Induction and prepatterning of the ze-
brafish pectoral fin bud requires axial retinoic acid signaling. Development 133,
2649–2659. 40. Kersten, S., Seydoux, J., Peters, J.M., et al. (1999). Peroxisome proliferator-activated
receptor alpha mediates the adaptive response to fasting. J. Clin. Invest. 103,
1489–1498. 13. Kim, D., Chen, R., Sheu, M., et al. (2019). Noncoding dsRNA induces retinoic acid syn-
thesis to stimulate hair follicle regeneration via TLR3. Nat. Commun. 10, 2811. 41. Nadarajapillai, K., Sellaththurai, S., Liyanage, D.S., et al. (2020). Molecular and func-
tional explication of thioredoxin mitochondrial-like protein (Trx-2) from big-belly sea-
horse (Hippocampus abdominalis) and expression upon immune provocation. Fish
Shellfish Immunol. 99, 495–504. 14. Lee, E., Ju, B.-G., and Kim, W.-S. (2012). Endogenous retinoic acid mediates the early
events in salamander limb regeneration. Anim. Cells Syst. 16, 462–468. 15. Cheroutre, H. (2016). Retinoic acid and retinoic acid receptors as pleiotropic modula-
tors of the immune system. Annu. Rev. Immunol. 34, 369–394. 42. Masonjones, H.D. (2001). The effect of social context and reproductive status on the
metabolic rates of dwarf seahorses (Hippocampus zosterae). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 129, 541–555. 16. REFERENCES 29. Lin, T., Liu, X., Xiao, D., et al. (2017). Plasma levels of immune factors and sex steroids
in the male seahorse Hippocampus erectus during a breeding cycle. Fish Physiol. Biochem. 43, 889–899. 1. Crespi, B., and Semeniuk, C. (2004). Parent-offspring conflict in the evolution of verte-
brate reproductive mode. Am. Nat. 163, 635–653. 2. Kawaguchi, M., Okubo, R., Harada, A., et al. (2017). Morphology of brood pouch
formation in the pot-bellied seahorse Hippocampus abdominalis. Zool. Lett. 3, 3–19. 30. Blum, N., and Begemann, G. (2015). Retinoic acid signaling spatially restricts osteo-
blasts and controls ray-interray organization during zebrafish fin regeneration. Development 142, 2888–2893. 3. Wilson, A.B., Vincent, A., Ahnesjö, I., et al. (2001). Male pregnancy in seahorses and
pipefishes (family Syngnathidae): rapid diversification of paternal brood pouch
morphology inferred from a molecular phylogeny. J. Hered. 92, 159–166. 31. Blum, N., and Begemann, G. (2012). Retinoic acid signaling controls the formation, pro-
liferation and survival of the blastema during adult zebrafish fin regeneration. Development 139, 107–116. 4. Stolting, K., and Wilson, A. (2010). Male pregnancy in seahorses and pipefish: beyond
the mammalian model. Bioessays 29, 884–896. 32. Campione, M., Steinbeisser, H., Schweickert, A., et al. (1999). The homeobox gene
Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping. Development 126, 1225–1234. 5. Lin, Q., Li, G., Qin, G., et al. (2012). The dynamics of reproductive rate, offspring survi-
vorship and growth in the lined seahorse, Hippocampus erectus Perry, 1810. Biol. Open 1, 391–396. 33. Hendee, K.E., Sorokina, E.A., Muheisen, S.S., et al. (2018). PITX2 deficiency and asso-
ciated human disease: insights from the zebrafish model. Hum. Mol. Genet. 27,
1675–1695. 6. Van Look, K.J.W., Dzyuba, B., Cliffe, A., et al. (2007). Dimorphic sperm and the unlikely
route to fertilisation in the yellow seahorse. J. Exp. Biol. 210, 432–437. 34. Cheung, L.Y.M., and Camper, S.A. (2020). PROP1-dependent retinoic acid signaling
regulates
developmental
pituitary
morphogenesis
and
hormone
expression. Endocrinology 161, bqaa002. 7. Carcupino, M., Baldacci, A., and Mazzini, M.P. (2005). Functional significance of the
male brood pouch in the reproductive strategies of pipefishes and seahorses: a
morphological and ultrastructural comparative study on three anatomically different
pouches. J. Fish Biol. 61, 1465–1480. 35. Wang, Y.J., Jia, G.X., Yan, R.G., et al. (2019). Testosterone-retinoic acid signaling di-
rects spermatogonial differentiation and seasonal spermatogenesis in the plateau
pika (Ochotona curzoniae). Theriogenology 123, 74–82. 8. Melamed, P., Xue, Y., Poon, J.F., et al. REFERENCES Udhane, S.S., Pandey, A.V., Hofer, G., et al. (2015). Retinoic acid receptor beta and an-
giopoietin-like protein 1 are involved in the regulation of human androgen biosyn-
thesis. Sci. Rep. 5, 10132. 43. Perera, N.C.N., Godahewa, G.I., Lee, S., et al. (2017). Manganese-superoxide dis-
mutase (MnSOD), a role player in seahorse (Hippocampus abdominalis) antioxi-
dant defense system and adaptive immune system. Fish Shellfish Immunol. 68,
435–442. 17. Crespo, D., Assis, L.H.C., van de Kant, H.J.G., et al. (2019). Endocrine and local
signaling interact to regulate spermatogenesis in zebrafish: follicle-stimulating hor-
mone, retinoic acid and androgens. Development 146, dev178665. 18. Mohanty, R., Das, S.K., and Patri, M. (2000). Modulation of benzo[a]pyrene induced
anxiolytic-like behavior by retinoic acid in zebrafish: involvement of oxidative stress
and antioxidant defense system. Neurotox. Res. 31, 493–504. 44. Roth, O., Solbakken, M.H., Torresen, O.K., et al. (2020). Evolution of male pregnancy
associated with remodeling of canonical vertebrate immunity in seahorses and pipe-
fishes. Proc. Natl. Acad. Sci. U S A 117, 9431. 19. Borgbo, T., Klucková, H., Macek, M., et al. (2017). The common follicle-stimulating hor-
mone receptor (FSHR) promoter polymorphism FSHR -29G > A affects androgen
production in normal human small antral follicles. Front. Endocrinol. (Lausanne)
8, 122. 45. Alonso-Alvarez, C., Bertrand, S., Devevey, G., et al. (2004). Increased susceptibility to
oxidative stress as a proximate cost of reproduction. Ecol. Lett. 7, 363–368. (
)
f 46. Wang, Y., Salmon, A.B., and Harshman, L.G. (2001). A cost of reproduction: oxidative
stress susceptibility is associated with increased egg production in Drosophila mela-
nogaster. Exp. Gerontol. 36, 1349–1359. 20. Raija, S., Haka-Risku, T., Prockop, D.J., et al. (1987). Complete primary structure of
the a1-chain of human basement membrane (type IV) collagen. FEBS Lett. 225,
188–194. 47. Nie, S., Huang, Y., Shi, M., et al. (2018). Protective role of ABCG2 against oxidative
stress in colorectal cancer and its potential underlying mechanism. Oncol. Rep. 40,
2137–2146. 21. Killen, P.D., Francomano, C.A., Yamada, Y., et al. (1987). Partial structure of the human
alpha 2(IV) collagen chain and chromosomal localization of the gene (COL4A2). Hum. Genet. 77, 318–324. 48. Wu, J., Hansen, J.M., Hao, L., et al. (2011). Retinoic acid stimulation of VEGF secretion
from human endometrial stromal cells is mediated by production of reactive oxygen
species. J. Physiol. (Lond.) 589 (Pt 4), 863–875. 22. Lebherzeichinger, D., Krenn, C.G., and Roth, G.A. (2013). Integrative Analysis of Metabolome and Transcriptome 23. La Marca, A., Carducci Artenisio, A., Stabile, G., et al. (2005). Evidence for cycle-depen-
dent expression of follicle-stimulating hormone receptor in human endometrium. Gynecol. Endocrinol. 21, 303–306. For each of the two processes (pouch formation and pregnancy), two separate
KEGG analyses were conducted for identified DEGs and SDMs. We then compared
the enriched KEGG pathways of the DEGs and SDMs identified for the same process
to identify the co-enriched pathways of transcriptome and metabolome analysis. Pear-
son correlation coefficients between DEGs and SDMs identified for comparison of the
same pouch process were further calculated. DEGs and SDMs with a correlation coef-
ficient >0.9 and p value < 0.01 were imported to Cytoscape (v.3.3.0),57 which visual-
ized and clarified the metabolomic and transcriptome data and built networks be-
tween genes and metabolites. Here, we focused on the entire correlation network
between the transcriptome and metabolome, rather than one specific correlation be-
tween a single transcript and metabolite. Moreover, according to the results published
previously,58,59 we did not further correct the p values. 24. Torlopp, A., Khan, M.A., Oliveira, N.M., et al. (2014). The transcription factor Pitx2 po-
sitions the embryonic axis and regulates twinning. Elife 3, e03743. 25. Aguirre, J., and Lambeth, J.D. (2010). Nox enzymes from fungus to fly to fish and
what they tell us about Nox function inmammals.Free Radic. Biol. Med.49, 1342–1353. l
d
d
(
)
h
h
l
d 26. Knowles, H., Li, Y., and Perraud, A.L. (2013). The TRPM2 ion channel, an oxidative
stress and metabolic sensor regulating innate immunity and inflammation. Immunol. Res. 55, 241–248. 27. Surai, A.P., Surai, P.F., Steinberg, W., et al. (2003). Effect of canthaxanthin content of
the maternal diet on the antioxidant system of the developing chick. Br. Poult. Sci. 44,
612–619. 28. Soga, T., Baran, R., Suematsu, M., et al. (2006). Differential metabolomics reveals
ophthalmic acid as an oxidative stress biomarker indicating hepatic glutathione con-
sumption. J. Biol. Chem. 281, 16768–16776. Tissue Sampling The seahorse dissection and sampling processes were performed on ice. After
dissection, UF-stage seahorse gonads were observed under a microscope, and only
male seahorses with testes were retained. Embryos were gently removed from preg-
nant seahorses before sampling. A total of 18 individual seahorses in each of the three
stages were used. Muscles from six seahorses from each stage were used to detect
RA, PRL, PR, and TE; another 12 seahorses in each stage were used for transcriptome
and metabolome analyses. From each animal, half of the pituitary gland, testis, and
brood pouch tissue were dissected, pooled, and quickly frozen in liquid nitrogen until
transcriptome analysis. For the UF stage, skin tissue was collected from the primordial
brood pouch area and designated as the pouch sample. After transcriptome sampling,
all remaining tissues from each animal were then frozen separately in liquid nitrogen
for metabolome analysis. Finally, 12 samples from each stage were used for metab- www.cell.com/the-innovation The Innovation 1, 100052, November 25, 2020 6 The Innovation Report SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j.xinn.2020. 100052. 57. Kohl, M., Wiese, S., and Warscheid, B. (2011). Cytoscape: software for visualization
and analysis of biological networks. Methods Mol. Biol. 696, 291–303. 58. Hao, R., Du, X., Yang, C., et al. (2019). Integrated application of transcriptomics and
metabolomics provides insights into unsynchronized growth in pearl oyster
Pinctada fucata martensii. Sci. Total Environ. 666, 46–56. REFERENCES Keratin 18 and heat-shock pro-
tein in chronic kidney disease. Adv. Clin. Chem. 62, 123–149. ll The Innovation 1, 100052, November 25, 2020 7 Innovation Report 49. Qin, G., Zhang, Y., Zhang, B., et al. (2020). Environmental estrogens and progestins
disturb testis and brood pouch development with modifying transcriptomes in
male-pregnancy lined seahorse Hippocampus erectus. Sci. Total Environ. 715,
136840. 59. Li, B., Song, K., Meng, J., et al. (2017). Integrated application of transcriptomics and
metabolomics provides insights into glycogen content regulation in the Pacific oyster
Crassostrea gigas. BMC Genomics 18, 713. ACKNOWLEDGMENTS 50. Schurch, N.J., Schofield, P., Gierlinski, M., et al. (2016). How many biological replicates
are needed in an RNA-seq experiment and which differential expression tool should
you use? RNA 22, 839–851. This research was supported by the National Natural Science Foundation of China
(41825013, 41806189), China; the K.C. Wong Education Foundation, China; the Key Special
Project for Introduced Talents Team of Southern Marine Science and Engineering Guangdong
Laboratory (Guangzhou) (GML2019ZD0407), China; the Guangdong Special Support Program
of Youth Scientific and Technological Innovation (2017TQ04Z269), China; and the ChinaPost-
doctoral Science Foundation grant (2019M663151), China. 51. Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359. 52. Lin, Q., Qiu, Y., Gu, R., et al. (2017). Draft genome of the lined seahorse, Hippocampus
erectus. Gigascience 6, 1–6. 53. Kim, D., Pertea, G., Trapnell, C., et al. (2013). TopHat2: accurate alignment of tran-
scriptomes in the presence of insertions, deletions and gene fusions. Genome
Biol. 14, R36. WEB RESOURCES 55. Wu, J., Mao, X., Cai, T., et al. (2006). KOBAS server: a web-based platform for auto-
mated
annotation
and
pathway
identification. Nucleic
Acids
Res. 34,
W720–W724. Sequencing raw data of transcriptome were deposited at NCBI under the accession num-
ber PRJNA628893. 56. Balmer, J.E., and Blomhoff, R. (2002). Gene expression regulation by retinoic acid. J. Lipid Res. 43, 1773–1808. The authors declare no conflicts of interest. 54. Wang, L., Feng, Z., Wang, X., et al. (2010). Degseq: an R package for identi-
fying differentially expressed genes from RNA-seq data. Bioinformatics 26,
136–138. DECLARATION OF INTERESTS The authors declare no conflicts of interest. LEAD CONTACT WEBSITE http://sourcedb.scsio.cas.cn/cn/rck/jcqn/202004/t20200410_5536905.html. The Innovation 1, 100052, November 25, 2020 www.cell.com/the-innovation 8
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Applying robust control theory to solve problems in bio-medical sciences: study of an apoptotic model
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Article Creative Commons: Attribution-No Derivative Works 3.0 Creative Commons: Attribution-No Derivative Works 3.0 Open Access Open Access Trujillo, M., Becerra, V. M. and Hadjiloucas, S. ORCID:
https://orcid.org/0000-0003-2380-6114 (2014) Applying robust
control theory to solve problems in bio-medical sciences: study
of an apoptotic model. Journal of Physics Conference Series,
490 (1). 012140. ISSN 1742-6588 doi:
https://doi.org/10.1088/1742-6596/490/1/012140 Available at
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1 E-mail: s.hadjiloucas@reading.ac.uk Abstract. Biological models of an apoptotic process are studied using models describing a
system of differential equations derived from reaction kinetics information. The mathematical
model is re-formulated in a state-space robust control theory framework where parametric and
dynamic uncertainty can be modelled to account for variations naturally occurring in biological
processes. We propose to handle the nonlinearities using neural networks. 1 To whom any correspondence should be addressed. 1. Introduction Robust control theory has been considered as a tool in the biomedical community for model
validation, optimal experiment design for hypothesis testing, systems oriented drug design, target
identification, or multi-target drug dosage optimization. Unfortunately practical application of this
method is limited to models that can be linearized without substantial loss of quantitative or qualitative
features. Since most problems arising in biomedical applications do not fall in this category, we
present a different perspective where traditional parameter sensitivity analysis is translated into
sensitivity of biological features that can be measured experimentally. In this study, biological models
of an apoptotic process are studied using models describing a system of differential equations derived
from reaction kinetics information. The mathematical model is re-formulated in a state-space robust
control theory framework where parametric and dynamic uncertainty can be modelled to account for
variations naturally occurring in biological processes. Since the standard robust control theory
framework is valid for linear systems only, we re-formulate the problem as piecewise linear allowing
for specific non-linearities. We propose to handle the nonlinearities using neural networks. The
approach overcomes the limitations imposed by forced linearization and extends the applicability of
robust control theory to problems encountered within the Bio-medical community. IP Address: 134.225.215.136
This content was downloaded on 14/03/2014 at 14:10 Please note that terms and conditions apply. 2nd International Conference on Mathematical Modeling in Physical Sciences 2013
IOP Publishing
Journal of Physics: Conference Series 490 (2014) 012140
doi:10.1088/1742-6596/490/1/012140 M Trujillo 1, V M Becerra and S Hadjiloucas
School of Systems Engineering, University of Reading, RG6 6AY, UK M Trujillo 1, V M Becerra and S Hadjiloucas
School of Systems Engineering, University of Reading, RG6 6AY, UK 2. Apoptosis model p p
Apoptosis is the programed death that regulates the disposal of cells that have been damaged or are no
longer required by an organism. Mis-regulation of this process can lead to pathological conditions
such as cancer when cells do not die or Alzheimer’s condition where cells die more than needed. Many models of apoptosis are available in the literature and choosing an adequate model can be a
difficult process. The current study focuses on the EARM (Extrinsic Apoptosis Reaction Model)
described in [1] because in the process of creating, evaluating, calibrating and evolving this model, as 1 To whom any correspondence should be addressed. 1 2nd International Conference on Mathematical Modeling in Physical Sciences 2013
IOP Publishing
Journal of Physics: Conference Series 490 (2014) 012140
doi:10.1088/1742-6596/490/1/012140 2nd International Conference on Mathematical Modeling in Physical Sciences 2013
IOP Publishing
Journal of Physics: Conference Series 490 (2014) 012140
doi:10.1088/1742-6596/490/1/012140 nces 2013
IOP Publishing
doi:10.1088/1742-6596/490/1/012140 doi:10.1088/1742-6596/490/1/012140 this is now broadly accepted in the literature as a comprehensive one, where the associated
experimental parameters are well calibrated. Furthermore, the model incorporates the latest hypotheses
associated to the different reaction mechanisms postulated in the literature. this is now broadly accepted in the literature as a comprehensive one, where the associated
experimental parameters are well calibrated. Furthermore, the model incorporates the latest hypotheses
associated to the different reaction mechanisms postulated in the literature. p
The model of the extrinsic apoptotic pathway published in [1] is formed by four sub-circuits that
interact. These describe the entire pathway leading to the apoptotic process. The first circuit starts by
the addition of tumor necrosis factor (TNF). The TNF-related apoptosis-inducing ligand (TRAIL),
binds to its receptor forming the death-inducing signalling complexes (DISC) and activating pro
caspase 8 (pro-C8). The second circuit represents the activation of pro-C8 produces caspase 8 (C8). C8 directly activates caspase-3 (C3) which is responsible for the cleavage of Poly (ADP-ribose)
polymerase (PARP), an essential cell substrate for cell survival. The third circuit is a feed-forward
loop where C8 promotes the cleavage of Bid to form tBid which activates Bax. Activated Bax
promotes the formation of pores in the mitochondria to translocate Smac and cytochrome c (CyC) to
the cytosol. Smac binds to X-linked IAP (XIAP) preventing it from inhibiting C3. CyC binds to Apaf
and then attracts caspase 9 (C9) to form the apoptosome which in turn produces more C3. 2. Apoptosis model The fourth
circuit is a feedback loop where the production of C3 activates caspase 6 (C6) to produce more C8. Figure 1. Simplified block diagram of the apoptosis model divided in four sub-circuits. The input is
the death inducing ligand TRAIL and the output is the amount of cleaved PARP. The four blocks are
the receptor complex, mitochondria, caspace cascade and feedback. Figure 1. Simplified block diagram of the apoptosis model divided in four sub-circuits. The input is
the death inducing ligand TRAIL and the output is the amount of cleaved PARP. The four blocks are
the receptor complex, mitochondria, caspace cascade and feedback. The model has 18 species with non-zero initial conditions and 40 additional species that
represent modified forms of the initial 18 species (cleaved and short-lived intermediate species). When
these species have moved across membranes, they are annotated differently. All species interact via 28
biochemical reactions that use 70 rate reactions including forward, reverse and catalytic rates for each
reaction. The biochemical reactions were then translated into 58 ordinary differential equations. y
q
It has been proposed and experimentally tested by the authors of the model that the apoptotic
network works as a variable delay snap-action switch described by the following function: ( ) [
( )
]
(1) (1) (1) where c is the amount of substrate cleaved at time t, f is the fraction cleaved at the end of the reaction,
Td is the delay between TRAIL addition and half the total substrate cleaved (cPARP) and Ts is the
switching time between initial and total effector caspase cleavage. This concept is illustrated with the
response of cPARP for an initial concentration of TRAIL (Figure 2a). where c is the amount of substrate cleaved at time t, f is the fraction cleaved at the end of the reaction,
Td is the delay between TRAIL addition and half the total substrate cleaved (cPARP) and Ts is the
switching time between initial and total effector caspase cleavage. This concept is illustrated with the
response of cPARP for an initial concentration of TRAIL (Figure 2a). The authors of the model found that Td depends on TRAIL dose, but Ts and f are independent of
TRAIL dose. The model parameters were calibrated to follow experimental values of Td, Ts and f. 3. Nonlinear robust control approach The most obvious application of robust control for the apoptosis network is to specify desirable
behaviour, translate it as performance functions, define boundaries for uncertain parameters and
dynamics and design a controller that achieves the required performance for all the possible uncertain
scenarios. However, the apoptosis pathway is not yet fully understood, many contradicting models
describe different possible hypotheses and experimental data cannot be yet generated to test them. The
model we selected has gone through various iterations of modelling, experimental data fitting and
calibration but since the experimental measurements of variables in individual cells destroy the cell, it
is not possible yet to monitor the same cell through all its stages in the process from reception of the
apoptosis-inducing ligand to cell death. Hence we focus in the use of robust control to provide further
understanding of the process, before we could design controllers to intervene at the cellular level. 2. Apoptosis model The
cell is destined to die when 10% of the total amount of PARP is cleaved therefore, the value of cPARP
= 0.1 PARP is the threshold necessary for cell death (Figure 2b). Experiments with TRIAL doses from
2 to 1,000 ng/ml corresponded to a variation in Td from 140 to 660 minutes. We assume that reducing
the TRIAL dose enough to prevent reaching the cPARP threshold will create a cell survival steady 2 2nd International Conference on Mathematical Modeling in Physical Sciences 2013
IOP Publishing
Journal of Physics: Conference Series 490 (2014) 012140
doi:10.1088/1742-6596/490/1/012140 doi:10.1088/1742-6596/490/1/012140 state for long enough to compare the set of parameter values key for differentiating death and survival
states. In figure 2b four species are shown as representative of the different stages of the apoptotic
process. The initiator caspase C8* is the only concentration that has a slow increment during the delay
period Td followed by a sudden increment after reaching the switching time Ts. Figure 2. a) cPARP trajectory in response to ligand treatment as described in (1), b) main responses of
apoptotic system species; initiator caspaces, mitochondria pores, effector caspaces and cleaved PARP. 0
2
4
6
8
10
x 10
5
cPARP
time (hrs)
cPARP response
0
1
2
3
4
5
6
0
2
4
6
8
10
x 10
4
time (hrs)
Main responses of apoptotic system
C8*(x5)
Mito pores
C3(x10)
cPARP(x0.1)
Cell survival
Cell death
cPARP threshold
Td
Ts
f
(a)
(b) 0
2
4
6
8
10
x 10
5
cPARP
time (hrs)
cPARP response
Td
Ts
f
(a) 0
1
2
3
4
5
6
0
2
4
6
8
10
x 10
4
time (hrs)
Main responses of apoptotic system
C8*(x5)
Mito pores
C3(x10)
cPARP(x0.1)
Cell survival
Cell death
cPARP threshold
(b) Figure 2. a) cPARP trajectory in response to ligand treatment as described in (1), b) main responses of
apoptotic system species; initiator caspaces, mitochondria pores, effector caspaces and cleaved PARP. (
) 3.1. General robust control problem Almost any dynamical system can be put in the general form described in figure 3a. In this general
form P is a plant consisting of the nominal dynamics of our system augmented with performance
functions defined by the user to force the system to satisfy design requirements, K is the controller to
be designed to satisfy performance and stability requirements for all the systems resulting from the
combination of the plant P with the uncertain block ∆ which contains the allowed variations in
parameter space and un-modelled dynamics that may affect the system. w is a vector of the external
inputs to the system, z are the outputs to be controlled, u are the inputs to the controller, y are the
measurements available from the system, wp and zp are the output and input to the uncertainty block. Figure 3. a) General plant P and controller K, b) Robust control problem for uncertainty ∆, c) Plant
and controller integrated in M for the analysis of robust performance
P
K
w
z
y
u
M
∆
w
z
wp
zp
a)
b)
P
K
∆
w
z
y
u
wp
zp
c) P
K
∆
w
z
y
u
wp
zp P
K
w
z
y
u
) M
∆
w
z
wp
zp
c) c) b) a) Figure 3. a) General plant P and controller K, b) Robust control problem for uncertainty ∆, c) Plant
and controller integrated in M for the analysis of robust performance 3 2nd International Conference on Mathematical Modeling in Physical Sciences 2013
IOP Publishing
Journal of Physics: Conference Series 490 (2014) 012140
doi:10.1088/1742-6596/490/1/012140 2nd International Conference on Mathematical Modeling in Physical Sciences 2013
IOP Publishing
Journal of Physics: Conference Series 490 (2014) 012140
doi:10.1088/1742-6596/490/1/012140 The mathematical representation of the perturbed plant P is given by The mathematical representation of the perturbed plant P is given by The mathematical representation of the perturbed plant P is given by {
̇ ( ) ( ) ( ) ( )
( ) ( )
(2) (2) where x is the state vector of the nominal system, ( ) are the dynamics of the system as described in
the nominal model, and are functions of x. 3.1. General robust control problem The nonlinear H∞ problem is to find a
controller action u that results in an L2-gain of the system P from w to z less or equal to a given
while maintaining system stability. This is an optimisation problem where the allowed disturbance has
to be maximized while the control energy has to be minimized. Solutions to this problem come from
theories such as differential games, disipativity, viscosity among others. The final result is a Hamilton-
Jacobi-Isaacs equation (HJIE) of the form: ( ) ( )
( ) [
( )
( ) ( )
( )] ( )
( ) ( ) ( )
(3) (3) Finding a storage function ( ) that satisfies the HJI in most cases is not straightforward. Once a
function ( ) satisfying equation 3 is found, the minimum energy control and the maximum
disturbance allowed for the system in figure 3 a) are given by: ( ) ( )
( ) ( ),
( )
( ) ( )
(4) (4) system given in figure 3 b) the perturbed plant becomes For the system given in figure 3 b) the perturbed plant becomes {
̇ ( ) ( ) ( ) [ ( ) ( )] ( )
[ ( ) ( )] ( )
( ) ( )
(5) {
̇ ( ) ( ) ( ) [ ( ) ( )] ( )
[ ( ) ( )] ( )
( ) ( )
(5) (5) where and are unknown functions that belong to the set of admissible uncertainties and
are the system parameters that may vary over time in a defined range [2]. For the purpose of this paper, we are not concerned with the design of a controller since we
consider the apoptosis network as a whole system with an integrated controller. Figure 3b shows the
general framework used in robust control theory for analysis rather than synthesis where P and K form
a single block M. This framework allows us to ask how big can the perturbation be and still permit the
system to achieve the desired performance. Then, by manipulating the weighting functions
(performance criteria) and the allowed uncertainties we can perform sensitivity analysis to measure the
impact of various parameters in the system, test different hypothesis for model validation/invalidation
and identify underlying mechanisms and interactions between species of the system. 3.2. References
[1]
Albeck J G, Burke J M, Spencer S L, Lauffenburger D A and Sorger P K 2008 PLoS Biology,
6(12): e299.
[2]
Abu-Khalaf M, Huang J and Lewis F L 2006 Nonlinear H2/H∞ Constrained Feedback Control
(Springer-Verlag London)
[3]
Gaudet S, Spencer S L, Chen W W, Sorger P K, 2012, PLoS Computational Biology, 1-15
[4]
Aliyu M D S 2011 Nonlinear H∞-control, Hamiltonian systems and Hamilton-Jacobi equation
(CRC Press, Taylor &Francis, Boca Raton FL, USA) 2nd International Conference on Mathematical Modeling in Physical Sciences 2013
IOP Publishing
Journal of Physics: Conference Series 490 (2014) 012140
doi:10.1088/1742-6596/490/1/012140 2nd International Conference on Mathematical Modeling in Physical Sciences 2013
IOP Publishing
Journal of Physics: Conference Series 490 (2014) 012140
doi:10.1088/1742-6596/490/1/012140 g
doi:10.1088/1742-6596/490/1/012140 can identify which once affect the most each feature. To study the effects that variations in the system
have on the defined features of interest we have to model such variations as uncertainties ∆ and
incorporate them in the system M. Parametric uncertainty is the variation in parameter space that we
want to explore and dynamic uncertainty accounts for the unknown dynamics taking place in the
system. When a mixture of both uncertainty types has to be analyzed then ∆ becomes a structured
uncertainty block that can be separated from the plant as in figure 3b. A solution for the HJIE from
equation 3 can be found indirectly by using iterative processes. The HJI is broken in a sequence of
linear partial differential equations and the aim becomes finding a possible storage function ( ). In
each iteration step, the performance of an initial stabilizing controller is improved until a satisfactory
solution to the HJI is found through several iterations. Even though finding ( ) for the linear partial
differential equations at each iteration is less complicated than finding it for the HJIE, nevertheless, it
is still a difficult task that needs to be solved. One possible way is by approximating ( ) with a
neural network at each iteration. A more complete description of the process can be found in [4]. 3.1. General robust control problem Performance functions, modeling uncertainty and solving the HJIE with Neural networks
The performance functions are used to modify the nominal system to include desired trajectories. In
the case of the apoptosis network, it was suggested in [3] that four features of the time trajectories
from cytosolic Smac and cPARP could define the difference between a normal and a pathological
response. These are: 1) tPARP which is the time it takes 50% of PARP to be cleaved, 2) tMOMP which is
the time it takes for half the Smac to translocate from the mitochondria, 3) tswitch which is the time
between the start and finish of PARP cleavage and 4) fPARP which is the amount of PARP cleaved after
the experiment or simulation. It is possible then to identify values of interest for these features and
define performance functions accordingly. By defining structural perturbations on various species we 4 4. Conclusion So far, because of its implementation complexity, robust control theory has failed to gain popularity as
a tool in the bio-medical field. Non-linear robust control theory is still under development and soft
computing alternatives are constantly introduced to overcome the complexity of solving problems that
cannot be tackled by linearization. The HJIE approach should be seen as an extension to linear -
control theory, providing a unifying approach in treating disturbances using a nonlinear counterpart of
the Riccati equations which are conventionally used to solve linear problems. Different soft computing
methodologies such as neural networks and fuzzy systems theory are worthwhile exploring to
overcome the difficulties of solving nonlinear HJI equations. A particular advantage in the general
formulation developed for the apoptotic process described in section 3.1 is that it clarifies the
parameters that need to be estimated (for example the different uncertainties introduced in the model
and their inter-relationship to other biochemical pathways). Furthermore, because of the nice stability and stabilizability properties of passive systems, such
as (global) asymptotic stabilizability by pure-gain output-feedback it is considerably desirable in
pharmacological or radiological intervention to render the associated biochemical non-linear systems
passive. In non-linear control theory, it is well understood that if the state space of a system is
reachable from the origin x=0, by an appropriate choice of an input u(t) so that its output is finite, then
it can be rendered dissipative. A further goal of the proposed formulation is, therefore, to identify the
control points in biochemical reactions that would render interconnected pathways passive through the
control of the supply rate (in our case the cPARP threshold in Fig. 2). Since most biochemical
pathways can be seen as interconnected dissipative sub-systems of processes described by storage
functions, the approach should have a wide domain of applications across Systems Biology. The
present work, therefore, serves also as an intellectual scaffolding upon which other Systems-Biology
problems can be formulated where seemingly non-linear processes can be controlled though the
control of the supply rate to an identified storage function of the system. References 5
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Supplementary Table 1 from Notch3 Pathway Alterations in Ovarian Cancer
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ENTARY TABLE S1. Notch Pathway Alterations in Patients with HGS-OvCa ENTARY TABLE S1. Notch Pathway Alterations in Patients with HGS-OvCa SUPPLEMENTARY TABLE S1. Notch Pathway Alterations in Patients with HGS-OvCa Supplementary Table 1a. Clinical Characteristics of Patients with Notch Pathway
Alterations ementary Table 1a. Clinical Characteristics of Patients with Notch Pathway
ti Supplementary Table 1b. Mutations of Notch2, Notch3 and Notch 4 in Patients with HGS-
OvCa
Strand
NOTCH2
4853
1
120292618 120292618
+
Silent
SNP
T
T
C
NOTCH2
4853
1
120262689 120262689
+
Missense_MutatiSNP
C
C
T
NOTCH2
4853
1
120269944 120269944
+
Missense_MutatiSNP
C
C
A
NOTCH2
4853
1
120294980 120294980
+
Missense_MutatiSNP
T
T
C
NOTCH3
4854
19
15137882
15137882
+
Missense_MutatiSNP
G
G
T
NOTCH3
4854
19
15139142
15139142
+
Missense_MutatiSNP
G
G
C
NOTCH3
4854
19
15142228
15142228
+
Silent
SNP
G
G
A
NOTCH3
4854
19
15142296
15142296
+
Silent
SNP
G
G
A
NOTCH3
4854
19
15159004
15159004
+
Silent
SNP
G
G
A
NOTCH3
4854
19
15139118
15139118
+
Silent
SNP
C
C
A
NOTCH3
4854
19
15142145
15142145
+
Missense_MutatiSNP
A
A
C
NOTCH3
4854
19
15161209
15161209
+
Missense_MutatiSNP
A
A
G
NOTCH4
4855
6
32280085
32280085
+
Missense_MutatiSNP
T
T
A
NOTCH4
4855
6
32296287
32296287
+
Silent
SNP
C
C
G
NOTCH4
4855
6
32296776
32296776
+
Missense_MutatiSNP
C
C
T
NOTCH4
4855
6
32298323
32298323
+
Missense_MutatiSNP
G
G
A
NOTCH4
4855
6
32271779
32271779
+
Missense_MutatiSNP
G
G
T
Variant-
Type
Reference-
Allele
Tumor-
Seq_Allele1
Tumor-
Seq_Allele2
Symbol
Gene
Id
Chromosome
Start-
position
End-
position
Variant-
Classification Supplementary Table 1b. Mutations of Notch2, Notch3 and Notch 4 in Patients with HGS-
OvCa ** Notch3 downregulation, and other alterations. *Notch 3 alterations ( amplification, upregulation, and mutation) nregulation, and other alterations. OvCa Supplementary Table 1c. Clinical Characteristics of Patients with NOTCH3 Alterations mentary Table 1c. Clinical Characteristics of Patients with NOTCH3 Alteration *Notch 3 alterations ( amplification, upregulation, and mutation ** Notch3 downregulation, and other alterations. ** Notch3 downregulation, and other alterations.
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https://openalex.org/W2922864376
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https://periodicals.karazin.ua/geoeco/article/download/12424/11819
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Ukrainian
| null |
Biostratigraphic partition of the Upper Jurassic and Lower Cretaceous (Oxfordian - Lower Berriasian) deposits of the Crimean Mountains for foraminiphers
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Vìsnik Harkìvsʹkogo nacìonalʹnogo unìversitetu ìmenì V.N. Karazìna. Serìâ Geologìâ. Geografìâ. Ekologìâ
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БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ
ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ
ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ За результатами форамініферового аналізу проведено детальне біостратиграфічне розчленування верхньої юри та
нижньої крейди (оксфорд-нижній беріас) Гірського Криму. В розрізах виділено п’ять форамініферових комплексів, які хара-
ктеризують верхньоюрський (нижньо- верхньооксфордський, нижньокімериджський, верхньотитонський) та нижньокрей-
довий (нижньоберіаський) вік порід. Аналіз форамініферових комплексів дозволив в кожному розрізі виявити біостратигра-
фічні підрозділи - зони: в нижньому оксфорді - Lenticulina quenstedti-Globuligerina oxfordiana; у верхньому окс-
форді - Lenticulina russiensis-Epistomina uhligi; у верхньому титоні - Anchispirocyclina lusitanica-Melathrokerion spirialis, у
нижньому беріасі (нижню зону) Protopeneroplis ultragranulatus-Siphoninella antiqua та верстви у нижньому кімериджі – з
Epistomina praetatariensis-Globuligerina parva. Виявлені форамініферові зони прослідковуються нами в різних структурно-
фаціальних зонах (зона Lenticulina quenstedti-Globuligerina oxfordiana – в Судацько-Феодосійській; зона - Lenticulina
russiensis-Epistomina uhligi – в Ай-Петрі-Бабуганській та Судацько-Феодосійській; верстви з Epistomina praetatariensis-
Globuligerina parva - в Судацько-Феодосійській; зона Anchispirocyclina lusitanica-Melathrokerion spirialis – в Сухоріченсько-
Байдарській, Демерджі-Карабійській та Судацько-Феодосійській; зона Protopeneroplis ultragranulatus-Siphoninella antiqua –
в Демерджі-Карабійській та Судацько-Феодосійській). Для кожної форамініферової зони наведена характеристика, яка
включає комплекс характерних видів, види, які зникають, з’являються та види, які продовжують своє існування, а також
окремі особливості. За літологічними особливостями та палеонтологічною характеристикою у відкладах досліджуваних
розрізів виявлені світи та підсвіти. В результаті дослідження уточнені біостратиграфічні характеристики розрізів та
доповнено палеонтологічну характеристику верхньоюрських та нижньокрейдових відкладів Гірського Криму. Ключові слова: оксфорд, кімеридж, титон, беріас, форамініферовий комплекс, форамініферова зона, структурно-фа-
ціальна зона, Гірський Крим. р
р
Ю. Б. Доротяк. БИОСТРАТИГРАФИЧЕСКОЕ РАСЧЛЕНЕНИЕ ВЕРХНЕЮРСКИХ И НИЖНЕМЕЛОВЫХ (ОКС-
ФОРД - НИЖНИЙ БЕРРИАС) ОТЛОЖЕНИЙ ГОРНОГО КРЫМА ЗА ФОРАМИНИФЕРАМИ. По результатам форами-
ниферового анализа проведено детальное биостратиграфическое расчленение верхней юры и нижнего мела (оксфорд-ниж-
ний берриас) Горного Крыма. В разрезах установлено пять фораминиферовых комплексов, которые характерезуют верхне-
юрский (нижне- верхнеоксфордский, нижнекимериджский, верхнетитонский) и нижнемеловой (нижнеберриасский) возраст
пород. Анализ фораминиферовых комплексов позволил в каждом разрезе выявить биостратиграфические подразделе-
ния - зоны: в нижнем оксфорде - Lenticulina quenstedti-Globuligerina oxfordiana; в верхнем оксфорде - Lenticulina russiensis-
Epistomina uhligi; в верхнем титоне - Anchispirocyclina lusitanica-Melathrokerion spirialis, в нижнем берриасе (нижнюю зону)
Protopeneroplis ultragranulatus-Siphoninella antiqua и слои в нижнем кимеридже – с Epistomina praetatariensis-Globuligerina
parva. Виявленные фораминиферовые зоны прослеживаются нами в разных структурно-фациальных зонах (зона Lenticulina
quenstedti-Globuligerina oxfordiana – в Судакско-Феодосийской; зона - Lenticulina russiensis-Epistomina uhligi – в Ай-Петри-
Бабуганской и Судакско-Феодосийской; слои с Epistomina praetatariensis-Globuligerina parva - в Судакско-Феодосийской; зона
Anchispirocyclina lusitanica-Melathrokerion spirialis – в Сухореченско-Байдарской, Демерджи-Карабийской и Судакско-Фео-
досийской; зона Protopeneroplis ultragranulatus-Siphoninella antiqua – в Демерджи-Карабийской и Судакско-Феодосийской). Вісник Харківського національного університету імені В.Н. Каразіна Вісник Харківського національного університету імені В.Н. Каразіна (
)
Юлія Богданівна Доротяк,
мол. наук. співроб., Інститут геологічних наук НАН України,
вул. О. Гончара, 55-б, м. Київ, 01601, Україна,
е-mail: dorotyak78@gmail.com, http://orcid.org/0000-0002-0498-1892 © Доротяк Ю. Б. БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ
ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ
ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ Чайковський публікують уточнену схему
стратиграфії юрських відкладів Гірського Криму
з пояснювальною запискою (наведений ярусний,
зональний поділ, 31 світа і 8 горизонтів) [25]. В
1993 р. складена В.В. Пермяковим, М.М. Пермя-
ковою, Б.П. Чайковським уточнена стратиграфі-
чна схема юрських відкладів Гірського Криму
(структурно-фаціальне районування, доповни-
лась зональна шкала, встановлені і виділені гори-
зонти, які є основними одиницями регіональних
стратиграфічних підрозділів) [26]. Формування мети статті. Метою статті є
стратиграфічне розчленування і кореляція верх-
ньоюрських та нижньоберіаських відкладів Гірсь-
кого Криму на підставі вивчення форамініфер, та
використання отриманих результатів для біостра-
тиграфічних розчленувань. Виклад основного матеріалу. Відклади окс-
фордського ярусу поширені на території Гірсь-
кого Криму досить широко і тісно пов’язані з під-
стилаючими верхньокеловейськими породами. За
амонітами для нижнього оксфорду Криму виді-
лено зону Cardioceras cordatum, якій видповідає
форамініферова зона Lenticulina quenstedti -
Globuligerina oxfordiana [30]. Вивчення беріаських відкладів Гірського
Криму пов′язують з вивченням фауни з «пачки
феодосійських мергелів» характерного маркую-
чого горизонту розрізу беріаса Східного Криму. В
1974 р. І.Г. Сазоновою і Н.Т. Сазоновим пропону-
ється перша схема зонального розчленування
примежових відкладів титону і беріасу за амоні-
тами [5; 29]. У 1984 р. В.В. Пермяковим [17] за
комплексом форамініфер, коралів, брахіопод,
двостулкових, брюхоногих та головоногих молю-
сків виділено двоякірну світу із стратотипу в Дво-
якірній долині, але сам стратотип не був описа-
ний. Починаючи з 1984 р. і до сьогодні прово-
дяться детальні дослідження примежових відкла-
дів юри та крейди в розрізах Гірського Криму, а
саме літологічні, магнітостратиграфічні [2], мак-
рофауністичні (амоніти, двостулкові молюски,
брахіоподи, белемніти, корали, криноідеі, аптихи)
[2-5], мікрофауністичні (форамініфери, остра-
коди) [4; 7, 9, 10, 12; 13, 14], палінологічні (спори
та пилок, диноцисти) [10] та дослідження наноп-
ланктону [18-23]. Відклади верхнього оксфорду пов’язані пос-
туповим переходом, як з підстиляючими нижньо-
оксфордськими, так і перекриваючими нижньокі-
мериджськими відкладами. В межах Гірського
Криму верхній оксфорд підрозділяється на дві
амонітові зони: Gregoryceras transversarium і
Epipeltoceras bimammatum. За форамініферами
виділено зону Lenticulina russiensis-Epistomina
uhligi, яка простежується впродовж всього пізд-
нього оксфорду [30]. Кімериджські відклади поширені на території
Гірського Криму і утворюють єдиний комплекс
порід з оксфордськими відкладами. Виділений
лише нижній під’ярус, в якому за амонітами виді-
лена лона Streblites tenuilobatus, а за форамініфе-
рами встановлені верстви з Epistomina praetata-
riensis - Globuligerina parva [30]. Відклади верх-
нього кімериджу впевнено не встановлені, це по-
яснюється розмивом або переривом в осадконако-
пиченні. БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ
ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ
ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ Для каждой фораминиферовой зоны приведена характеристика, которая включает комплекс характерных видов, исчезаю-
щие виды, появляющиеся, и виды, продолжающие свое существование, а также отдельные особенности. По литологичным
особенностям и палеонтологической характеристики в отложениях изучаемых разрезов виявлены свиты и подсвиты. В ре-
зультате исследования уточнена биостратиграфическая характеристика разрезов и дополнена палеонтологическая харак-
теристика верхнеюрских и нижнемеловых отложений Горного Крыма. Ключевые слова: оксфорд, кимеридж, титон, берриас, фораминиферовый компле
турно-фациальная зона, Горный Крым. Вступ. Відклади верхньої юри і крейди в Гір-
ському Криму поширені досить широко і предста-
влені – оксфордським, кімериджським, титонсь-
ким і беріаським ярусами. Ступінь вивченості цих
відкладів неоднакова. Найбільш детально вивчені
відклади оксфордського, титонського та беріась-
кого ярусів. За основу біостратиграфічного по-
ділу верхньоюрських та нижньокрейдових відкла-
дів Гірського Криму нами взята «Стратиграфічна
схема верхньоюрських та нижньокрейдових відк-
ладів Гірського Криму» 2013 р. [20]. дових відкладів Гірського Криму висвітлюється в
численних роботах, та особливо детально в узага-
льнюючих працях, а саме «Стратиграфія УРСР. Том 7. Юра» [27], «Геологія СРСР. Т. 8. Крим»
[6], «Стратиграфія та форамініфери верхньої юри
та нижньої крейди Криму» [15], «Нижня крейда
заходу і півдня України» [16], «Юрські відклади
півдня України» [17], «Беріас Гірського Криму»
[5], «Стратиграфія верхнього протерозою та фа-
нерозою України» [20] та в статтях і тезах [1,3,14]. В 1969 р Є.А. Успенською розроблена стра-
тиграфічна схема верхньоюрських відкладів
Криму за амонітами [27]. В 1970 р. Є.А. Успен-
https://doi.org/10.26565/2410-7360-2018-49-04 В 1969 р Є.А. Успенською розроблена стра-
тиграфічна схема верхньоюрських відкладів
Криму за амонітами [27]. В 1970 р. Є.А. Успен-
https://doi.org/10.26565/2410-7360-2018-49-04 Аналіз останніх досліджень і публікацій. Історія вивчення верхньоюрських та нижньокрей- © Доротяк Ю. Б. - 49 -
- 49 - Серія «Геологія. Географія. Екологія», випуск 49 У зв’язку із складною геологічною будовою
досліджуваної території та тектонічним району-
ванням до цього часу залишається багато невирі-
шених питань, в тому числі і остаточно не встано-
влена межа примежових відкладів титону та бері-
асу. Базою для остаточних висновків може слугу-
вати детальний біостратиграфічних аналіз відкла-
дів межі юри та крейди Гірського Криму. ською і В.В. Пермяковим удосконалюється стра-
тиграфічна схема (наведено районування Гірсь-
кого Криму зональний поділ, літологічна і фауні-
стична характеристика, виділені світи - ескі-орди-
нська, бітацька, уточнено положення перерв) [28]. В 1984 р. В.В. Пермяковим і М.В. Ваніною публі-
кується стратиграфічна схема юрських відкладів
Гірського Криму (затверджена УРМСК в 1982 р.),
яка складається з загальної, регіональної шкал і
кореляції місцевих стратиграфічних підрозділів
[17]. В 1991 р. В.В. Пермяков, М.М. Пермякова,
Б.П. БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ
ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ
ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ та продовжують своє існування поо-
динокі
нижньооксфордські
види
Epistomina
nemunensis
G r i g . ,
Saracenaria
cornucopiae
(S c h w a g e r ). Відклади
зони
Lenticulina
russiensis-Epistomina uhligi простежені у нижній
частині манджильської світи. (р
, )
В Ай-Петрі-Бабуганській СФЗ відклади вер-
хнього окфорду представлені мергелем сірим гли-
нистим щільним нешаруватим. Форамініферові
угрупування багаточисельні, представлені бенто-
сними формами. За кількістю видів форми з сек-
реційною черепашкою (68%) переважають над аг-
лютинуючими (32%). Характерними видами для
данної зони є Texstularia jurassica G ü m b e l .,
Epistomina stelligeraformis M j a t l . Виявлені ску-
пчення Marsonella doneziana D a і n , Lagena
apiculata (R e u s s ), Epistomina limbata K a p t .,
E. stelligeraformis M j a t l . В угрупуваннях прису-
тні бореальні форми - Lenticulina polovinkaensis
Put.,
L. sibirensis
(Kosyr.),
L. nordformis
Put. et Rom. та продовжують своє існування поо-
динокі
нижньооксфордські
види
Epistomina
nemunensis
G r i g . ,
Saracenaria
cornucopiae
(S c h w a g e r ). Відклади
зони
Lenticulina
russiensis-Epistomina uhligi простежені у нижній
частині манджильської світи. (р
)
В Ай-Петрі-Бабуганській СФЗ відклади вер-
хнього окфорду представлені мергелем сірим гли-
нистим щільним нешаруватим. Форамініферові
угрупування багаточисельні, представлені бенто-
сними формами. За кількістю видів форми з сек-
реційною черепашкою (68%) переважають над аг-
лютинуючими (32%). Характерними видами для
данної зони є Texstularia jurassica G ü m b e l .,
Epistomina stelligeraformis M j a t l . Виявлені ску-
пчення Marsonella doneziana D a і n , Lagena
apiculata (R e u s s ), Epistomina limbata K a p t .,
E. stelligeraformis M j a t l . В угрупуваннях прису-
тні бореальні форми - Lenticulina polovinkaensis
Put.,
L. sibirensis
(Kosyr.),
L. nordformis
Put. et Rom. та продовжують своє існування поо-
динокі
нижньооксфордські
види
Epistomina
nemunensis
G r i g . ,
Saracenaria
cornucopiae
(S c h w a g e r ). Відклади
зони
Lenticulina
russiensis-Epistomina uhligi простежені у нижній
частині манджильської світи. В нижньому беріасі за амонітами виділяють
зону Berriasella jacobi в межах, якої встановлено
за
планктонними
форамініферами
шари
з
Globuligerina gulekhensis, в нижній частині яких
виділені зони за бентосними форамініферами: ни-
жня
зона
Protopeneroplis
ultragranula-
tus - Siphoninella antiqua і верхня зона Quadratina
tunassica - Siphoninella antiqua. Сукупність цих зон
відповідає об'єму амонітовій зоні Berriasella jacobi
[5; 30]. [5; 30]. ОКСФОРДСЬКИЙ ЯРУС. БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ
ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ
ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ Але, в останні роки отримані деякі до-
кази присутності верхнього кімериджу в розрізах
Ялтинського амфітеатру [1; 14], та у південно-схі-
дному Криму [3], проте стратиграфія верхнього
кімериджу на теперішній час недостатньо обґрун-
тована і в стратиграфічній схемі 2013 року не ви-
діляється. На сьогодні стратиграфічні схеми верхньоюр-
ських та нижньокрейдових відкладів Гірського
Криму удосконалені, зкорельовані з МСШ
2016 р., відповідно змінено назву деяких амоніто-
вих зон, уточнено вік та обсяг окремих світ, роз-
ширена палеонтологічна характеристика, прове-
дена кореляція з суміжними регіонами. У схему
введена шкала за форамініферами, яка розроблена
по верхній юрі К.І. Кузнецовою, а по нижній
крейді Т.М. Горбачик. Макрофауна (амоніти, дво-
стулкові молюски, гастроподи і корали) за даними
В.В. Пермякова, Р.Й. Лещуха, В.В. Аркадьєва,
Є.Ю. Барабошкіна [30]. Титонські відклади широко поширені на тери-
торії Гірського Криму та залягають без чітко ви-
раженого кутового неузгодження на кімериджсь-
ких відкладах і, можливо, раннього титону, при-
падає перерва в осадконакопиченні. Стратиграфі-
чно відклади нижнього титону розділені на дві
амонітові лони: Kossmatia richteri, яка співпадає з
верствами з Epistomina ventriosa - Texstularia
densa,
і
лона
Semiformiceras
semiforme
– - 50 - Вісник Харківського національного університету імені В.Н. Каразіна Бабуганській СФЗ - відслонення 49 км дороги
Ялта-Севастополь та в Судацько-Феодосійській
СФЗ, а саме у розрізах відслонень поблизу с. Бо-
гатівка на півострові Меганом, св. № 3 на г. Кара-
даг (рис. 1, 2) [8; 11]. Бабуганській СФЗ - відслонення 49 км дороги
Ялта-Севастополь та в Судацько-Феодосійській
СФЗ, а саме у розрізах відслонень поблизу с. Бо-
гатівка на півострові Меганом, св. № 3 на г. Кара-
даг (рис. 1, 2) [8; 11]. відповідає зоні Astacolus laudatus - Epistomina
omninoreticulata [30]. У верхньому титонському
під’ярусі за форамініферами виділено зону
Anchispirocyclina
lusitanica
-
Melathrokerion
spirialis, яка співпадає з амонітовою лоною
Paraulacosphinctes transitorius [30]. (р
, )
В Ай-Петрі-Бабуганській СФЗ відклади вер-
хнього окфорду представлені мергелем сірим гли-
нистим щільним нешаруватим. Форамініферові
угрупування багаточисельні, представлені бенто-
сними формами. За кількістю видів форми з сек-
реційною черепашкою (68%) переважають над аг-
лютинуючими (32%). Характерними видами для
данної зони є Texstularia jurassica G ü m b e l .,
Epistomina stelligeraformis M j a t l . Виявлені ску-
пчення Marsonella doneziana D a і n , Lagena
apiculata (R e u s s ), Epistomina limbata K a p t .,
E. stelligeraformis M j a t l . В угрупуваннях прису-
тні бореальні форми - Lenticulina polovinkaensis
Put.,
L. sibirensis
(Kosyr.),
L. nordformis
Put. et Rom. БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ
ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ
ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ Нижній окс-
фордський під’ярус. Зона Lenticulina quenstedti-
Globuligerina oxfordiana. Нижньооксфордські від-
клади досліджувалися в Судацько-Феодосійській
структурно-фаціальній зоні (далі скорочення
СФЗ) в розрізах відслонень поблизу с. Богатівка
на півострові Меганом, Янишарській бухті та в
св. № 2 на г. Карадаг (рис. 1, 2) [11]. Відклади
представлені зеленувато-сірими темно-сірими
алевритистими, піскуватими глинами. Угрупо-
вання форамініфер виділені з глинистих фацій. Дані угруповання представлені бентосними (93%)
і планктонними (7%) формами. За кількістю видів
форми з секреційною черепашкою (60%) перева-
жають над аглютинуючими (33%), але за кількі-
стю екземплярів їх співвідношення зворотнє, в де-
яких розрізах, а саме св. 2 на г. Карадаг, відсло-
нення поблизу с. Богатівка. Одним з важливих
компонентів даного фауністичного угрупування є
присутність у відкладах нижнього оксфорду пла-
нктонних форм. Globuligerina oxfordiana (Grig.) –
це вид вузького стратиграфічного і широкого гео-
графічного поширення. Він зустрічається у розрі-
зах нижнього оксфорду в обмеженій кількості
(10-30 екз, на 200 г сухої породи). Угрупування
малочисельні, рідше багаточисельні. Характер-
ними видами для данної зони є Lenticulina
attenuatа (Kub. et Zw.), Epistomina nemunensis
Grig., Spirillina kübleri M j a t l Globuligerina
oxfordiana
(G r i g .). Виявлені
скупчення
Ammodiscus
cf. uglicus
ehremeevae
D a i n ,
Psammatodendron
cf. dichotomicum
Neagu,
Epistomina nemunensis Grig., Ophthalmidium
dilatatum P a a l z ., O. tenuisimum , O. cf. stuifense
(Paalz.), Sigmoilina milioliniformis (P a a l z .). Від-
клади зони Lenticulina quenstedti-Globuligerina
oxfordiana простежені у верхній частині судацької
світи. [ ;
]
ОКСФОРДСЬКИЙ ЯРУС. Нижній окс-
фордський під’ярус. Зона Lenticulina quenstedti-
Globuligerina oxfordiana. Нижньооксфордські від-
клади досліджувалися в Судацько-Феодосійській
структурно-фаціальній зоні (далі скорочення
СФЗ) в розрізах відслонень поблизу с. Богатівка
на півострові Меганом, Янишарській бухті та в
св. № 2 на г. Карадаг (рис. 1, 2) [11]. Відклади
представлені зеленувато-сірими темно-сірими
алевритистими, піскуватими глинами. Угрупо-
вання форамініфер виділені з глинистих фацій. Дані угруповання представлені бентосними (93%)
і планктонними (7%) формами. За кількістю видів
форми з секреційною черепашкою (60%) перева-
жають над аглютинуючими (33%), але за кількі-
стю екземплярів їх співвідношення зворотнє, в де-
яких розрізах, а саме св. 2 на г. Карадаг, відсло-
нення поблизу с. Богатівка. Одним з важливих
компонентів даного фауністичного угрупування є
присутність у відкладах нижнього оксфорду пла-
нктонних форм. Globuligerina oxfordiana (Grig.) –
це вид вузького стратиграфічного і широкого гео-
графічного поширення. Він зустрічається у розрі-
зах нижнього оксфорду в обмеженій кількості
(10-30 екз, на 200 г сухої породи). Угрупування
малочисельні, рідше багаточисельні. БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ
ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ
ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ Янишарська бухта,
17. м. Св. Іллі, 18. Двоякірна бухта, 19. с. Богатівка (п-в Меганом). – відслонення з фауною; – відслонення пусті; – свердловини Рис. 1. Карта-схема фактичного матеріалу. Рис. 1. Карта-схема фактичного матеріалу. Умовні позначення: Умовні позначення:
Структурно-фаціальні зони: І – Сухоріченсько-Байдарська, ІІ – Ай-Петрі-Бабуганська,
ІІІ – Демерджі-Карабійська, ІV – Судацько-Феодосійська. Розрізи: 1. с. Тилове, 2 с. Орлине, 3. 49 км дороги Ялта-Севастополь, 4. Богата Ущелина,
5. Ай-Петринська Яйла, 6. м. Іограф, 7. г. Ай-Петрі, 8. Урочище Панагії, 9. г. Карабі-Яйла,
10. с. Красноселівка (р. Тонасу), 11. с. Красноселівка (с. Кучук-Узень), 12. хр. Суук-Су (св. 47),
13. смт Богатоє (св. 54), 14. г. Карадаг (св. 2), 15. г. Карадаг (св. 3), 16. Янишарська бухта,
17. м. Св. Іллі, 18. Двоякірна бухта, 19. с. Богатівка (п-в Меганом). – відслонення з фауною; – відслонення пусті; – свердловини Умовні позначення:
Структурно-фаціальні зони: І – Сухоріченсько-Байдарська, ІІ – Ай-Петрі-Бабуганська,
ІІІ – Демерджі-Карабійська, ІV – Судацько-Феодосійська. Розрізи: 1. с. Тилове, 2 с. Орлине, 3. 49 км дороги Ялта-Севастополь, 4. Богата Ущелина,
5. Ай-Петринська Яйла, 6. м. Іограф, 7. г. Ай-Петрі, 8. Урочище Панагії, 9. г. Карабі-Яйла,
10. с. Красноселівка (р. Тонасу), 11. с. Красноселівка (с. Кучук-Узень), 12. хр. Суук-Су (св. 47),
13. смт Богатоє (св. 54), 14. г. Карадаг (св. 2), 15. г. Карадаг (св. 3), 16. Янишарська бухта,
17. м. Св. Іллі, 18. Двоякірна бухта, 19. с. Богатівка (п-в Меганом). – відслонення з фауною; – відслонення пусті; – свердловини Структурно-фаціальні зони: І – Сухоріченсько-Байдарська, ІІ – Ай-Петрі-Бабуганська,
ІІІ – Демерджі-Карабійська, ІV – Судацько-Феодосійська. Розрізи: 1. с. Тилове, 2 с. Орлине, 3. 49 км дороги Ялта-Севастополь, 4. Богата Ущелина,
5. Ай-Петринська Яйла, 6. м. Іограф, 7. г. Ай-Петрі, 8. Урочище Панагії, 9. г. Карабі-Яйла,
10. с. Красноселівка (р. Тонасу), 11. с. Красноселівка (с. Кучук-Узень), 12. хр. Суук-Су (св. 47),
13. смт Богатоє (св. 54), 14. г. Карадаг (св. 2), 15. г. Карадаг (св. 3), 16. Янишарська бухта,
17. м. Св. Іллі, 18. Двоякірна бухта, 19. с. Богатівка (п-в Меганом). – відслонення з фауною; – відслонення пусті; – свердловини Zw.), Spirillina kübleri Mjatl. Разом з тим вияв-
лені скупчення Rhizammina indivisa Brady,
Psammatodendron cf. dichotomicum Neagu. Відк-
лади
верств
з
Epistomina
praetatariensis-
Globuligerina parva простежені у верхній частині
манджильської світи. переважають над аглютинуючими (2%). Характе-
рними видами є Texstularia densa Gofman,
Ophthalmidium
sigmoiliniformis
(A n t . ),
Lenticulina
nodosa
(R e u s s ),
L. uspenskajae
K . K u z ., L. БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ
ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ
ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ Характер-
ними видами для данної зони є Lenticulina
attenuatа (Kub. et Zw.), Epistomina nemunensis
Grig., Spirillina kübleri M j a t l Globuligerina
oxfordiana
(G r i g .). Виявлені
скупчення
Ammodiscus
cf. uglicus
ehremeevae
D a i n ,
Psammatodendron
cf. dichotomicum
Neagu,
Epistomina nemunensis Grig., Ophthalmidium
dilatatum P a a l z ., O. tenuisimum , O. cf. stuifense
(Paalz.), Sigmoilina milioliniformis (P a a l z .). Від-
клади зони Lenticulina quenstedti-Globuligerina
oxfordiana простежені у верхній частині судацької
світи. В Судацько-Феодосійській СФЗ верхньоо-
ксфордські відклади представлені сланцевими
глинами, глинистими алевролітами та мергелями
зеленувато-сірими піщанистими, з яких виділені
форамініферові угрупування. Угрупування мало-
чисельні, представлені бентосними формами. За
кількістю видів форми з секреційною черепаш-
кою (98%) переважають над аглютинуючими
(2%). Характерними видами є Epistomina volgensis
Mjatl., E. uhligi Mjatl. Виявлені скупчення
Epistomina limbata K a p t . В верхньооксфордсь-
ких угрупуваннях продовжують існувати пооди-
нокі
верхньокеловейські
Lenticulina
tumida
(M j a t l . ) ,
L. parmula
G o f . ,
Epistomina
mosquensis
U h l i g . та
нижньооксфордські
Epistomina
nemunensis
G r i g . ,
Saracenaria
cornucopiae
(S c h w a g e r ). Відклади
зони
Lenticulina russiensis - Epistomina uhligi просте-
жені у нижній частині манджильської світи. КІМЕРИДЖСЬКИЙ ЯРУС. Нижній кіме-
риджський
під’ярус. Верстви
Epistomina
praetatariensis-Globuligerina parva. Відклади ниж-
нього кімериджу досліджувалися в Судацько-Фе-
одосійській СФЗ в розрізі відслонення поблизу
с. Богатівка на півострові Меганом (Рис. 1, 2) [11]. Відклади представлені товщею глин з сидери-
тами, з яких виділені форамініферові угрупу-
вання. Дані угрупування малочисельні, представ-
лені бентосними формами, серед яких аглютину-
ючі форми представлені більшістю екземплярів
(74%) в порівнянні з секреційними (26%). Харак-
терними видами є Lenticulina simplex (Kubl. et Верхній
оксфордський
під’ярус. Зона
Lenticulina russiensis-Epistomina uhligi. Верхньоо-
ксфордські відклади досліджувалися в Ай-Петрі- Верхній
оксфордський
під’ярус. Зона
Lenticulina russiensis-Epistomina uhligi. Верхньоо-
ксфордські відклади досліджувалися в Ай-Петрі- - 51 - Серія «Геологія. Географія. Екологія», випуск 49 Рис. 1. Карта-схема фактичного матеріалу. Умовні позначення:
Структурно-фаціальні зони: І – Сухоріченсько-Байдарська, ІІ – Ай-Петрі-Бабуганська,
ІІІ – Демерджі-Карабійська, ІV – Судацько-Феодосійська. Розрізи: 1. с. Тилове, 2 с. Орлине, 3. 49 км дороги Ялта-Севастополь, 4. Богата Ущелина,
5. Ай-Петринська Яйла, 6. м. Іограф, 7. г. Ай-Петрі, 8. Урочище Панагії, 9. г. Карабі-Яйла,
10. с. Красноселівка (р. Тонасу), 11. с. Красноселівка (с. Кучук-Узень), 12. хр. Суук-Су (св. 47),
13. смт Богатоє (св. 54), 14. г. Карадаг (св. 2), 15. г. Карадаг (св. 3), 16. БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ
ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ
ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ immensa K. K u z ., Astacolus laudatus
G o f m a n , Epistomina ventriosa Esp. et Sigal,
Pseudonodosaria
tutkovskii
(M j a t l . ),
Globospirillina caucasica G o f . , Trocholina alpina
(Leupold), T. solecensis Biel. et Poz., Ramulina
spinata
A n t . ,
Discorbis
agalarovae
Ant. D. crimicus S c h k h i n a . Виявлені скупчення
Epistomina
ventriosa
E s p . e t S i g a l ,
Globospirillina caucasica Go f . , Spirillina kübleri
M j a t l . В угрупуваннях ще продовжують існу-
вання деякі кімериджські види Lenticulina cf. essica Putrja, L. cf. acutata P u t r j a , L. cf. pertiosa
P u t r j a , а також з'являються поодинокі нижньо-
беріаські, а саме Ammobaculites cf. incostans
gracielis (B r a t . e t B r a n d ), Stomatostoecha cf. enisalensis Gorb., Dentalina cf. nana R e u s s ,
Siphoninella antique G o r b . , Trocholina cf. burlini
G o r b . Відклади
зони
Anchispirocyclina
lusitanica-Melathrokerion spirialis простежені у
верхній частині деймен-деринської світи. ТИТОНСЬКИЙ ЯРУС. Верхній титонсь-
кий під’ярус. Зона Anchispirocyclina lusitanica-
Melathrokerion spirialis. У вивчених автором роз-
різах
мікропалеонтологічно
охарактеризовані
тільки верхньотитонські відклади. Дані відклади
досліджувалися
в
Сухоріченсько-Байдарській
СФЗ (відслонення с. Орлине, с. Тилове), в Демер-
джі-Карабійській СФЗ (відслонення на околицях
с. Боксан північний схил г. Карабі-Яйла) і в Суда-
цько-Феодосійській СФЗ (відслонення с. Красно-
селівка, Двоякірна бухта та свердловина № 54
район смт Богате) (Рис. 1, 2) [7; 9; 10; 12]. ТИТОНСЬКИЙ ЯРУС. Верхній титонсь-
кий під’ярус. Зона Anchispirocyclina lusitanica-
Melathrokerion spirialis. У вивчених автором роз-
різах
мікропалеонтологічно
охарактеризовані
тільки верхньотитонські відклади. Дані відклади
досліджувалися
в
Сухоріченсько-Байдарській
СФЗ (відслонення с. Орлине, с. Тилове), в Демер-
джі-Карабійській СФЗ (відслонення на околицях
с. Боксан північний схил г. Карабі-Яйла) і в Суда-
цько-Феодосійській СФЗ (відслонення с. Красно-
селівка, Двоякірна бухта та свердловина № 54
район смт Богате) (Рис. 1, 2) [7; 9; 10; 12]. ТИТОНСЬКИЙ ЯРУС. Верхній титонсь-
кий під’ярус. Зона Anchispirocyclina lusitanica-
Melathrokerion spirialis. У вивчених автором роз-
різах
мікропалеонтологічно
охарактеризовані
тільки верхньотитонські відклади. Дані відклади
досліджувалися
в
Сухоріченсько-Байдарській
СФЗ (відслонення с. Орлине, с. Тилове), в Демер-
джі-Карабійській СФЗ (відслонення на околицях
с. Боксан північний схил г. Карабі-Яйла) і в Суда-
цько-Феодосійській СФЗ (відслонення с. Красно-
селівка, Двоякірна бухта та свердловина № 54
район смт Богате) (Рис. БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ
ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ
ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ 1, 2) [7; 9; 10; 12]. В Сухоріченсько-Байдарській СФЗ відклади
представлені перешаруванням алевритистих вап-
няків, сидеритів і глин. Форамініферові угрупу-
вання виділені з глин. Угрупування малочисельні,
представлені бентосними формами. За кількістю
видів форми з секреційною черепашкою (98%) - 52 - Вісник Харківського національного університету імені В.Н. Каразіна В Демерджі-Карабійській СФЗ відклади пред-
ставлені перешаруванням глинистих і піскуватих
вапняків, карбонатних глин. Форамініферові
угрупування виділені з глин. Угрупування мало-
чисельні, представлені бентосними формами. За
кількістю видів форми з секреційною черепаш-
кою (92%) переважають над аглютинуючими
(8%). Характерними видами є Stomatostoecha
compressa
Gorb.,
Texstularia
densa
Gof.,
Melathrokerion spirialis
Gorb. , Belorussiella
taurica G o r b . , Lenticulina. vistulae B i e l . E t
P o z . ,
L. immensa
K . K u z . ,
Epistomina
ventriosa Esp. et Sigal, Globospirillina caucasica
Gof. , Trocholina alpina (Leup.). В угрупуваннях
з′являються поодинокі нижньоберіаські види:
Lenticulina cf. neocomiana (R o m . ), Trocholina
molesta G o r b . Разом з тим виявлені скупчення
Epistomina ventriosa Esp. et Sigal, Trocholina molesta G o r b . Відклади зони Anchispirocyclina
lusitanica-Melathrokerion spirialis простежені у ни-
жній частині беденекирської світи. molesta G o r b . Відклади зони Anchispirocyclina
lusitanica-Melathrokerion spirialis простежені у ни-
жній частині беденекирської світи. В Судацько-Феодосійській СФЗ відклади
представлені вапняками глинистими, сірими, пе-
літоморфними, які поступово заміщуються вапня-
ками оолітовими, глинами алевритистими вапни-
стими, строкатими, дуже міцними, темно-сірими,
зеленувато-сірими глинами з частими прошар-
ками світло-сірих і рожевих вапняків, глинами
алевролітовими темно-зеленувато-сірими плитча-
стими з прошарками вапняків брунатно-сірих мі-
цних детритових. Форамініферові угрупування
виділені з глинистих фацій. Угрупування багато-
чисельні, рідше малочисельні, представлені бен-
тосними (99%) і планктонними (1%) формами. За
кількістю видів форми з секреційною черепаш-
кою (84%) переважають над аглютинуючими Рис. 2.1. Біостратиграфічна схема верхньоюрських-нижньокрейдових відкладів
(оксфорд-нижній беріас) Гірського Криму Рис. 2.1. Біостратиграфічна схема верхньоюрських-нижньокрейдових відкладів
(оксфорд-нижній беріас) Гірського Криму Рис. 2.1. Біостратиграфічна схема верхньоюрських-нижньокрейдових відкладів
(оксфорд-нижній беріас) Гірського Криму - 53 - Серія «Геологія. Географія. Екологія», випуск 49 Рис. 2.2. Біостратиграфічна схема верхньоюрських-нижньокрейдових відкладів
(оксфорд-нижній беріас) Гірського Криму
(15%) В данному угрупованні виявлені планк-
(Leup ) Discorbis crimicus Schok
D agalarovae Рис. 2.2. Біостратиграфічна схема верхньоюрських-нижньокрейдових відкладів
(оксфорд-нижній беріас) Гірського Криму Рис. 2.2. Біостратиграфічна схема верхньоюрських-нижньокрейдових відкладів
(оксфорд-нижній беріас) Гірського Криму (Leup.), Discorbis crimicus Schok., D. agalarovae
Ant., D. infracretaceous Schok. В угрупуваннях
з′являються поодинокі нижньоберіаські види:
Gaudryina cf. alternans Gorb., Paleotexstularia
crimica Gorb., Dorothia cf. praeoxycona Moul.,
Verneuilina
angularis
Gorb.,
Lenticulina cf. neocomiana (R o m . ), L. macra Gorb., Trocholina
molesta G o r b . , T. burlini Gorb., T. elongata
(Leup.), Siphoninella antique Gorb. Разом з тим ви-
явлені скупчення Anchispirocyclina lusitanica
(Egger), Lenticulina macra Gorb., Marginulina cf. mollis K. Kuz., Epistomina ventriosa Epit. Вісник Харківського національного університету імені В.Н. Каразіна et Sig.,
Globospirillina caucasica (Hoff.), Spirillina kübleri (Leup.), Discorbis crimicus Schok., D. agalarovae
Ant., D. infracretaceous Schok. В угрупуваннях
з′являються поодинокі нижньоберіаські види:
Gaudryina cf. alternans Gorb., Paleotexstularia
crimica Gorb., Dorothia cf. praeoxycona Moul.,
Verneuilina
angularis
Gorb.,
Lenticulina cf. neocomiana (R o m . ), L. macra Gorb., Trocholina
molesta G o r b . , T. burlini Gorb., T. elongata
(Leup.), Siphoninella antique Gorb. Разом з тим ви-
явлені скупчення Anchispirocyclina lusitanica
(Egger), Lenticulina macra Gorb., Marginulina cf. mollis K. Kuz., Epistomina ventriosa Epit. et Sig.,
Globospirillina caucasica (Hoff.), Spirillina kübleri (15%). В данному угрупованні виявлені планк-
тонні форми (Globuligerina gulekhensis Gorb.), які
представлені поодинокими видами. Характер-
ними видами є Glomospira multivoluta Rom.,
Anchispirocyclina
lusitanica
(Egger),
Stomatostoecha compressa Gorb., Melathrokerion
spirialis Gorb., M. eospirialis Gorb., Texstularia
densa Gof., Belorussiella taurica Gorb., Lenticulina
immensa K. Кuz., Astacolus laudatus (Reuss),
Saracenaria
tsaramandrosoensis
Epit. et Sig.,
Pseudonodosaria tutkovskii (Mjatl.), Ophthalmidium
sigmoiliniformis (Ant.), Ramulina spinata Ant.,
Globospirillina caucasica (Goff.), Trocholina alpinа - 54 - Вісник Харківського національного університету імені В.Н. Каразіна існик Харківського національного університе spirialis
Gorb.,
Belorussiella
taurica
Gorb.,
Verneuilina angularis Gorb., V. subminuta Gorb.,
Lenticulina cf. ambanjabensis
Espit. et Sig.,
Discorbis praelongus Gorb., D. crimicus Schok.,
D. infracretaceous Schok., Pseudolamarckina reussi
(A n t . ),
Globospirillina
neocomiana
(Moul.),
Siphoninella
antique
Gorb.,
Protopeneroplis
ultragranulatus Gorb., Trocholina alрina (Leup.),
T. molesta Gorb. T. burlini Gorb., T. elongatа
(Leup.). В угрупуваннях з′являються поодинокі
крейдові види: Triplasia emslandensis acuta
Bart. et Br., Gaudryina neocomica Chal., G. gradata
Bert.,
Gaudryiadhella
onachensis
(Sig.),
Quinqueloculina
infravalanginiana
Bart.,
Lenticulina neocomiana Gorb., L. macra Gorb., L. cf. onachensis onachensis, L. cf. guttata guttata (ten
Dam), L. cf. circumcidanea (Bert.), Saracenaria
cretacea Gorb., S cf. navicula (Orb.), Vaginulina
recta Reuss, V. truncata Reuss, V. cf. hagenovi
(Reuss), Vaginulinopsis cf. grata (Reuss), Nodosaria
grassulariformis Bass., N. cf. concinna Reuss,
N. paupercula Reuss та ін. Також продовжують іс-
нувати поодинокі титонські види - Ammobaculites
hagni Bhal. et Ab., Ammodiscus cf. veteranus Kosyr.,
Gaudryina vadaszi Cush. et Glaz., Ophthalmidium
sigmoiliniformis
(Ant.),
Astacolus praesibirensis
Kos., A. aquilonicus (Mjatl.), A. laudatus (Reuss),
Lenticulina uspenskajae K. Kuz., L. gregaria Putrja,
L. cf. tumidiuscula Pjat., L. cf. tanatcheva Putrja,
Epistomina ventriosa
Espit. et Sig.,
Discorbis
agalarovae Ant., Trocholina solecensis Biel. et Poz. Разом з тим виявлені скупчення Glomospira
gordialis
(Jon. et Par.),
Epistomina
caracаlla
anterion Bart. et Br., Discorbis crimicus Schok.,
Globospirillina
caucasica
(Hoff.),
Lenticulina
neocomiana Gorb., L. macra Gorb., Trocholina
molesta Gorb. T. elongatа (Leup.). Вісник Харківського національного університету імені В.Н. Каразіна Відклади зони
Protopeneroplis ultragranulatus-Siphoninella antiqua
простежені у верхній частині двоякірної світи. (Mjatl.), Trocholina elongata (Leup.). Відклади
зони Anchispirocyclina lusitanica-Melathrokerion
spirialis простежені у нижній частині двоякірної
світи. БЕРІАСЬКИЙ ЯРУС. Нижній беріаський
під’ярус. Зона
Protopeneroplis
ultragranulatus - Siphoninella antiqua. Нижньобері-
аські відклади досліджувались в Демерджі-Кара-
бійській СФЗ (відслонення на околицях с. Боксан
північний схил г.Карабі-Яйла) і в Судацько-Фео-
досійській СФЗ (відслонення с. Красноселівка,
Двоякірна бухта, мис Св. Іллі та свердловинах
№ 54 район смт Багате, № 47 хр. Суук-Су) (Рис. 1,
2) [7; 9; 10; 12]. В Демерджі-Карабійській СФЗ відклади пред-
ставлені глинами карбонатними, з яких виділені
форамініферові угрупування. Угрупування мало-
чисельні, представлені бентосними формами. За
кількістю видів форми з секреційною черепаш-
кою (72%) переважають над аглютинуючими
(28%). Характерними видами є Triplasia cf. emslandensis
acuta
B a r t . e t B r a n d ,
Lenticulina macra G o r b . , Discorbis crimicus
S c h o h . , Trocholina molesta G o r b . , T. durlini
G o r b . , T. alpina (L e u p . ). В угрупуваннях ще
продовжують існувати поодинокі титонські види:
Stomatostoecha compressa Gorb., Ammobaculites
hagni
B h a l l a e t A b b a s ,
A. infravolgensis
M j a t l . , A. subaequalis M j a t l . , Melathrokerion
spirialis
G o r b . ,
Texstularia
densa
G o f .,
Quinqueloculina podlubiensis T e r e s . , Lenticulina
uspenskajae K. Kuz., Epistomina cf. ventriosa
E s p . e t S i g . Відклади зони Protopeneroplis
ultragranulatus-Siphoninella antiqua простежені у
верхній частині беденекірської світи. р
р
В Судацько-Феодосійській СФЗ відклади
представлені глинами алевритистими, строка-
тими, щільними, вапнистими, пісковиками сі-
рими дрібнозернистими глинистими, вапняками
глинистими, сірими, алевролітами піскуватими та
пісковиками глинистими дрібнозернистими та
глинами темно-сірими, перешаруванням зелену-
вато-сірих тонкоплитчастих глин, алевролітів, ко-
ричневато-сірих масивних вапняків і мергелів те-
мно-зеленувато-сірих міцних плитчастих, вапня-
ком темно-брунатним дрібнозернистим з тонкими
лінзами чорної і сірої глини. Форамініферові
угрупування виділені з алевролітів, мергелів,
глин, вапняків. Угрупування багаточисельні, рі-
дше малочисельні, представлені бентосними
(99%) і планктонними (1%) формами. За кількі-
стю видів форми з секреційною черепашкою
(82%) переважають над аглютинуючими (17%). Планктоні форми представлені поодинокі видами
Globuligerina gulekhensis (Gorb. et Por.)., G. cf. caucasica Gorb. et Por.. Характерними видами є
Stomatostoecha enisalensis Gorb., Melathrokerion Висновки. Література р
ур
1. Анікеєва О.В., Жабіна Н.М. Умови седиментації верхньоюрських відкладів Гірського Криму (Ялтинський ам-
фітеатр) [Текст] / О.В. Анікеєва, Н.М. Жабіна // Викопна фауна і флора України: Палеоекологічний та стра-
тиграфічний аспекти. – Київ, 2009. – С. 99-103. 2. Аркадьев В.В. Новые данные по биостратиграфии берриасских отложений бассейна р. Тонасу (Горный
Крым) [Текст] / В.В. Аркадьев, Т.Н. Богданова, С.В. Лобачева // Второе всерос. совещ. «Меловая система
России: проблемы стратиграфии палеогеографии». – СПб., 2005. – С. 111-135. 3. Аркадьев В.В., Рогов М.А. Новые данные по биостратиграфии и аммонитам верхнего кимериджа и титона
Восточного Крыма [Текст] / В.В. Аркадьев, М.А. Рогов // Стратиграфия. Геол. корреляция. – 2006 (а). – Т. 14,
№ 2. – С. 90-104. 4. Аркадьев В.В. Биостратиграфия пограничних отложений юры и мела Восточного Крыма [Текст] / В.В. Ар-
кадьев, А.А. Федорова, Ю.Н. Савельева, Е.М. Тесакова // Стратиграфия. Геологическая корреляция. 2006. (б)
– Т. 14, № 3. – С. 84-112. ,
5. Аркадьев В.В. Берриас Горного Крыма [Текст] / В.В. Аркадьев, Т.Н. Богданова, А.Ю. Гужиков, С.В. Лобачева
Н.В. Мышкина, Е.С. Платонов, Ю.Н. Савельева, О.В. Шурекова, Б.Т. Янин. – Санкт-Петербург: Лема, 2012. – 473 с. 6. Геология СССР. Т. 8. Крым. Ч. 1. Геологическое описание [Текст] / под. ред. А.В. Сидоренко. – М.: Недра,
1969. – 575 с. 7. Доротяк Юлія Стратифікація титон-беріаських відкладів південно-східної частини Гірського Криму за фо-
рамініферами [Текст] / Юлія Доротяк // Палеонтол. зб. – Львів, 2007. – № 39. – C. 125-130. 8. Доротяк Ю.Б. Розподіл форамініфер в оксфордських відкладах Гірського Криму [Текст] / Ю.Б. Доротяк //
Палеонтологічні дослідження в Україні: історія, сучасний стан та перспективи: Зб. наук. праць ІГН НАН
України. – Київ, 2007. – С. 134-139. 9. Доротяк Ю.Б. Фораминиферовые комплексы пограничных титон-берриасских отложений Горного Крыма
[Текст] / Ю.Б. Доротяк // Біостратиграфічні основи побудови стратиграфічних схем фанерозою України:
Зб. наук. праць ІГН НАН України. – Київ, 2008. – С. 78-82. 10. Доротяк Ю.Б. Xарактеристика пограничных отложений юры и мела в Горном Крыму (фораминиферы, изве-
стковый н аннопланктон, диноцисты, палинокомплексы) [Текст] / Ю.Б. Доротяк, А.В. Матвеев, Е.А. Шевчук
// Викопна фауна і флора України: Палеоекологічний та стратиграфічний аспекти: Зб. наук. праць ІГН НАН
України. – Київ, 2009. – С. 108-117. 11. Доротяк Ю.Б. Палеогеографія пізньокеловейсько – ранньокімериджського басейну Гірського Криму [Текст]
/ Ю.Б. Доротяк // Проблеми обґрунтування регіональних стратонів фанерозою України: Матеріали XXXVII
сесії Палеонтол. т-ва НАН України. – Київ, 2016. – С. 47-49. 12. Доротяк Ю.Б., Клименко Ю.В. Вісник Харківського національного університету імені В.Н. Каразіна За результатами досліджень авто-
ром в розрізах Гірського Криму за форамініфе-
рами встановлені біостратиграфічні підрозділи
зони:
в
нижньому
оксфорді
-
Lenticulina
quenstedti-Globuligerina oxfordiana; у верхньому
оксфорді - Lenticulina russiensis-Epistomina uhligi;
у верхньому титоні - Anchispirocyclina lusitanica-
Melathrokerion spirialis та у нижньому беріасі, ни-
жню
зону
-
Protopeneroplis
ultragranulatus-
Siphoninella antiqua; і верстви в нижньому кімери-
джі
з
Epistomina
praetatariensis-Globuligerina
parva. Зона нижнього оксфорду Lenticulina
quenstedti-Globuligerina oxfordiana є в верхній ча-
стині судацької світи Судацько-Феодосійській
СФЗ; зона верхнього оксфорду Lenticulina
russiensis-Epistomina uhligi простежується в ниж-
ній частині яйлинської світи в Ай-Петрі-Бабуган-
ській та в нижній частині манджильської світи в Висновки. За результатами досліджень авто-
ром в розрізах Гірського Криму за форамініфе-
рами встановлені біостратиграфічні підрозділи
зони:
в
нижньому
оксфорді
-
Lenticulina
quenstedti-Globuligerina oxfordiana; у верхньому
оксфорді - Lenticulina russiensis-Epistomina uhligi;
у верхньому титоні - Anchispirocyclina lusitanica-
Melathrokerion spirialis та у нижньому беріасі, ни-
жню
зону
-
Protopeneroplis
ultragranulatus-
Siphoninella antiqua; і верстви в нижньому кімери-
джі
з
Epistomina
praetatariensis-Globuligerina
parva. Зона нижнього оксфорду Lenticulina
quenstedti-Globuligerina oxfordiana є в верхній ча-
стині судацької світи Судацько-Феодосійській
СФЗ; зона верхнього оксфорду Lenticulina
russiensis-Epistomina uhligi простежується в ниж-
ній частині яйлинської світи в Ай-Петрі-Бабуган-
ській та в нижній частині манджильської світи в - 55 - Серія «Геологія. Географія. Екологія», випуск 49 Судацько-Феодосійській СФЗ; зона верхнього ти-
тону Anchispirocyclina lusitanica-Melathrokerion
spirialis простежується у верхній частині деймен-
деринської світи в Сухоріченсько-Байдарській, у
нижній частині беденекирської світи в Демерджі-
Карабійській та у нижній частині двоякірної світи
Судацько-Феодосійській СФЗ; зона нижнього бе-
ріасу Protopeneroplis ultragranulatus-Siphoninella
antiqua – простежується у верхній частині бедене-
кірської світи Демерджі-Карабійській та у верх-
ній частині двоякірної світи Судацько-Феодо- сійській СФЗ; верстви нижнього кімериджу з
Epistomina praetatariensis-Globuligerina parva про-
стежуються у верхній частині манджильської
світи в Судацько-Феодосійській СФЗ. В резуль-
таті проведених мікрофауністичних досліджень
верхньоюрських та нижньокрейдових (оксфорд-
нижній беріас) відкладів у досліджених автором
розрізах встановлені біостратиграфічні підроз-
діли за форамініферами та доповнено палеонтоло-
гічну характеристику даних відкладів Гірського
Криму. UDC 56.076:551.762(477) (
)
Yuliya Dorotyak,
Junior Researcher, Institute of Geological Sciences of NAS of Ukraine,
55-b O. Honchara St., Kyiv, 01601, Ukraine,
е-mail: dorotyak78@gmail.com, http://orcid.org/0000-0002-0498-1892 (
)
Yuliya Dorotyak,
Junior Researcher, Institute of Geological Sciences of NAS of Ukraine,
55-b O. Honchara St., Kyiv, 01601, Ukraine,
е-mail: dorotyak78@gmail.com, http://orcid.org/0000-0002-0498-1892 (
)
Yuliya Dorotyak,
Junior Researcher, Institute of Geological Sciences of NAS of Ukraine,
55-b O. Honchara St., Kyiv, 01601, Ukraine, y
е-mail: dorotyak78@gmail.com, http://orcid.org/0000-0002-0498-1892 Література Форамініфери і спікули губок з пограничних відкладів юри та крейди східної
частини Гірського Криму [Текст] / Ю.Б. Доротяк, Ю.В. Клименко // 40 років Палеонтологічному товариству
України: Матеріали XXXIX сесії Палеонтол. т-ва НАН України. – Київ, 2017. – С. 89-91. 13. Жабіна Н.М. До питання проведення межі юри та крейди в Криму за форамініферами [Текст] / Н.М. Жабіна
// Матеріали палентологічного товариства НАН України. Біосфера і геологічні катастрофи. – Київ, 1997. –
С. 33-34. 14. Жабіна Н.М., Анікеєва О.В. Нові дані, щодо віку двоякірної світи (Південно-східний Крим) [Текст] / Н.М. Жа-
біна, О.В. Анікеєва // Палеонтологічні дослідження в удосконаленні стратиграфічних схем фанерозойських
відкладів: Матеріали XXXVII сесії Палеонтол. т-ва НАН України. – Київ, 2012. – С. 41-43. ва К.И., Горбачик Т.Н. Стратиграфия и фораминиферы верхней юры и нижнего мела Крыма [Текст
знецова, Т.Н. Горбачик; под. ред. Вахрамеев В.А. – М. : Наука, 1985. – 132 с. Й
Й 15. Кузнецова К.И., Горбачик Т.Н. Стратиграфия и фораминиферы верхней юры и нижнего мела Крыма [Текст]
/ К.И. Кузнецова, Т.Н. Горбачик; под. ред. Вахрамеев В.А. – М. : Наука, 1985. – 132 с. 16 Л
Р Й Н
йд
д і і д
У
ї
[Т
] / Р Й Л
К
Н
Д
1992
208 / К.И. Кузнецова, Т.Н. Горбачик; под. ред. Вахрамеев В.А. – М. : Наука, 1985. – 132 с. 16 Лещух Р Й Нижня крейда заходу і півдня України [Текст] / Р Й Лещух
К : Наук Думка 1992
208 с у
ц
,
р
;
р
р
у
,
16. Лещух Р.Й. Нижня крейда заходу і півдня України [Текст] / Р.Й. Лещух. – К.: Наук. Думка, 1992. – 208 с. Й
Й щу
р
у
р
[
]
щу
у
Ду
17. Лещух Р.Й., Юрські відклади півдня України [Текст] / Р.Й. Лещух, В.В. Пермяков, Б.М. Полухтович. – Львів:
Євросвіт, 1999. – 336 с. р
18. Матвеев А.В. Известковый наннопланктон из пограничных отложений юры и мела Восточного Крыма
[Текст] / А.В. Матвеев // Матеріали XXXIX сесії палеонтологічного товариства НАН України. – Київ. 2017. – С. 92-93. - 56 - Вісник Харківського національного університету імені В.Н. Каразіна 19. Матвеев А.В. Титон центральной части Горного Крыма [Текст] / А.В. Матвеев // Матеріали міжнародної
науково-практичної конференції «Регіон-2008: Стратегія оптимального розвитку». – Харків. 2008. – С. 341-344. 20. Матвеев А.В. Известковий нанопланктон титона Восточного Крыма [Текст] / А.В. Матвеев // Збірник нау-
кових праць Інституту геологічних наук НАН України. Викопна фауна і флора України: Палеоекологічний та
стратиграфічний аспекти. – Київ, 2009. – С. 104-107. 21. Матвеев А.В. Стратиграфия нижнего мела юга Украины по известковому наннопланкт
А.В. Матвеев // Геол. журн. 2014. № 4. – С. 67-74. 22. Матвеев А.В. Известковый нанопланктон нижнего берриаса Горного Крыма [Текст] / А.В. Матвеев // Мело-
вая система России и ближнего зарубежья: Проблемы стратиграфии и палеогеографи под ред. Е.Ю. Бара-
бошкина, И.В. Благовещенского. V Всерос. совещ. – Ульяновск: УлГУ, 2010. – С. 251-256. 23. Матвеев А.В. Стратиграфия нижнего мела Юго-Восточного Крыма по известковому наннопланктону
[Текст] / А.В. Матвеев // Современная микропалеонтология. Труды Всероссийского микропалеонтологиче-
ского совещания. – Москва. 2012. – С. 313-316. щ
24. Матлай Л.М. Стратиграфічне розчленування примежових розрізів юри і крейди Східного Криму за вапняко-
вим нанопланктоном [Текст] / Л.М. Матлай // Доп. НАН України. 2011. № 1. – С. 106-111. 25. Пермяков В.В., Пермякова М.Н., Чайковский П.П. Новая схема стратиграфии юрских отложений Горного
Крыма [Текст] / В.В. Пермяков, М.Н. Пермякова, П.П. Чайковский // Препр. АН Укр. Ин-т геол. наук; 91-12. – Киев, 1991. – 38 с. 26. Пермяков В.В. Новые местные стратиграфические подразделения в юре Горного Крыма [Текст] / В. Пе-
рмяков, М.Н. Пермякова, О.Г. Седенко, П.П. Чайковский // Геологічний журнал. – Київ, 1993. № 2. - С. 79-81. 26. Пермяков В.В. Новые местные стратиграфические подразделения в юре Горного Крыма [Текст] / В. Пе-
рмяков, М.Н. Пермякова, О.Г. Седенко, П.П. Чайковский // Геологічний журнал. – Київ, 1993. № 2. - С. 79-81. 27. Стратиграфія юрських відкладів УРСР. Т. 7. Юра. [Текст] / За ред. І.М. Ямниченка. – Київ: Наук. думка, 27. Стратиграфія юрських відкладів УРСР. Т. 7. Юра. [Текст] / За ред. І.М. Ямниченка. – Київ
1969. – 216 с. 28. Стратиграфическая схема юрских отложений Украины [Текст]. – Киев: Наук. думка, 1970. – 28 с. 28. Стратиграфическая схема юрских отложений Украины [Текст]. – Киев: Наук. думка, 1970. – 28 с. 29 С
ф
й У
б
[Т
]
К
Н тратиграфическая схема юрских отложений Украины [Текст]. – Киев: Наук. думка, 1970. – 28 с. р
р ф
р
р
[
]
у
у
29. Стратиграфическая схема меловых отложений Украины и объяснительная записка. Вісник Харківського національного університету імені В.Н. Каразіна [Текст]. – Киев: Наук. думка, 1971. – 91 с. 30. Стратиграфія верхнього протерозою та фанерозою України. Т. 1. Стратиграфія верхнього протерозою,
палеозою та мезозою України [Текст] / Гол. ред. П.Ф. Гожик, ІГН НАН України. – Київ: Логос, 2013. – 638 с. UDC 56.076:551.762(477) BIOSTRATIGRAPHIC PARTITION OF THE UPPER JURASSIC
AND LOWER CRETACEOUS (OXFORDIAN - LOWER BERRIASIAN)
DEPOSITS OF THE CRIMEAN MOUNTAINS FOR FORAMINIPHERS Introduction. Upper Jurassic and Lower Cretaceous deposits in the Crimean Mountains are widespread
and are represented by all stages - Oxfordian, Kimmeridgian, Tithonian, and Berriasian. These deposits have
been unevenly studied. The most studied deposits are the Oxfordian, Tithonianian and Berriasian stages. The
biostratigraphic division of the Upper Jurassic and Lower Cretaceous deposits of the Mountain Crimea is based
on "Stratigraphic scheme of the Upper Jurassic and Lower Cretaceous deposits of the Mountain Crimea" 2013. been unevenly studied. The most studied deposits are the Oxfordian, Tithonianian and Berriasian stages. The
biostratigraphic division of the Upper Jurassic and Lower Cretaceous deposits of the Mountain Crimea is based
on "Stratigraphic scheme of the Upper Jurassic and Lower Cretaceous deposits of the Mountain Crimea" 2013. Purpose. Detailed biostratigraphic devision of the Upper Jurassic and Lower Cretaceous (Oxfor-
dian - Lower Berriasian) of the Mountain Crimea as results of foraminiferal analysis. g p
pp
p
Purpose. Detailed biostratigraphic devision of the Upper Jurassic and Lower Cretaceous (Oxfor-
dian - Lower Berriasian) of the Mountain Crimea as results of foraminiferal analysis. Results of the study. There are five foraminiferal complexes in sections that characterize Upper Jurassic
(Lower Upper Oxfordian, Lower Kimmeridgian, Upper Tithonian) and Lower Cretaceous (Lower Berriasian)
age. Analysis of foraminiferal complexes allowed us to establish biostratigraphical zones in each section: ox-
fordiana in the Lower Oxfordian Lenticulina quenstedti-Globuligerina; in the Upper Oxfordian Lenticulina
russiensis-Epistomina uhligi; in the Upper Tithonian Anchispirocyclina lusitanica-Melathrokerion spirialis; in
the Lower Berriasian (lower zone) Protopeneroplis ultragranulatus-Siphoninella antiqua and layers in the
Lower Kimmeridgian with Epistomina praetatariensis-Globuligerina parva. Specified foraminiferal zones
were traced by us in different structural-facial zones (zone Lenticulina quenstedti-Globuligerina oxfordiana - in
Sudak-Feodosia; zone Lenticulina russiensis-Epistomina uhligi - in Ai-Petri-Babugansk and Sudak-Feodosia;
strata layers with Epistomina praetatariensis-Globuligerina parva - in Sudak-Feodosia; zone Anchispirocyclina
lusitanica-Melathrokerion spirialis - in Sukhorychensk-Baidar, Demerdzhi-Karabijskaya and Sudak-Feodo-
siya; the zone of Protopeneroplis ultragranulatus-Siphoninella antiqua in Demerdzhi-Karabijskaya and Sudak- - 57 - рія «Геологія. Географія. Екологія», випуск 4 Feodosiya). Characteristic features of each foraminiferа zone include a complex of species, disappearing spe-
cies, and species that continue to exist, as well as particular features of the complexes. According to lithological
peculiarities and paleontological characteristics, suite and sub suite are defined. Feodosiya). Characteristic features of each foraminiferа zone include a complex of species, disappearing spe-
cies, and species that continue to exist, as well as particular features of the complexes. References f
1. Anikeyeva O.V., Zhabina N.M. (2009). Sedimentation conditions of the Upper Jurassic deposits in the Crimean Moun-
tains, Yalta Amphitheater. Fossil flora and fauna of Ukraine: paleoecological and stratigraphic aspects: Proceedings
of the Institute of Gological Sciences of the NAS of Ukraine. Kyiv, 99-103. f
f
g
f
f
y
2. Arkadiev V.V., Bogdanova T.N., Guzhikov A.Yu., Lobacheva S.V., Myshkina N.V., Platonov E.S., Savelyeva Yu.N.,
Shurekova O.V., Yanin B.T. (2012). Berriasian stage of the Mountainous Crimea. V.V. Arkadiev, T.N. Bogdanova
(Editors). St.-Petersburg: Lema, 473. 3. Arkadyev V.V., Rogov M.A. (2006). New data on biostratigraphy and ammonites of the Upper Kimmeridgian and
Tithonian of the Eastern Crimea. Stratigraphy. Geol. correlation.T. 14, No. 2, 90-104. 4. Geology of the USSR. (1969) T. 8. Crimea. Part 1. Geological description: Sidorenko A.V. (Ch. ed.). Moscow: Nedra,
575. lia (2007). The stratification of the Tithonian-Berriassian sediments of the foraminifers in the South-East
rimean Mountain Paleontologichnyy zbirnyk №39 125 130 5. Dorotyak Yulia (2007). The stratification of the Tithonian-Berriassian sediments of the foraminifers in
part of the Crimean Mountain. Paleontologichnyy zbirnyk, № 39, 125-130. 6. Dorotyak Yu.B. (2007). The distribution of foraminifers in Oxford sediments of the Mountainous Crimea. Paleonto-
logical studies in Ukraine: history, present-day state and prospects: Proceedings of the Institute of Geological Sci-
ences of the NAS of Ukraine. Kyiv, 134-139. 7. Dorotyak Yu.B. (2008). Phoraminiferۥs complex of the frontier Tithonian-Berriassian sediments of the Crimean Moun-
tains. Biostratigraphic fundamentals of creating the stratigraphic schemes of the Phanerozoic of Ukraine: Proceed-
ings of the Institute of Geological Sciences of the NAS of Ukraine. Kyiv, 78-82. 8. Dorotyak Yu.B., Matveev A.V., Shevchuk Е.A. (2009). Characteristics of the conterminal scurfs drainage area of Ju-
rassic and Cretaceous in the Crimean Mountains (foraminifers, cacareous nanoplankton, dynocysts, palinocom-
plexes). Fossil flora and fauna of Ukraine: Paleoecological and stratigraphic aspects: Proceedings of the Institute of
Geological Sciences of the NAS of Ukraine. Kyiv, 108-117. 9. Dorotyak Yu.B. (2016). Paleogeography of the Late Callovian - Earley Kimmeridgian Basin of the Crimean Moun-
tains. Problems of justification of regional stratigraphic units of Phanerozoic of Ukraine: Proceedings of XXXVII
session of Paleontological society of NAS of Ukraine. Kyiv, 47-49. f
g
y f
f
y
10. Dorotyak Yu.B., Klimenko Yu.V. (2017). Foraminifers and sponges from the boundary sediments of the Jurassic and
Cretaceous of the eastern part of the Crimean Mountains. BIOSTRATIGRAPHIC PARTITION OF THE UPPER JURASSIC
AND LOWER CRETACEOUS (OXFORDIAN - LOWER BERRIASIAN)
DEPOSITS OF THE CRIMEAN MOUNTAINS FOR FORAMINIPHERS According to lithological
peculiarities and paleontological characteristics, suite and sub suite are defined. Conclusions. As a result of the study, the biostratigraphic characteristics of the sections have been spec-
ified and the paleontological characteristics of the Upper Jurassic and Lower Cretaceous deposits of the Moun-
tain Crimea have been supplemented. Keywords: Oxfordian, Kimmeridgian, Tithonian, Berriasian, foraminiferal complexes, foraminiferal
zone, structural-facial zone, Crimean Mountains Keywords: Oxfordian, Kimmeridgian, Tithonian, Berriasian, foraminiferal complexes, foraminiferal
zone, structural-facial zone, Crimean Mountains References Paleontological society of Ukraine is 40 years: Proceedings
of XXXIX session of Paleontological society of NAS of Ukraine. Kyiv, 89-91. f
f
g
y f
f
y
11. Zhabina N.M., Anikeyeva O.V. (2012). New data on the age of the biaxial world (Southeast Crimea). Proceedings of
XXXIV session of Paleontological society of NAS of Ukraine. Kyiv, 41-43. 12. Kuznetsova K.I., Gorbachik T.H. (1985). Upper Jurassic and Lower Cretaceous stratigraphy and foraminifers of the
Crimea. Vachrameev (responsible). Moscow: Nayka, 132. 12. Kuznetsova K.I., Gorbachik T.H. (1985). Upper Jurassic and Lower Cre
Crimea. Vachrameev (responsible). Moscow: Nayka, 132. p
y
. Leschukh R.Y. (1992). Lower Cretaceous of the West and the South of Ukraine. Kyiv: Nauk.Dumka. 208. 14. Leschukh R.Y., Permyakov V.V., Polukhtovich B.M. (1999). Jurassic deposits of southern Ukraine. Lviv: Eurovision,
336. 15. Permyakov V.V, Permyakova M.N., Tchaikovsky P.P. (1991). New scheme of stratigraphy of the Jurassic deposits of
the Crimean Mountains. Pres. AU Ukr. Institute of Geological Sciences; 91-12. Kyiv, 38. V.V., Permyakova M.N., Sedenko O.G., Tchaikovsky P.P. (1993). New local stratigraphic subdivisions in
Mountains. Geol journ. № 2, 79-81. 16. Permyakov V.V., Permyakova M.N., Sedenko O.G., Tchaikovsky P.P. (1993). New local stratigraphic
the Crimean Mountains. Geol journ. № 2, 79-81. the Crimean Mountains. Geol journ. № 2, 79 81. 17. Stratigraphy of the Jurassic deposits in the Ukrainian SSR. Vol. VII. Jurassic. (1969). Kyiv: Naukova Dumka,
101-124. 17. Stratigraphy of the Jurassic deposits in the Ukrainian SSR. Vol. VII. Jurassic. (1969). Kyiv: N
101-124. of the Jurassic deposits in the Ukrainian SSR. Vol. VII. Jurassic. (1969). Kyiv: Naukova Dumka, 18. Stratigraphic scheme of the Jurassic sediments of Ukraine. (1970). Kyiv: Naukova Dumka, 28. g
p
f
f
(
y
19. Stratigraphic scheme of Cretaceous deposits of Ukraine and explanatory note. (1971). Kyiv: Na 20. Hozhyk P.F. ed. (2013). Stratigraphy of Upper Proterozoic and Phanerozoic of Ukraine. Vol.1. Stratigraphy of Upper
Proterozoic, Paleozoic and Mesozoic of Ukraine. Kyiv Institute of Geological Sciences of the NAS of Ukraine. Logos,
637. - 58 -
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A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension
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Corresponding author(s):
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Our web collection on statistics for biologists contains articles on many of the points above. Life sciences study design arch | reporting summary he therapeutic efficacy of the Ky3 antibody was tested using antibody derived from 2 separate manufacturing batches in greater tha
istinct studies. nvestigators were blinded to treatment groups. All animals were assigned a unique ID and phenotype recorded in a bespoke researc
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database. Sheffield investigators were blinded to antibody labels until after all the phenotype data were collected. Sample size
Data exclusions
Replication
Randomization
Blinding
Sample size was calculated using in house data obtained from previous studies. A sample size of at least 4 animals was used to provide greater
than 95% power to detect a difference in RVSP of 10 mmHg with a SD of 3 mmHg with 95% confidence. Additional animals were studied in
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distinct studies. Animals were randomised to treatment regime and investigators blinded to treatment labels
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Intraoperative blood transfusion volume is an independent risk factor for postoperative acute kidney injury in type A acute aortic dissection
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© The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
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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. Liu et al. BMC Cardiovasc Disord (2020) 20:446
https://doi.org/10.1186/s12872-020-01727-3 Liu et al. BMC Cardiovasc Disord (2020) 20:446
https://doi.org/10.1186/s12872-020-01727-3 Open Access Abstract Background: Type A acute aortic dissection is a life-threatening disease associated with adverse clinical outcomes. Acute kidney injury (AKI) is common after surgery. However, the relationship between intraoperative blood transfu-
sion and postoperative AKI remains unclear. Methods: The records of 130 patients who underwent type A acute aortic dissection surgery from January 2015 to
December 2018 were retrospectively analyzed. According to the Kidney Disease Improving Global Outcomes criteria,
postoperative AKI was defined based on serum creatinine concentration. Multivariable logistic regression analysis was
applied to estimate the independent association between intraoperative blood transfusion volume and the risk of
postoperative AKI. Results: Postoperative AKI was observed in 82 patients (63.08%). The in-hospital mortality was 16.15% (n = 21). Multi-
variate logistic regression showed that the amount of intraoperative blood transfusion was independently associated
with the risk of postoperative AKI in a dose-dependent manner. Every 200 ml increment of blood transfusion volume
was associated with a 31% increase in AKI risk (odds ratio 1.31 and 95% confidence interval 1.01–1.71). Conclusions: Intraoperative transfusion volume may increase the incidence of postoperative AKI. The mechanism
and influence of transfusion thresholds on AKI need to be explored in the future. Keywords: Blood transfusion, Acute kidney injury, Type A acute aortic dissection dissection, patients often suffer from unstable hemody-
namics, coagulopathy, and organ malperfusion. Acute
kidney injury (AKI) is a common complication and has
been reported as an important risk factor for mortality
in patients undergoing cardiac surgery [1, 2]. More than
30% of patients undergoing cardiac surgery with cardio-
pulmonary bypass (CPB) are likely to develop AKI and
approximately 3% of patients need renal replacement
therapy (RRT) for severe AKI. Various risk factors for
AKI, including volume depletion, hypotension, anemia,
and blood transfusion, were identified in reports [3, 4]. Freeland and colleagues reported that blood transfusion Intraoperative blood transfusion volume
is an independent risk factor for postoperative
acute kidney injury in type A acute aortic
dissection Yanli Liu1†, Yuqiang Shang2†, Ding Long1 and Li Yu1* Background Acute aortic dissection remains a clinical emergency
characterized by anterior chest or back pain and has
high morbidity and mortality, especially for type A acute
aortic dissection. Unlike other elective cardiac sur-
gery, in emergency aortic repair for type A acute aortic Full list of author information is available at the end of the article *Correspondence: yuliwhzxyy@163.com
†Yanli Liu and Yuqiang Shang have contributed equally to this work
1 Intensive Care Unit, The Central Hospital of Wuhan, Tongji Medical
College, Huazhong University of Science and Technology, Wuhan, Hubei,
China
Full list of author information is available at the end of the article Outcome variables
A AKI was categorized in accordance with the Kidney Dis-
ease: Improving Global Outcomes criteria. Postopera-
tive AKI was defined by the changes in serum creatinine
within 48 h after surgery and classified as stage 1, stage
2, and stage 3 on the basis of postoperative serum cre-
atinine. Stage 1 AKI showed more than 50% increase
or 0.3 mg/dl increase in serum creatinine. Stage 2 AKI
showed 100% increase in serum creatinine. Stage 3 AKI
showed 200% or higher or 4 mg/dl increase in serum
creatinine or initiation of renal replacement therapy
[13]. The last serum creatinine value before surgery was
defined as the baseline serum creatinine value. The indi-
cations for RRT included acidosis, electrolyte distur-
bance and fluid overload. The initiation timing of RRT
was determined by the clinician. Our primary endpoint
was the occurrence of AKI within 48 h after surgery. Sec-
ondary endpoints were stages 2–3 AKI within 48 h after Liu et al. BMC Cardiovasc Disord (2020) 20:446 Liu et al. BMC Cardiovasc Disord (2020) 20:446 Page 2 of 8 was an independent predictor of postoperative AKI in
patients undergoing coronary artery bypass grafting, aor-
tic or mitral valve surgery, or combined coronary artery
bypass grafting and valve surgery [5]. Previous studies
have also probed into the relationship between blood
transfusion and AKI, suggesting a 10–20% increase in the
risk of AKI in patients with cardiac surgery after being
given each unit of perioperative blood transfusion [6–10]. Reasonable perioperative management might curb the
development of kidney injury [11, 12]. However, the rela-
tionship between intraoperative blood transfusion and
postoperative AKI in patients undergoing type A acute
aortic dissection surgery remains unclear. For this reason,
we conducted a retrospective cohort study on patients
with type A acute aortic dissection surgery performed
with CPB, and further explored the dose–response asso-
ciation between intraoperative blood transfusion volume
and postoperative AKI. (Ethics approval NO: 2020.163). Data were anonymous,
and the requirement for informed consent was waived. The following patients were not eligible for participation:
death within 24 h after surgery; renal artery dissection
or occlusion; preoperative shock (a systolic blood pres-
sure < 90 mmHg); dialysis-dependent before surgery; no
serum creatinine measures after surgery; missing blood
transfusion volume. Finally, a total of 130 patients among
158 participants were eligible for the study (Fig. 1). Surgical technique All participants underwent right axillary artery can-
nulation for CPB. CPB was initiated after systemic hep-
arinization maintaining an activated clotting time longer
than 480 s. Cooling was initiated after CPB was estab-
lished. The ascending aorta was clamped and cold blood
cardioplegia was infused through the coronary ostia to
accomplish cardiac arrest. During CPB, the mean arte-
rial pressure was maintained between 50 and 70 mmHg. An arterial blood gas monitoring was performed every
30 min and the required hemoglobin level was at least
80 g/L. Whether to replace an aortic valve depended on
the condition of the aortic valve. If there was moderate
or severe aortic regurgitation, Bentall procedure was per-
formed. If the severity of aortic regurgitation was mild,
only ascending aorta replacement was performed. For
patients with aortic arch involvement in the dissection,
total arch replacement combined with stented elephant
trunk implantation or hybrid operation was performed. Moderate hypothermia circulatory arrest or deep hypo-
thermia circulatory arrest and selective cerebral per-
fusion were necessitated in this procedure. Moderate
hypothermia was defined as nasopharyngeal tempera-
ture between 22 and 25 °C, while deep hypothermia was
defined as nasopharyngeal temperature between 18 and
21 °C. After selective cerebral perfusion was terminated,
rewarming was initiated. Types of intraoperative blood
transfusion included erythrocytes, fresh frozen plasma,
platelets and cryoprecipitate. The blood transfusion vol-
ume depended on intraoperative bleeding and hemo-
globin concentration during CPB. Study population
Th
d The study was a retrospective cohort design, includ-
ing patients who underwent type A acute aortic dis-
section surgery in the Center Hospital of Wuhan from
January 2015 to December 2018. The diagnosis of type
A acute aortic dissection was in accordance with con-
trast-enhanced computed tomography. This study was
approved by the ethics committees of this hospital Fig. 1 Flowchart of patients underwent type A acute aortic dissection surgery Liu et al. BMC Cardiovasc Disord (2020) 20:446 Page 3 of 8 Page 3 of 8 regression was applied to estimate the risk of postop-
erative AKI. The variables used in multivariable analysis
included clinical factors that are known to increase post-
operative AKI to control confounding bias. Nonlinear
relationship was explored between intraoperative blood
transfusion volume and AKI risk following emergent tho-
racic aortic surgery via the smoothing plot generated by
generalized additive model with an adjustment for poten-
tial confounders. All of the analyses were performed
using R (https://www.R-project.org, version 3.5.2) and
EmpowerStats (https://www.empowerstats.com, X&Y
Solutions, Inc., Boston, MA) with a two-sided signifi-
cance threshold of P < 0.05. surgery, prolonged ventilation, length of stay in the ICU,
incidence of renal replacement therapy, adult respira-
tory distress syndrome (ARDS), perioperative myocardial
infarction, stroke, redo surgery, and in-hospital death. Baseline characteristics A total of 130 patients with type A acute aortic dissection
were included in this study (Table 1) with average age
54.74 ± 11.84 years old. 101 patients (77.69%) were males. The major comorbidities included hypertension (62.31%),
diabetes (6.15%), coronary artery disease (5.38%), chronic
obstructive pulmonary disease (4.62%), and stroke
(6.15%).CPB time was 193 (154–233) min, aortic cross
clamp time was 101 (81–124) min, and circulatory arrest
time was 16 (0–24) min. 82 patients (63.08%) developed
AKI, 31 patients (23.85%) on stage 1 AKI, and 51 patients
(39.23%) on stage 2 or 3 AKI. Demographic and perioperative characteristics strati-
fied according to Improving Global Outcomes criteria
[13] were shown in Table 2. The type of procedure, pump
time, and the incidence of blood transfusion were associ-
ated with AKI development after surgery. No significant
difference was found between patients with AKI and
those without AKI in terms of age, sex, diabetes, and Cir-
culatory arrest time. Clinical datah Worse in-hospital outcomes were observed in patients
with AKI, which contributed to approximately 14.62%
of in-hospital death. Patients with AKI had long intuba-
tion time and ICU stay, and patients with stage 3 AKI
were susceptible to renal replacement therapy. The major
adverse events, including perioperative ARDS, periopera-
tive myocardial infarction, stroke, and redo surgery, were
more frequent in patients with AKI than those without. The demographic characteristics were collected dur-
ing patient hospitalization from the electronic medical
record system. Information of preoperative hemoglobin,
eGFR, preoperative and postoperative serum creatinine
levels, type of surgical procedure, time on bypass, aortic
cross clamp time, circulatory arrest time, the amount and
type of intraoperative blood transfusion, reoperation and
other events after surgery were also collected. Statistical analysish BMC Cardiovasc Disord (2020) 20:446 Page 4 of 8 Table 1 Demographic characteristics of patients at baseline
AKI, acute kidney injury; COPD, chronic obstructive pulmonary; CPB, cardiopulmonary bypass; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate;
ICU, intensive care unit; SD, standard deviation; SBP, systolic blood pressure
Characteristics
All patients (n = 130)
Age (years), mean (SD)
54.74 (11.84)
Gender, n (%)
Male
101 (77.69)
Female
29 (22.31)
Hypertension, n (%)
81 (62.31)
Diabetes mellitus, n (%)
8 (6.15)
COPD, n (%)
6 (4.62)
Stroke, n (%)
8 (6.15)
Coronary artery disease, n (%)
7 (5.38)
SBP (mmHg), mean (SD)
133.63 (28.41)
DBP (mmHg), mean (SD)
72.92 (19.31)
eGFR [ml/(min.1.73m2)], mean (SD)
91.86 (40.27)
Preoperative hemoglobin (g/l), mean (SD)
122.96 (21.58)
Emergency operation, n (%)
88 (67.69)
CPB time (min), median (IQR)
193 (154–233)
Aortic cross clamp time (min), median (IQR)
101 (81–124)
Circulatory arrest time (min), median (IQR)
16 (0–24)
Total arch replacement, n (%)
76 (58.46)
Semi-arch replacement, n (%)
3 (2.31)
Stented elephant trunk, n (%)
72 (55.38)
Bentall procedure, n (%)
55 (42.31)
Ascending aorta replacement, n (%)
73 (56.15)
David procedure, n (%)
2 (1.54)
Intraoperative erythrocytes use (ml), median (IQR)
2300 (1300–3675)
Intraoperative fresh frozen plasma use (ml), median (IQR)
900 (550–1775)
Intraoperative platelets use (ml), median (IQR)
1200 (600–1200)
Intraoperative cryoprecipitate use (ml), median (IQR)
500 (213–519)
Intraoperative blood transfusion (ml), median (IQR)
4975 (2756–6819)
Postoperative AKI, n (%)
82 (63.08)
Length of ICU (day)
5.0 (3.0–10.0)
Length of mechanical ventilation (day)
2.5 (1.0–6.0)
In-hospital mortality, n (%)
21 (16.15) Table 1 Demographic characteristics of patients at baseline
AKI, acute kidney injury; COPD, chronic obstructive pulmonary; CPB, cardiopulmonary bypass; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate;
ICU intensive care unit; SD standard deviation; SBP systolic blood pressure
Characteristics
All patients (n = 130)
Age (years), mean (SD)
54.74 (11.84)
Gender, n (%)
Male
101 (77.69)
Female
29 (22.31)
Hypertension, n (%)
81 (62.31)
Diabetes mellitus, n (%)
8 (6.15)
COPD, n (%)
6 (4.62)
Stroke, n (%)
8 (6.15)
Coronary artery disease, n (%)
7 (5.38)
SBP (mmHg), mean (SD)
133.63 (28.41)
DBP (mmHg), mean (SD)
72.92 (19.31)
eGFR [ml/(min.1.73m2)], mean (SD)
91.86 (40.27)
Preoperative hemoglobin (g/l), mean (SD)
122.96 (21.58)
Emergency operation, n (%)
88 (67.69)
CPB time (min), median (IQR)
193 (154–233)
Aortic cross clamp time (min), median (IQR)
101 (81–124)
Circulatory arrest time (min), median (IQR)
16 (0–24)
Total arch replacement, n (%)
76 (58.46)
Semi-arch replacement, n (%)
3 (2.31)
Stented elephant trunk, n (%)
72 (55.38)
Bentall procedure, n (%)
55 (42.31)
Ascending aorta replacement, n (%)
73 (56.15)
David procedure, n (%)
2 (1.54)
Intraoperative erythrocytes use (ml), median (IQR)
2300 (1300–3675)
Intraoperative fresh frozen plasma use (ml), median (IQR)
900 (550–1775)
Intraoperative platelets use (ml), median (IQR)
1200 (600–1200)
Intraoperative cryoprecipitate use (ml), median (IQR)
500 (213–519)
Intraoperative blood transfusion (ml), median (IQR)
4975 (2756–6819)
Postoperative AKI, n (%)
82 (63.08)
Length of ICU (day)
5.0 (3.0–10.0)
Length of mechanical ventilation (day)
2.5 (1.0–6.0)
In-hospital mortality, n (%)
21 (16.15) Table 1 Demographic characteristics of patients at baseline Table 1 Demographic characteristics of patients at baseline AKI, acute kidney injury; COPD, chronic obstructive pulmonary; CPB, cardiopulmonary bypass; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate;
ICU, intensive care unit; SD, standard deviation; SBP, systolic blood pressure complications, including bleeding, ARDS, acute myo-
cardial infarction, stroke, AKI, and infection. Statistical analysish Postoper-
ative AKI is a common complication of cardiac surgery
with CBP. Volume depletion, hypotension, anemia, and
blood transfusion increase the incidence of AKI [3, 4]. In this study, We explored the relationship between
intraoperative blood transfusion volume and postop-
erative AKI for patients with type A acute aortic dis-
section surgery and found that intraoperative blood
transfusion volume was an independent risk factor for
postoperative AKI. Over the last decade, the risk of
blood transfusion for AKI has been the focus of many
observational studies. Freeland found that patients with
postoperative AKI seemed to have a high incidence of increased with an increment of blood transfusion volume,
and the curve tends to go down when the amount of blood
transfusion is above 4000 ml (Fig. 2). In dose–response
analyses, every 200 ml increment of blood transfusion vol-
ume could lead to a 31% (odds ratio 1.31 and 95% confi-
dence interval 1.01–1.71) increase in AKI risk when the
amount of blood transfusion was below 4000 ml. However,
null volume association was observed when the amount of
blood transfusion was more than 4000 ml (Table 3). Statistical analysish No difference was observed in the pre-transfusion Hgb
level between patients with and without AKI. Patients
with AKI received much more amount of blood transfu-
sion than those without AKI. A nonlinear relationship
between intraoperative blood transfusion volume and AKI
was explored. We observed the risk of postoperative AKI The parameters were summarized as mean ± standard
deviation (SD) for normally distributed continuous vari-
ables, median with interquartile range for continuous
variables with skewed distribution, and frequency (per-
centage) for categorical variables. Multivariable logistic Liu et al. Discussion Type A acute aortic dissection is a severe disease
related to increased morbidity and mortality. Patients
undergoing cardiac surgery suffer from a series of Liu et al. Discussion BMC Cardiovasc Disord (2020) 20:446 Page 5 of 8 Table 2 Comparison of demographic, preoperative, intraoperative, and postoperative characteristics between patients
with AKI and those without AKI
AKI, acute kidney injury; ARDS, adult respiratory distress syndrome; COPD, chronic obstructive pulmonary; CPB, cardiopulmonary bypass; DBP, diastolic blood
pressure; eGFR, estimated glomerular filtration rate; ICU, intensive care unit; SD, standard deviation; SBP, systolic blood pressure
No AKI
Stage 1 AKI
Stage 2 or 3 AKI
P value
N
48
31
51
–
Age (years), mean (SD)
53.15 (11.61)
57.13 (11.22)
54.78 (12.39)
0.35
Male, n (%)
33 (68.75%)
27 (87.10%)
41 (80.39%)
0.16
Hypertension, n (%)
22 (45.83%)
25 (80.65%)
34 (66.67%)
0.006
Diabetes mellitus, n (%)
1 (2.08%)
3 (9.68%)
4 (7.84%)
0.37
COPD, n (%)
2 (4.17%)
1 (3.23%)
3 (5.88%)
1.00
Stroke, n (%)
3 (6.25%)
2 (6.45%)
3 (5.88%)
1.00
Coronary artery disease, n (%)
1 (2.08%)
4 (12.90%)
2 (3.92%)
0.12
SBP (mmHg), mean (SD)
131.90 (22.70)
125.39 (27.54)
140.27 (32.43)
0.06
DBP (mmHg), mean (SD)
71.02 (16.53)
66.71 (21.57)
78.49 (19.15)
0.018
eGFR [ml/(min.1.73m2)], mean (SD)
100.57 (30.65)
71.53 (22.21)
94.84 (51.22)
0.007
Preoperative hemoglobin (g/l), mean (SD)
125.87 (18.39)
120.77 (22.74)
124.53 (22.44)
0.61
Emergency operation, n (%)
21 (43.75%)
25 (80.65%)
42 (82.35%)
< 0.001
CPB time (min), median (IQR)
176 (120–221)
173 (132–212)
210 (185–244)
0.001
Aortic cross clamp time (min), median (IQR)
96 (76–118)
96 (74–107)
120 (95–127)
0.004
Circulatory arrest time (min), median (IQR)
12 (0–20)
15 (0–24)
18 (8–24)
0.26
Nasopharyngeal temperature (°C), mean (SD)
20.9 (2.1)
20.8 (2.1)
20.4 (2.5)
> 0.05
Total arch replacement, n (%)
20 (41.67%)
18 (58.06%)
38 (74.51%)
0.004
Semi-arch replacement, n (%)
2 (4.17%)
1 (3.23%)
0 (0.00%)
0.35
Stented elephant trunk, n (%)
19 (39.58%)
17 (54.84%)
36 (70.59%)
0.008
Bentall procedure, n (%)
27 (56.25%)
14 (45.16%)
14 (27.45%)
0.014
Ascending aorta replacement, n (%)
21 (43.75%)
17 (54.84%)
35 (68.63%)
0.044
Intraoperative erythrocytes use (ml), median (IQR)
1750 (1050–3375)
2200 (1400–2775)
2950 (1950–4350)
0.007
Intraoperative fresh frozen plasma use (ml), median (IQR)
850 (550–1200)
1000 (575–1550)
1000 (550–1925)
0.55
Intraoperative platelets use (ml), median (IQR)
600 (300–1200)
1200 (900–1200)
1200 (750–1200)
0.006
Intraoperative cryoprecipitate use (ml), median (IQR)
350 (0–500)
475 (250–525)
500 (250–560)
0.11
Intraoperative blood transfusion(ml), median (IQR)
2825 (1988–5538)
4400 (2750–5800)
4850 (3650–6875)
0.007
ARDS, n (%)
8 (16.67%)
13 (41.94%)
31 (60.78%)
< 0.001
Perioperative myocardial infarction, n (%)
0 (0.00%)
0 (0.00%)
2 (3.92%)
0.342
Redo surgery, n (%)
2 (4.17%)
3 (9.68%)
13 (25.49%)
0.007
Length of ICU (day), median (IQR)
4.00 (2.75–5.25)
6.00 (3.50–6.50)
9.00 (3.00–14.50)
0.001
Length of mechanical ventilation (day), median (IQR)
2.00 (1.00–3.00)
2.00 (1.00–4.00)
5.00 (2.00–7.50)
< 0.001
In-hospital mortality, n (%)
2 (4.17%)
5 (16.13%)
14 (27.45%)
0.005 Table 2 Comparison of demographic, preoperative, intraoperative, and postoperative characteristics between patients
with AKI and those without AKI f demographic, preoperative, intraoperative, and postoperative characteristics between patients
h
AKI AKI, acute kidney injury; ARDS, adult respiratory distress syndrome; COPD, chronic obstructive pulmonary; CPB, cardiopulmonary bypass; DBP, diastolic blood
pressure; eGFR, estimated glomerular filtration rate; ICU, intensive care unit; SD, standard deviation; SBP, systolic blood pressure blood transfusion [5]. Discussion Koch et al. reported that periop-
erative red blood cell transfusion was the independent
predictor of postoperative AKI in patients undergoing
isolated coronary artery bypass grafting [7]. Mean-
while, blood transfusion was strongly associated with
AKI in a retrospective cohort study of patients with
acute coronary syndrome undergoing PCI, which was
consistent with our study to some extent [3]. However,
some studies reported null association between blood
transfusion and postoperative AKI, presumably due
to the limited sample size and heterogeneous patients’
characteristics [14, 15]. To the best of our knowledge, the current study is
the first to investigate the association of intraopera-
tive blood transfusion volume with postoperative AKI
in patients undergoing type A acute aortic dissection
surgery. We observed a nonlinear relationship between
intraoperative blood transfusion volume and postop-
erative AKI. A 31% increment of the postoperative AKI
risk was observed for every 200 ml of blood transfu-
sion received when the total volume was lower than
4000 ml. However, no difference was observed when
the blood transfusion volume was higher than 4000 ml. Different from the nonlinear association reported in Liu et al. BMC Cardiovasc Disord (2020) 20:446 Page 6 of 8 Fig. 2 The nonlinear relationship between intraoperative blood
transfusion volume and postoperative AKI. The results were
generated utilizing generalized additive model and adjusted for age,
gender, hypertension, diabetes mellitus, eGFR, emergency operation,
CPB time, aortic cross clamp time, nasopharyngeal temperature,
total arch replacement, semi-arch replacement, stented elephant
trunk, Bentall procedure, ascending aorta replacement, intraoperative
erythrocytes use, intraoperative platelets use and redo surgery. The
red line indicates the risk of AKI and the blue dot line indicates 95%
confidence intervals was correlated with worse in-hospital outcomes. These
associations were confirmed in prior studies [16, 17].h i
The mechanisms through which blood transfusion
volume increased the risk of postoperative AKI are not
illuminated, but several potential explanations are con-
sidered. The pathogenesis of AKI is mostly related to
inflammation, renal hypoxia, impairment of tissue oxy-
gen delivery, and oxidative stress. Patients who undergo
cardiac surgery with CPB are subjected to the initiation
phase of ischemia–reperfusion kidney injury and the
extension phase of kidney injury. The initiation phase
is characterized by renal artery vasoconstriction and
increased oxygen consumption. Discussion Patients in the initia-
tion phase have an increased risk of aggravation to the
extension phase, which may lead to AKI development if
they cannot recover or if they suffer from other exter-
nal risk factors, such as blood transfusion, ischemia, and
anemia [10, 18, 19]. Red blood cell transfusion is com-
mon during cardiac surgery with CPB. Red blood cells
during storage undergo a series of changes, including
decreased deformability, increased fragility, progressive
hemolysis, and accumulation of free hemoglobin and
iron that may accelerate organ tissue dysfunction [20–
22]. Macrophages under normal circumstances dispose
of red blood corpuscles and release iron to the circula-
tion bound to transferrin. However, a certain amount of
free hemoglobin and iron released by macrophages fails
to bind to the iron binding sites on the iron-carrier pro-
tein transferrin in circulation after blood transfusion
and is toxic to the kidneys [10, 23]. Also, dysregulation
of renal haemodynamics is typical of AKI. Platelets play
a crucial part in renal haemodynamic processes by regu-
lating the endothelial vascular permeability. Activation of
platelets could release granules and microvesicles, which
are associated with the pathophysiology of AKI [24, 25]. Other evidence further links blood transfusion and AKI. Patients with persistent positive fluid balance are likely
inclined to suffer from kidney injury. Increased kidney
interstitial pressure and kidney venous pressure due to Fig. 2 The nonlinear relationship between intraoperative blood
transfusion volume and postoperative AKI. The results were
generated utilizing generalized additive model and adjusted for age,
gender, hypertension, diabetes mellitus, eGFR, emergency operation,
CPB time, aortic cross clamp time, nasopharyngeal temperature,
total arch replacement, semi-arch replacement, stented elephant
trunk, Bentall procedure, ascending aorta replacement, intraoperative
erythrocytes use, intraoperative platelets use and redo surgery. The
red line indicates the risk of AKI and the blue dot line indicates 95%
confidence intervals Fig. 2 The nonlinear relationship between intraoperative blood
transfusion volume and postoperative AKI. The results were
generated utilizing generalized additive model and adjusted for age,
gender, hypertension, diabetes mellitus, eGFR, emergency operation,
CPB time, aortic cross clamp time, nasopharyngeal temperature,
total arch replacement, semi-arch replacement, stented elephant
trunk, Bentall procedure, ascending aorta replacement, intraoperative
erythrocytes use, intraoperative platelets use and redo surgery. The
red line indicates the risk of AKI and the blue dot line indicates 95%
confidence intervals our study, Karkouti et al. Discussion Fluid overload causes
visceral and peripheral edema, which leads to difficult
organic functioning and delays the AKI diagnosis due to
the dilution of serum creatinine [26, 27]. A randomized
trial concluded that goal-directed therapy decreased
blood transfusion and the incidence of AKI attributing
to this effect to improved fluid management [28]. Thus,
the important risk factors may be interrelated to AKI
development. the excess fluid that overwhelms the limited capacity
of the kidneys lead to the decrease in glomerular filtra-
tion rate. Fluid accumulation in patients may be asso-
ciated with increased mortality. Fluid overload causes
visceral and peripheral edema, which leads to difficult
organic functioning and delays the AKI diagnosis due to
the dilution of serum creatinine [26, 27]. A randomized
trial concluded that goal-directed therapy decreased
blood transfusion and the incidence of AKI attributing
to this effect to improved fluid management [28]. Thus,
the important risk factors may be interrelated to AKI
development. Abbreviations
AKI A
k d 7. Koch CG, Li L, Duncan AI, Mihaljevic T, Cosgrove DM, Loop FD, et al. Morbidity and mortality risk associated with red blood cell and blood-
component transfusion in isolated coronary artery bypass grafting. CRIT
Care Med. 2006;34(6):1608–16. Abbreviations
AKI: Acute kidney injury; ARDS: Adult respiratory distress syndrome; COPD:
Chronic obstructive pulmonary; CPB: Cardiopulmonary bypass; DBP: Diastolic
blood pressure; eGFR: Estimated glomerular filtration rate; ICU: Intensive care
unit; RRT: Renal replacement therapy; SD: Standard deviation; SBP: Systolic
blood pressure. 8. De Santo L, Romano G, Della CA, de Simone V, Grimaldi F, Cotrufo M,
et al. Preoperative anemia in patients undergoing coronary artery
bypass grafting predicts acute kidney injury. J Thorac Cardiovasc Surg. 2009;138(4):965–70. References 1. Li Y, Yang N, Duan W, Liu S, Yu S, Yi D. Acute aortic dissection in China. Am
J Cardiol. 2012;110(7):1056–61. 1. Li Y, Yang N, Duan W, Liu S, Yu S, Yi D. Acute aortic dissection in China. Am
J Cardiol. 2012;110(7):1056–61. 2. Ko T, Higashitani M, Sato A, Uemura Y, Norimatsu T, Mahara K, et al. Impact of acute kidney injury on early to long-term outcomes in patients
who underwent surgery for type a acute aortic dissection. Am J Cardiol. 2015;116(3):463–8. 3. Karrowni W, Vora AN, Dai D, Wojdyla D, Dakik H, Rao SV. Blood transfusion
and the risk of acute kidney injury among patients with acute coronary
syndrome undergoing percutaneous coronary intervention. Circ Cardio-
vasc Interv. 2016;9(9):e003279. Competing interests Competing interests
The authors declare that they have no competing interests. Discussion found a linear relationship
between blood transfusion volume and risk of AKI in
a randomized goal-directed fluid resuscitation study
[10]. In their study, non-cardiac surgery patients were
included and the amount of blood transfusion was rela-
tively small, which might account for the difference. f
AKI is a common adverse event for type A acute aor-
tic dissection. Our study also evaluated the relation-
ship between postoperative AKI and in-hospital major
adverse events. An association was found between the
incidence of AKI and major adverse events. Patients
with AKI were associated with long ventilation time
and prolonged ICU stay. Moreover, the severity of AKI Table 3 Multivariable logistic regression analysis to estimate the independent association between blood transfusion
volume and the risk of postoperative AKI
Model 1: Crude model
Model 2: Adjusted for age, gender, hypertension, and diabetes mellitus
Model 3: Additional adjust for eGFR, emergency operation, CPB time, aortic cross clamp time, nasopharyngeal temperature, total arch replacement, semi-arch
replacement, stented elephant trunk, Bentall procedure, ascending aorta replacement, intraoperative erythrocytes use, intraoperative platelets use and redo surgery
Variable
Model 1
P value
Model 2
P value
Model 3
P value
Blood transfusion volume ≤ 4000 ml
Increase per 200 ml
1.29 (1.08, 1.53)
0.004
1.30 (1.07, 1.57)
0.008
1.31 (1.01, 1.71)
0.044
Blood transfusion volume > 4000 ml
Increase per 200 ml
1.02 (0.96, 1.07)
0.57
1.01 (0.96, 1.07)
0.70
1.02 (0.96, 1.08)
0.61 Table 3 Multivariable logistic regression analysis to estimate the independent association betwe
volume and the risk of postoperative AKI j
g , g
, yp
,
Model 3: Additional adjust for eGFR, emergency operation, CPB time, aortic cross clamp time, nasopharyngeal temperature, total arch replacement, semi-arch
replacement, stented elephant trunk, Bentall procedure, ascending aorta replacement, intraoperative erythrocytes use, intraoperative platelets use and redo surgery Liu et al. BMC Cardiovasc Disord (2020) 20:446 Liu et al. BMC Cardiovasc Disord (2020) 20:446 Liu et al. BMC Cardiovasc Disord (2020) 20:446 Page 7 of 8 University of Science and Technology for his contribution to the statistical
analysis. University of Science and Technology for his contribution to the statistical
analysis. the excess fluid that overwhelms the limited capacity
of the kidneys lead to the decrease in glomerular filtra-
tion rate. Fluid accumulation in patients may be asso-
ciated with increased mortality. Ethics approval and consent to participate t
cs app o a a d co se t to pa t c pate
This study was approved by the ethics committees of the central hospital of
Wuhan (Ethics approval NO: 2020.163). The requirement for informed consent
was waived. Author details
1 Author details
1 Intensive Care Unit, The Central Hospital of Wuhan, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan, Hubei, China. 2 Department of Cardiothoracic Surgery, The Central Hospital of Wuhan, Tongji
Medical College, Huazhong University of Science and Technology, Wuhan,
Hubei, China. Received: 20 February 2020 Accepted: 6 October 2020 Received: 20 February 2020 Accepted: 6 October 2020 Authors’ contributions YL and LYL conceived and supervised the study; LYL and SYQ designed experi-
ments; LYL, SYQ and LD performed information acquisition and examination
analysis; LYL wrote the manuscript; LD and YL made manuscript revisions. All
authors read and approved the final manuscript. Conclusions Our study showed that the amount of intraoperative
blood transfusion was an independent risk factor for
postoperative AKI in patients with type A acute aor-
tic dissection. Intraoperative transfusion volume might
increase the incidence of postoperative AKI, and the
mechanism underlying transfusion thresholds on AKI
should be further investigated. 4. Mehta RL, Burdmann EA, Cerda J, Feehally J, Finkelstein F, Garcia-Garcia G,
et al. Recognition and management of acute kidney injury in the Inter-
national Society of Nephrology 0by25 Global Snapshot: a multinational
cross-sectional study. Lancet. 2016;387(10032):2017–25. 5. Freeland K, Hamidian JA, Duvall LM, Mancini MC. Postoperative blood
transfusion is an independent predictor of acute kidney injury in cardiac
surgery patients. J Nephropathol. 2015;4(4):121–6. 6. Brown JR, Kramer RS, MacKenzie TA, Coca SG, Sint K, Parikh CR. Determi-
nants of acute kidney injury duration after cardiac surgery: an externally
validated tool. Ann Thorac Surg. 2012;93(2):570–6. Availability of data and materials y
The datasets used and/or analysed during the current study are available from
the corresponding author on reasonable request. Study limitationsh This study is subjected to several limitations. Firstly, the
causality could not be established because of the nature
of the observational study. Thus, further prospective
intervention study is needed. Secondly, type A acute aor-
tic dissection surgery is a complicated procedure. We are
often forced into the situation where multiple transfu-
sions are required.The appropriate timing of blood trans-
fusion could not be determined, which is an interesting
topic to study in the future. Thirdly, because the sample
size for this study is relative small, perhaps the influ-
ence of blood transfusion volume becomes less impor-
tant. Fourthly, multiple factors may cause postoperative
AKI after aortic surgery, including preoperative shock
or hypotension, renal artery dissection or occlusion,
anemia, surgical trauma, prolonged use of CPB, hypo-
thermia, and extensive blood product, during the study
period, it is very difficult to take all factors into consider-
ation. Fortunately, most important variables are included
in our study to discuss the incidence of AKI. Fifthly,
the residual confounding and bias could not be totally
addressed in the observational study. We gratefully thank Dr. Liangkai Chen from the Department of Nutrition and
Food Hygiene, Hubei Key Laboratory of Food Nutrition and Safety Huazhong Acknowledgements Boyle JM, Moualla S, Arrigain S, Worley S, Bakri MH, Starling RC, et al. Risks and outcomes of acute kidney injury requiring dialysis after cardiac
transplantation. Am J Kidney dis. 2006;48(5):787–96. 14. Boyle JM, Moualla S, Arrigain S, Worley S, Bakri MH, Starling RC, et al. Risks and outcomes of acute kidney injury requiring dialysis after cardiac
transplantation. Am J Kidney dis. 2006;48(5):787–96. 24. Yamamoto T, Tada T, Brodsky SV, Tanaka H, Noiri E, Kajiya F, et al. Intravital
videomicroscopy of peritubular capillaries in renal ischemia. Am J Physiol
Renal Physiol. 2002;282(6):F1150–5. p
y
15. Ho J, Reslerova M, Gali B, Nickerson PW, Rush DN, Sood MM, et al. Serum
creatinine measurement immediately after cardiac surgery and predic-
tion of acute kidney injury. Am J Kidney Dis. 2012;59(2):196–201. 25. Jansen M, Florquin S, Roelofs J. The role of platelets in acute kidney injury. Nat Rev Nephrol. 2018;14(7):457–71. 26. Grams ME, Estrella MM, Coresh J, Brower RG, Liu KD. Fluid balance,
diuretic use, and mortality in acute kidney injury. Clin J Am Soc Nephrol. 2011;6(5):966–73. 26. Grams ME, Estrella MM, Coresh J, Brower RG, Liu KD. Fluid balance,
diuretic use, and mortality in acute kidney injury. Clin J Am Soc Nephrol. 2011;6(5):966–73. 16. Zhou H, Wang G, Yang L, Shi S, Li J, Wang M, et al. Acute kidney injury
after total arch replacement combined with frozen elephant trunk
implantation: incidence, risk factors, and outcome. J Cardiothor Vasc
Anesthesia. 2018;32(5):2210–7. 27. Avila MO, Rocha PN, Zanetta DM, Yu L, Burdmann EA. Water balance,
acute kidney injury and mortality of intensive care unit patients. J Bras
Nefrol. 2014;36(3):379–88. 27. Avila MO, Rocha PN, Zanetta DM, Yu L, Burdmann EA. Water balance,
acute kidney injury and mortality of intensive care unit patients. J Bras
Nefrol. 2014;36(3):379–88. 17. Hoste EA, Clermont G, Kersten A, Venkataraman R, Angus DC, De
Bacquer D, et al. RIFLE criteria for acute kidney injury are associated with
hospital mortality in critically ill patients: a cohort analysis. Crit Care. 2006;10(3):R73. 28. Weber CF, Gorlinger K, Meininger D, Herrmann E, Bingold T, Moritz A,
et al. Point-of-care testing: a prospective, randomized clinical trial of
efficacy in coagulopathic cardiac surgery patients. Anesthesiology. 2012;117(3):531–47. 28. Weber CF, Gorlinger K, Meininger D, Herrmann E, Bingold T, Moritz A,
et al. Point-of-care testing: a prospective, randomized clinical trial of
efficacy in coagulopathic cardiac surgery patients. Anesthesiology. 2012;117(3):531–47. 18. Acknowledgements Redfors B, Bragadottir G, Sellgren J, Sward K, Ricksten SE. Acute renal
failure is NOT an “acute renal success”—a clinical study on the renal
oxygen supply/demand relationship in acute kidney injury. Crit Care Med. 2010;38(8):1695–701. Acknowledgements We gratefully thank Dr. Liangkai Chen from the Department of Nutrition and
Food Hygiene, Hubei Key Laboratory of Food Nutrition and Safety Huazhong Liu et al. BMC Cardiovasc Disord (2020) 20:446 Liu et al. BMC Cardiovasc Disord (2020) 20:446 Page 8 of 8 9. Bove T, Calabro MG, Landoni G, Aletti G, Marino G, Crescenzi G, et al. The
incidence and risk of acute renal failure after cardiac surgery. J Cardio-
thorac Vasc Anesth. 2004;18(4):442–5. 20. Tinmouth A, Fergusson D, Yee IC, Hebert PC. Clinical consequences of red
cell storage in the critically ill. Transfusion. 2006;46(11):2014–27. 21. Donadee C, Raat NJ, Kanias T, Tejero J, Lee JS, Kelley EE, et al. Nitric
oxide scavenging by red blood cell microparticles and cell-free
hemoglobin as a mechanism for the red cell storage lesion. Circulation. 2011;124(4):465–76. 10. Karkouti K. Transfusion and risk of acute kidney injury in cardiac surgery. Br J Anaesth. 2012;109(Suppl 1):i29–38. 10. Karkouti K. Transfusion and risk of acute kidney injury in cardiac surgery. Br J Anaesth. 2012;109(Suppl 1):i29–38. pp
11. Rosner MH, Okusa MD. Acute kidney injury associated with cardiac
surgery. Clin J Am Soc Nephrol. 2006;1(1):19–32. 11. Rosner MH, Okusa MD. Acute kidney injury associated with cardiac
surgery. Clin J Am Soc Nephrol. 2006;1(1):19–32. 22. Almac E, Ince C. The impact of storage on red cell function in blood
transfusion. Best Pract Res Clin Anaesthesiol. 2007;21(2):195–208. 12. Vanmassenhove J, Kielstein J, Jorres A, Biesen WV. Management of
patients at risk of acute kidney injury. Lancet. 2017;389(10084):2139–51. 12. Vanmassenhove J, Kielstein J, Jorres A, Biesen WV. Management of
patients at risk of acute kidney injury. Lancet. 2017;389(10084):2139–51. 12. Vanmassenhove J, Kielstein J, Jorres A, Biesen WV. Management of
patients at risk of acute kidney injury. Lancet. 2017;389(10084):2139–51. 13. Khwaja A. KDIGO clinical practice guidelines for acute kidney injury. Nephron Clin Pract. 2012;120(4):c179–84. 23. Lasocki S, Longrois D, Montravers P, Beaumont C. Hepcidin and
anemia of the critically ill patient: bench to bedside. Anesthesiology. 2011;114(3):688–94. p
y
j
y
13. Khwaja A. KDIGO clinical practice guidelines for acute kidney injury. Nephron Clin Pract. 2012;120(4):c179–84. y
y
13. Khwaja A. KDIGO clinical practice guideline
Nephron Clin Pract. 2012;120(4):c179–84. 13. Khwaja A. KDIGO clinical practice guidelin
Nephron Clin Pract. 2012;120(4):c179–84. 24. Yamamoto T, Tada T, Brodsky SV, Tanaka H, Noiri E, Kajiya F, et al. Intravital
videomicroscopy of peritubular capillaries in renal ischemia. Am J Physiol
Renal Physiol. 2002;282(6):F1150–5. 14. Publisher’s Note
S
N Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations. 19. Ho J, Lucy M, Krokhin O, Hayglass K, Pascoe E, Darroch G, et al. Mass
spectrometry-based proteomic analysis of urine in acute kidney injury
following cardiopulmonary bypass: a nested case-control study. AM J
Kidney Dis. 2009;53(4):584–95. •
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https://openalex.org/W3014263424
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https://europepmc.org/articles/pmc7279193?pdf=render
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English
| null |
A common polymorphism in <i>SNCA</i> is associated with accelerated motor decline in <i>GBA</i>-Parkinson’s disease
|
Journal of neurology, neurosurgery and psychiatry
| 2,020
|
cc-by
| 2,573
|
A common polymorphism in
SNCA is associated with
accelerated motor decline in
GBA-Parkinson’s disease A growing number of genetic susceptibility
factors have been identified for Parkinson’s
disease (PD). The combination of inherited
risk variants is likely to affect not only risk
of developing PD but also its clinical course. Variants in the GBA gene are particularly
common, being found in approximately 5%
to 10% of patients, and they lead to more
rapid disease progression.1 However, the
effect of concomitant genetic risk factors on
disease course in GBA-PD is not known. Sequencing of the GBA gene was carried
out in 114 patients in the CamPaIGN
cohort, as described here.7 A further
16 patients underwent targeted genetic
screening for common GBA variants using
the Illumina Multi-Ethnic Genotyping
Array (MEGA) chip. Genetic analysis of
the SNCA rs356219 polymorphism had
also been previously performed in 124
patients from the cohort.2 GBA variants
were identified in 20 patients, which were
included in survival analyses. Of these, 7
carried the high-risk (G/G) SNCA geno-
type, with 13 carrying low-risk (G/A or
A/A) genotypes. There were no differences
in age at diagnosis nor in baseline assess-
ments including total and part III Unified
Parkinson’s Disease Rating Scale scores,
Hoehn and Yahr scores, MMSE scores or
levodopa equivalent doses between these
SNCA genotype groups. All carriers of the
G/G genotype were male, compared with
53.8% in the low-risk group (p=0.03)
(online supplementary table 1). The aggregation of α-synuclein, encoded
by the SNCA gene, is central to the patho-
genesis of PD. The SNCA rs356219 A/G
polymorphism alters the risk of developing
PD, with homozygotes for guanine (G/G)
having an increased risk compared with
carriers of an adenine (G/A or A/A) at this
locus.2 The relationship between glucoce-
rebrosidase (the enzyme encoded by the
GBA gene) and α-synuclein is complex. These proteins have been shown to interact
directly in vitro, as well as to influence the
intracellular levels and processing of each
other, potentially in a bidirectional feedback
loop.3 4 Interestingly, a recent genome-wide
association study found that the presence
of this SNCA polymorphism was associated
with an increased likelihood of developing
PD in GBA mutation carriers.5 We therefore
hypothesised that the presence of the SNCA
rs356219 polymorphism would accelerate
the clinical course of GBA variant-associated
PD. Here, we report on the effect of this
SNCA polymorphism on clinical outcomes
within the GBA-PD population. This cohort contained four patients with
the non-coding c. 762–18 T>A variant
(online supplementary table 2). Letter G/G carriers reached HY3 within 3 years of
diagnosis. informed consent was obtained from all
subjects. Newly diagnosed patients were
followed up with assessments every 2 years
for up to 18 years. Time to development
of dementia (defined as Mini-Mental State
Examination (MMSE) score of 24 or less,
with fulfilment of Diagnostic and Statistical
Manual of Mental Disorders IV criteria),
progression to postural instability (Hoehn
and Yahr stage three (HY3)) and death were
determined. To account for potential confounders,
time to HY3 was also compared using a Cox
regression model controlling for sex and
age at diagnosis. The G/G SNCA genotype
was associated with a HR for progression to
HY3 of 3.8 (95% CI 0.9 to 16.1), p=0.07,
adjusted p=0.21) when controlling for
these confounders. Because all the SNCA
G/G carriers were male, we also performed a
survival analysis in which only male subjects
were included, to further control for sex as
a potential confounder. An increased risk
of progression to HY3 was again observed
in G/G carriers in comparison to A carriers
(p=0.036, adjusted p=0.11; online supple-
mentary figure 1). SNCA genotype had no
effect on progression to HY3 in non-carriers
of GBA variants (n=85) in the CamPaIGN
cohort (p=0.88, adjusted p=2.65; online
supplementary figure 2). A common polymorphism in
SNCA is associated with
accelerated motor decline in
GBA-Parkinson’s disease While this
has been reported to be a potential risk
factor for PD,8 its importance is not clear. We therefore also performed analysis after
exclusion of these patients and found that
progression to HY3 was greater in the G/G
carrier group, with a HR of 5.3 (95% CI
1.1 to 26.2; p=0.041, adjusted p=0.12). To our knowledge, no previous studies
have investigated the influence of concom-
itant genetic risk factors on the progression
of GBA-PD. Here we report that the SNCA
rs356219
polymorphism
significantly
affects motor progression in GBA-PD,
with the G/G genotype associated with a
particularly aggressive disease course. This
effect was not observed in patients with PD
without GBA variants, suggesting that it was
relatively specific to GBA-PD. Survival analyses for the aforementioned
outcome measures were performed, with
statistical significance determined using
log-rank tests. P-values were adjusted for
multiple comparisons using the Bonfer-
roni method. Among carriers of GBA vari-
ants, there were no differences between
the high-risk and low-risk SNCA groups
for time to dementia (p=0.29, adjusted
p=0.86) or death (p=0.43, adjusted
p=1.28) (figure 1A,B). Progression to
HY3, however, was significantly faster in
the G/G SNCA group (p=0.02, adjusted
p=0.07), with mean time to development
of postural instability 2.0 years (95% CI
1.3 to 2.7) compared with 4.9 years (95%
CI 2.5 to 7.3) in A carriers (figure 1C). All This new study therefore raises the inter-
esting possibility that GBA variants and
the G/G SNCA rs356219 polymorphism
act synergistically to accelerate pathology
and clinical progression in PD. GBA muta-
tions are thought to increase the risk of
PD predominantly through perturbations
in
the
lysosome-autophagy
system—a
system important in α-synuclein clearance. Longitudinal data from GBA-variant
carriers were analysed from the community-
based
‘Cambridgeshire
Incidence
of
Parkinson’s disease from General Prac-
tice to Neurologist’ (CamPaIGN) cohort
(n=142).6 This study was approved by
the local ethics committee and written Figure 1 Survival analyses in GBA-PD patients comparing those with high-risk and low-risk variants in the SNCA rs356219 polymorphism. Kaplan-Meier
curves for time to (A) dementia, (B) death and (C) postural instability. A, adenine, G, guanine; H&Y3, Hoehn and Yahr stage three; PD, Parkinson’s disease. Figure 1 Survival analyses in GBA-PD patients comparing those with high-risk and low-risk variants in the SNCA rs356219 polymorphism. Kaplan-Meier
curves for time to (A) dementia, (B) death and (C) postural instability. A, adenine, G, guanine; H&Y3, Hoehn and Yahr stage three; PD, Parkinson’s disease. PostScript PostScript References 1 Cilia R, Tunesi S, Marotta G, et al. Survival and
dementia in GBA-associated Parkinson’s disease: the
mutation matters. Ann Neurol 2016;80:662–73. Twitter Thomas Foltynie @foltynie Twitter Thomas Foltynie @foltynie Twitter Thomas Foltynie @foltynie 2 Goris A, Williams-Gray CH, Clark GR, et al. Tau and
alpha-synuclein in susceptibility to, and dementia in,
Parkinson’s disease. Ann Neurol
2007;62:145–53. 2 Goris A, Williams-Gray CH, Clark GR, et al. Tau and
alpha-synuclein in susceptibility to, and dementia in,
Parkinson’s disease. Ann Neurol
2007;62:145–53. Contributors TBS wrote the manuscript. TBS, MC,
SW-R, GL and TF performed the research. TF, CRS,
RAB and CHW-G developed the original concepts and
provided critical review of the manuscript. Contributors TBS wrote the manuscript. TBS, MC,
SW-R, GL and TF performed the research. TF, CRS, Contributors TBS wrote the manuscript. TBS, MC,
SW-R, GL and TF performed the research. TF, CRS,
RAB and CHW-G developed the original concepts and
provided critical review of the manuscript. RAB and CHW-G developed the original concepts and
provided critical review of the manuscript. RAB and CHW-G developed the original concepts and
provided critical review of the manuscript. 3 Mazzulli JR, Xu Y-H, Sun Y, et al. Gaucher disease
glucocerebrosidase and α-synuclein form a
bidirectional pathogenic loop in synucleinopathies. Cell
2011;146:37–52. 3 Mazzulli JR, Xu Y-H, Sun Y, et al. Gaucher disease
glucocerebrosidase and α-synuclein form a
bidirectional pathogenic loop in synucleinopathies. Cell
2011;146:37–52. Funding The CamPaIGN study has received financial
support from the Wellcome Trust, the Medical Research
Council, Parkinson’s UK and the Patrick Berthoud Trust. CHW-G is supported by a Research Councils UK/UK
Research and Innovation Fellowship awarded by the
Medical Research Council (MR/R007446/1). RAB is
supported by the Wellcome Trust Stem Cell Institute
(Cambridge 203151/Z/16/Z). RAB is a National Institute
for Health Research (NIHR) Senior Investigator (NF-
SI-0616-10011). TBS received financial support from
the Cure Parkinson’s Trust. The study is also supported
by the NIHR Cambridge Biomedical Research Centre
Dementia and Neurodegeneration Theme (reference
number 146281). CRS’ work is supported in part by
National Institutes of Health grants R01AG057331,
U01NS100603, R01AG057331, and the American
Parkinson Disease Association. Illumina MEGA Chip
genotyping was made possible by a philanthropic
investment from Dooley LLC (to Brigham & Women’s
Hospital and CRS). 4 Yap TL, Velayati A, Sidransky E, et al. Membrane-
bound α-synuclein interacts with glucocerebrosidase
and inhibits enzyme activity. Mol Genet Metab
2013;108:56–64. 5 Blauwendraat C, Reed X, Krohn L, et al. PostScript Thomas B Stoker ,1,2 Marta Camacho,1
Sophie Winder-Rhodes,1 Ganqiang Liu,3,4,5
4 5
6 Clemens R Scherzer,4,5 Thomas Foltynie ,6
Roger A Barker,1,2 Caroline H Williams-Gray1 1John van Geest Centre for Brain Repair, Department
of Clinical Neurosciences, University of Cambridge,
Cambridge, UK 1John van Geest Centre for Brain Repair, Department
of Clinical Neurosciences, University of Cambridge,
Cambridge, UK To cite Stoker TB, Camacho M, Winder-Rhodes S, et al. J Neurol Neurosurg Psychiatry 2020;91:673–674. Received 9 October 2019
Revised 18 February 2020
Accepted 9 March 2020
Published Online First 2 April 2020 2Wellcome Trust ‑ Medical Research Council Stem Cell
Institute, University of Cambridge, Cambridge, UK
3School of Medicine, Sun Yat-Sen University, 2Wellcome Trust ‑ Medical Research Council Stem Cell
Institute, University of Cambridge, Cambridge, UK
3School of Medicine, Sun Yat-Sen University,
Guangzhou, Guangdong, China
4Advanced Center for Parkinson’s Disease Research,
Harvard Medical School, Brigham and Women’s
Hospital, Boston, Massachusetts, USA
5Precision Neurology Program, Harvard Medical School,
Brigham and Women’s Hospital, Boston, Massachusetts
USA J Neurol Neurosurg Psychiatry 2020;91:673–674. doi:10.1136/jnnp-2019-322210 Hospital, Boston, Massachusetts, USA
5 ORCID iDs 5Precision Neurology Program, Harvard Medical School,
Brigham and Women’s Hospital, Boston, Massachusetts,
USA 5Precision Neurology Program, Harvard Medical School,
Brigham and Women’s Hospital, Boston, Massachusetts
USA Thomas B Stoker http://orcid.org/0000-0001-5186-
7630 Thomas B Stoker http://orcid.org/0000-0001-5186-
7630 6Department of Clinical and Movement Neurosciences,
UCL Institute of Neurology, London, UK 6Department of Clinical and Movement Neurosciences,
UCL Institute of Neurology, London, UK Thomas Foltynie http://orcid.org/0000-0003-0752-
1813 Thomas Foltynie http://orcid.org/0000-0003-0752-
1813 We acknowledge that our sample size is
small, but these preliminary observations
raise the possibility that GBA variants and
the G/G SNCA rs356219 polymorphism
synergistically alter motor progression in
PD. In this study we have considered the
time from diagnosis to the development
of important clinical milestones in PD. The time to diagnosis may vary between
patients, so it should be recognised that this
does not necessarily reflect disease dura-
tion. Time from disease onset to the devel-
opment of clinical milestones may be more
representative of disease duration, but onset
is very difficult to ascertain accurately, given
that many patients experience prodromal
symptoms that are not initially attributed
to PD. Correspondence to Dr Thomas B Stoker, John van
Geest Centre for Brain Repair, Department of Clinical
Neurosciences, University of Cambridge, Cambridge,
Cambridgeshire CB2 0PY, UK; tbs26@cam.ac.uk Correspondence to Dr Thomas B Stoker, John van
Geest Centre for Brain Repair, Department of Clinical
Neurosciences, University of Cambridge, Cambridge,
Cambridgeshire CB2 0PY, UK; tbs26@cam.ac.uk PostScript Furthermore, glucocerebrosidase and α-sy-
nuclein have been shown to interact directly
in vitro and to coexist in Lewy bodies of
patients with PD, with a greater propor-
tion of Lewy bodies containing glucocere-
brosidase in patients with GBA mutations
compared with those in patients with PD
not carrying a GBA variant.4 9 It has been
speculated that mutant glucocerebrosidase
potentiates the aggregation of α-synuclein,
and it is therefore feasible that SNCA vari-
ants such as the rs356219 polymorphism
alters the degree to which GBA variants
predispose to PD pathology and disease
progression. It has also been suggested that
α-synuclein impedes the transit of glucoce-
rebrosidase from the endoplasmic reticulum
to the lysosome, further supporting the idea
that the relationship between these two
proteins is directly important in the patho-
genesis of GBA-PD.3 The proposed syner-
gistic interaction between variants in the
GBA gene and SNCA gene therefore could
also potentially be explained by differences
in the degree to which α-synuclein impairs
normal glucocerebrosidase processing asso-
ciated with specific SNCA variants. 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/. of our study is that all subjects in the G/G
group were male, and though this was
accounted for in the Cox regression model
and by performing the additional male-
only analysis, it would be important to
investigate the relationship between these
two genetic risk factors in a large cohort,
to reduce the effect of such confounders
and to allow for the stratification of G/G
carriers into those with severe and those
with mild GBA variants. © Author(s) (or their employer(s)) 2020. Re-use
permitted under CC BY. Published by BMJ. © Author(s) (or their employer(s)) 2020. Re-use
permitted under CC BY. Published by BMJ. ►
►Additional material is published online only. To
view please visit the journal online (http://dx.doi.org/
10.1136/jnnp-2019-322210). A common polymorphism in
SNCA is associated with
accelerated motor decline in
GBA-Parkinson’s disease J Neurol Neurosurg Psychiatry June 2020 Vol 91 No 6 673 PostScript References Genetic
modifiers of risk and age at onset in GBA associated
Parkinson’s disease and Lewy body dementia. Brain
2020;143:234–48. There is a degree of genotype–phenotype
correlation in GBA-PD, with severe muta-
tions accelerating disease course to a greater
extent than less severe variants.10 However,
because our sample size was small, it was
not possible to stratify the patients with
GBA-PD into those with non-severe and
severe GBA variants, so our GBA-PD
population was genetically heterogeneous
(online supplementary table 2). Mean age
at diagnosis was approximately 5 years later
in G/G carriers which may potentially have
contributed to their accelerated disease
course. However, there were no differences
in time to dementia or death, and when age
at diagnosis was accounted for in the Cox
regression model, the accelerated progres-
sion to HY3 persisted. Another limitation 6 Williams-Gray CH, Mason SL, Evans JR, et al. The
campaign study of Parkinson’s disease: 10-year
outlook in an incident population-based cohort. J
Neurol Neurosurg Psychiatry
2013;84:1258–64. 7 Winder-Rhodes SE, Evans JR, Ban M, et al. Glucocerebrosidase mutations influence the natural
history of Parkinson’s disease in a community-based
incident cohort. Brain 2013;136:392–9. 7 Winder-Rhodes SE, Evans JR, Ban M, et al. Glucocerebrosidase mutations influence the natural
history of Parkinson’s disease in a community-based
incident cohort. Brain 2013;136:392–9. 8 Moors TE, Paciotti S, Ingrassia A, et al. Characterization
of brain lysosomal activities in GBA-Related and
sporadic Parkinson’s disease and dementia with Lewy
bodies. Mol Neurobiol 2019;56:1344–55. Competing interests None declared. Competing interests None declared. Patient consent for publication Not required. Provenance and peer review Not commissioned;
externally peer reviewed. Provenance and peer review Not commissioned;
externally peer reviewed. 9 Goker-Alpan O, Stubblefield BK, Giasson BI, et al. Glucocerebrosidase is present in α-synuclein
inclusions in Lewy body disorders. Acta Neuropathol
2010;120:641–9. 9 Goker-Alpan O, Stubblefield BK, Giasson BI, et al. Glucocerebrosidase is present in α-synuclein
inclusions in Lewy body disorders. Acta Neuropathol
2010;120:641–9. 10 Liu G, Boot B, Locascio JJ, et al. Specifically
neuropathic Gaucher’s mutations accelerate cognitive
decline in Parkinson’s. Ann Neurol 2016;80:674–85. 674 J Neurol Neurosurg Psychiatry June 2020 Vol 91 No 6
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Climate change will likely facilitate invasion of Asian orchid Eulophia graminea into new areas
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Biological invasions
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cc-by
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Biol Invasions (2024) 26:1969–1983
https://doi.org/10.1007/s10530-024-03290-w Biol Invasions (2024) 26:1969–1983
https://doi.org/10.1007/s10530-024-03290-w ORIGINAL PAPER Climate change will likely facilitate invasion of Asian orchid
Eulophia graminea into new areas Marta Kolanowska · Agnieszka Rewicz ·
James D. Ackerman Received: 29 November 2023 / Accepted: 5 March 2024 / Published online: 2 April 2024
© The Author(s) 2024 Received: 29 November 2023 / Accepted: 5 March 2024 / Published online: 2 April 2024
© The Author(s) 2024 Abstract Climate change plays an increasing role in
the global biodiversity crisis. Alteration in local cli-
matic conditions not only can negatively affect native
biodiversity but also can accelerate the introduction
and spread of invasive species. In this study the eco-
logical niche modelling approach was used to evalu-
ate possible changes in the distribution of suitable
niches of invasive orchid Eulophia graminea within
its native (Asia) and non-native geographical range
(America, Australia). We mapped the current poten-
tial range of this species and analysed three various
projections of future climate (for 2100) each with
four different climate change scenarios (SSPs). Cal-
culated niche overlap indexes indicated low similarity
of niches occupied by native and invasive populations
of E. graminea and Australian populations seem to be
the most unique, while American and Asian groups
share partially similar niches. The occurrence of the
American population of E. graminea was correlated especially with the temperature seasonality, while the
Asian and Australian populations with annual pre-
cipitation and precipitation of the wettest quarter. As
indicated in our analyses within Asia and America,
E. graminea does not occupy all climatically suit-
able niches. On the other hand, in Australia the spe-
cies studied already occupies all appropriate niche
space. Climate change will likely be favorable for
species studied to expand its range if the biotic com-
ponents of its niche space (e.g., mycorrhizal fungi)
will respond similarly. The most significant range
expansion is predicted to occur in Australia which is
interesting considering the marginally suitable habi-
tats that E. graminea currently occupies. Keywords Climate projection · Intercontinental
invasion · Niche modelling · Shared socioeconomic
pathways Introduction Orchidaceae is the second largest family of flow-
ering plants with more than 31,000 species (POWO
2023). Representatives of this group are found around
the world, except polar regions and very dry deserts
(Dressler 1981). Nonetheless, the highest orchid
diversity is in the humid tropics (Vitt et al. 2023). Despite the great variation in morphology and physi-
ology observed within Orchidaceae (Zhang et al. 2018), relatively few species are considered to be
invasive or weedy (Ackerman 2007; Daehler 1998;
Randall 2017). In all biological invasions, there are barriers to
overcome at every stage of an invasion (Blackburn
et al. 2011). Major constraints to orchid establish-
ment and spread involve symbioses. The first barrier
is seed production. Fruit set in orchids is pollination
limited, which is exacerbated by generally having
one or very few pollinators (Ackerman et al. 2023;
Tremblay et al. 2005). Although fruits can contain
hundreds to millions of tiny, wind-dispersed seeds
which to a certain extent compensates for low fruit set
(Arditti and Ghani 2000; Sonkoly et al. 2016), disper-
sal is strongly leptokurtic and establishment is seed
limited (Ackerman et al. 1996; Brzosko et al. 2017). Like most plants, orchids that have become invasive
usually arrived at novel locations through the horti-
cultural trade (Rojas-Sandoval and Ackerman 2021;
van Kleunen et al. 2018), but subsequent stages of an
invasion, establishment and spread, generally require
seed production. If a novel location lacks a fauna that
can provide pollinator services, then self-pollination
and vegetative propagation are the only means for
population growth and dispersal. Less than a quarter
of orchid species have the capacity to autonomously
self-pollinate (Ackerman et al. 2023), further con-
straining which orchids would be able to become
established and spread. Biological invasions are complex ecological pro-
cesses which depend on propagule pressure, species
invasive potential, and the susceptibility of commu-
nities to invasion (Chaffin et al. 2016; Chown et al. 2015; Lowry et al. 2012; Simberloff et al. 2013). Moreover, this process is currently accelerated by
globalization which circumvents natural biogeo-
graphic barriers and increases propagule pressure,
and accidental or intentional releases of non-native
species (Hänfling and Kollmann 2002; Meyerson
and Mooney 2007). Global warming can accelerate
the spread of invasive species (Demertzis and Ili-
adis 2018) which can exacerbate climate change by
increasing wetland methane and terrestrial nitrous
oxide emissions (Bezabih Beyene et al. 2022). Introduction Supplementary Information The online version
contains supplementary material available at https://doi.
org/10.1007/s10530-024-03290-w. Human activities are responsible for the accumula-
tion of alien plant species across most regions of the
world, a process that continues to this day (Seebens
et al. 2017). Most of these species have been inten-
tionally introduced for agriculture, forage, forestry,
but most commonly as ornamentals (Beaury et al. 2021; Dodd et al. 2015; Rojas-Sandoval and Acker-
man 2021). The latter pathway is particularly egre-
gious as domestic gardens select species with traits M. Kolanowska (*) · A. Rewicz
Department of Geobotany and Plant Ecology, University
of Lodz, Ul. Banacha 12/16, 90‑237 Lodz, Poland
e-mail: marta.a.kolanowska@gmail.com M. Kolanowska (*) · A. Rewicz
Department of Geobotany and Plant Ecology, University
of Lodz, Ul. Banacha 12/16, 90‑237 Lodz, Poland
e-mail: marta.a.kolanowska@gmail.com J. D. Ackerman
Department of Biology, University of Puerto Rico,
17 Avenue Universidad Suite 1701, San Juan,
PR 00925‑2537, USA ol.: (01 123456789)
3 1970 M. Kolanowska et al. fundamental niche shift (Müller-Schärer et al. 2004),
release from dispersal barriers (Smith et al. 2020)
or biotic constraints (realized niche shifts) (Mitchell
et al. 2006). Also the spread of exotics is expected to
be influenced by the physiological response of alien
and native species to environmental changes and the
subsequent changes in ecological interactions (Robin-
son et al. 2020).l that are associated with invasion success (Guo et al. 2019). Unsurprisingly, many plants escape cultiva-
tion, spread into novel areas thereby becoming inva-
sive with potential negative impacts on the envi-
ronment, local biota, and human well-being (van
Kleunen et al. 2018). Non-indigenous plants, once introduced may
become invasive without continued intervention by
humans if environmental and biotic conditions are
conducive for establishment and spread, even into
undisturbed native vegetation (Mashhadi and Rado-
sevich 2004) where they can be a major threat to
native biodiversity and alter ecosystem functions
(Lowry et al. 2012; Simberloff et al. 2013; Vitousek
and Walker 1989). On the other hand, invasions may
also create novel communities under human-impacted
conditions that may provide ecosystem services such
as soil conservation, nutrient cycling, wildlife habi-
tat, carbon storage, watershed protection, and miti-
gate species extinctions (Lugo 2009). Whether or not
the net outcome of biological invasions is negative,
innocuous, or positive is likely context dependent,
either environmentally, biologically, sociologically, or
combinations of them all (Daehler 2003; Osborne and
Gioria 2022). List of localities List of localities Localities of E. graminea were compiled from the
Global Biodiversity Information Facility (GBIF
2022), herbarium specimens and field excursions. Only records which could be georeferenced with the
precision of 1 km were used in ENM analyses and the
duplicate presence records (records within the same
grid cell) were removed using MaxEnt. The complete
list of localities used in our study is available as Sup-
plementary Table S1. Due to the lack of sufficient
location data, the samples from South Africa were not
included in further analyses. Introduction Forecasting biological invasions is crucial for
managing non-native species but any predictions of
potential spread of exotics, should consider potential
species niche shifts resulting from evolved environ-
mental tolerances (fundamental niche) or the pres-
ence of novel conditions in the invaded range (real-
ized niche) (Tingley et al. 2014). The niche shift in
newly occupied regions (Rodder and Lotters 2009;
Stiels et al. 2015; Zhu et al. 2017) can result from The second hurdle to establishment success upon
arriving at a novel location is the need to form an
association with orchid mycorrhizal fungi (OMF). Orchid seeds are extremely small, lack endosperm 1
Vol:. 3
(1234567890) Climate change will likely facilitate invasion of Asian orchid Eulophia graminea into new… 1971 The reasons for the geographical differences in inva-
siveness are unknown.f and are dependent on OMF to provide resources for
successful germination (McCormick et al. 2018). Often this symbiosis is maintained into adulthood,
although the OMF that provide for successful germi-
nation are not necessarily the same that associate with
adult plants (Bayman et al. 2016; Phillips et al. 2020;
Zhang et al. 2018). While some OMF have a very
broad distribution, on a local scale they are not ubiq-
uitous so that local orchid populations may associate
with different sets of fungi that may involve niche
and/or spatial segregation (Fernández et al. 2023;
McCormick et al. 2018; Swift et al. 2019), although
this is not always the case (Suarez et al. 2016). The
spatial distribution of the fungi may be dependent on
biophysical factors which, in turn, would affect the
distribution of their orchid symbionts (Izuddin et al. 2019; Jacquemyn et al. 2016). The aim of this study is to evaluate differences
in environmental niche preferences of E. graminea
in various geographical regions, to visualize current
non-native potential range of this orchid and to esti-
mate the invasive potential of this species under vari-
ous climate change scenarios using ecological niche
modelling (ENM). Machine learning-based models
are commonly used to estimate distribution of suita-
ble niches of invasive species and predict their further
spread in non-native areas (Cunze et al. 2020; Fand
et al. 2020; Paclibar and Tadiosa 2019; Stiels et al. 2011). Methods q
y
)
A rapidly spreading orchid species which is
already present on several continents (Ackerman
and González-Orellana 2021; Juárez Gutiérrez et al. 2023) is Eulophia graminea Lindl. (chinese crown
orchid, grass leaved Eulophia). This terrestrial spe-
cies is native to southern and central Asia. It produces
rounded pseudobulbs and long, thin, linear leaves. The long inflorescence of E. graminea is composed of
numerous flowers with greenish tepals and 3-lobed,
white-pinkish lip. As summarized by Chang et al. (2010), there are several characters of E. graminea
that likely facilitate its invasiveness. The quick ger-
mination and fast rhizome production enhance seed-
ling survival and the short juvenile stage increases the
expansive potential of this orchid. While pollinator
availability is an important factor affecting long-term
survival of most orchids, flowers of E. graminea are
autogamous (Chang et al. 2010). Its dispersibility is
exemplified by being one of the few orchid species
to colonize remnants of Krakatau 25 years after the
1883 explosion (Partomihardjo 2003).i Climatic niche similarity We used a principal components analysis (PCA) to
assess niche variability within the native and non-
native distribution of E. graminea populations (from
America, Asia and Australia). Each population was
characterized by a set of 21 traits of which 19 were
related to the climate data (Fick and Hijmans 2017),
one described land cover (European Commission
2003), and one soil type (Hengl et al. 2017). To
reduce bias, samples were spatially filtered at 5 km. Calculations were made with the software pack-
ages PAST ver. 4.03 (PAST PAleontological STatis-
tics Version 3.20). The data matrix (Supplementary
Table S2) was transformed (square root) before per-
forming the ordination analysis. The first report of the chinese crown orchid outside
its native range came from Australian Northern Ter-
ritories (Macrae 2002; Pemberton 2013). Later it was
recorded in South Africa (O’Conner et al. 2006) and
USA (Pemberton et al. 2008). While the populations
in Australia and Africa have not significantly spread
since initial observations, its rapid range expansion
in Florida has continued and has reached into the
West Indies (Ackerman and González-Orellana 2021;
Juárez Gutiérrez et al. 2023; Singhurst et al. 2020). 1
Vol.: (01 3
123456789) 1972 M. Kolanowska et al. for four Shared Socio-economic Pathways (SSPs):
1–2.6, 2–4.5, 3–7.0 and 5–8.5 (Li et al. 2021; McGee
et al. 2000; Meinshausen et al. 2020). SSPs are tra-
jectories adopted by the Intergovernmental Panel
on Climate Change (IPCC), comprising narrative
descriptions of future world development (Riahi et al. 2017). SSP storylines describe contrasting visions of
future society and the assumed climate change chal-
lenges, with global warming in 2100 ranging from a
low of 3.1 °C to a high of 5.1 °C above pre-industrial
levels. O’Neill et al. (2017). Three different simula-
tions of future climate developed by Coupled Model
Intercomparison Project Phase 6 (CNRM), Goddard
Institute for Space Studies (GISS), and Institute for
Numerical Mathematics (INM) were used. We chose
these projections because they present the most differ-
ent simulations of maximum temperature and precipi-
tation within study areas (Supplementary Figure S4). Climatic niche modelling The modelling of the current and future distribu-
tion of the species studied was done using the
maximum entropy method implemented in Max-
Ent version 3.3.2 (Elith et al. 2011; Phillips et al. 2006; Phillips and Dudik 2008), which is based on
presence-only observations. Bioclimatic variables
in 30 arc-seconds of interpolated climate surface
downloaded from WorldClim v. 2.1 were used for
the modelling (Fick and Hijmans 2017). The study
area was divided into three geographical regions—
America (0.15°S–44.16°N, 127.64–58.20°W), Asia
(10.99°S–41.80°N, 67.54–159.78°E), and Australia
(10.79–17.39°S, 125.98–138.09°E). Pearsons’ correlation coefficient was computed
using SDMtoolbox 2.3 for ArcGIS (Brown 2014;
Brown et al. 2017) (Supplementary Table S3) and
highly correlated (> 0.8) variables were removed
from ENM analyses to prevent problems associated
with auto-correlation. The final list of bioclimatic
variables used in the analyses is provided in Table 1. In all analyses the maximum number of itera-
tions was set to 10,000 and convergence threshold to
0.00001. The neutral (= 1) regularization multiplier
value and auto features were used. The “random seed"
option provided a random test partition and back-
ground subset for each run and 20% of the samples We predicted the future extent of E. graminea cli-
matic niches for 2080–2100 by using four projections Table 1 Climatic variables used in ENM analyses (marked with +)
Variable code
Variable description
Geographical region
America
Australia
Asia
bio1
Annual mean temperature
+
+
+
bio2
Mean diurnal range [mean of monthly (max temp–min temp)]
+
+
+
bio3
Isothermality (bio2/bio7) (× 100)
+
+
+
bio4
Temperature seasonality (standard deviation × 100)
bio5
Max temperature of warmest month
+
bio6
Min temperature of coldest month
bio7
Temperature annual range (bio5-bio6)
bio8
Mean temperature of wettest quarter
+
+
bio9
Mean temperature of driest quarter
+
bio10
Mean temperature of warmest quarter
bio11
Mean temperature of coldest quarter
bio12
Annual precipitation
+
+
+
bio13
Precipitation of wettest month
bio14
Precipitation of driest month
+
+
+
bio15
Precipitation seasonality (coefficient of variation)
+
+
+
bio16
Precipitation of wettest quarter
bio17
Precipitation of driest quarter
+
bio18
Precipitation of warmest quarter
+
+
+
bio19
Precipitation of coldest quarter Table 1 Climatic variables used in ENM analyses (marked with +) Table 1 Climatic variables used in ENM analyses (marked with +)
Variable code
Variable description 1
Vol:. Climatic niche modelling ( Climate change will likely facilitate invasion of Asian orchid Eulophia graminea into new… 1973 were used as test points. The run was performed as a
bootstrap with 100 replicates. The output was set to
logistic. We used the “fade by clamping” function in
MaxEnt was used to prevent extrapolations outside
the environmental range of the training data (Owens
et al. 2013). All analyses of GIS data were carried
out using ArcGis 10.6 (Esri, Redlands, CA, USA). The evaluation of the created models was made using
the area under the curve (AUC) (Mason and Graham
2002) and True Skill Statistic (TSS) (Čengić et al. 2020; Shabani et al. 2016). Results Differences in occupied niches between geographical
groups Differences in occupied niches between geographical
groups The PCA analyses revealed the highest popula-
tion density of E. graminea from America clustered
in the center of the PCA plot, with distinct separate
populations originating from Australia. Populations
of the orchid from Asia were the most dispersed and
occupied the right part of the PCA plot (Fig. 1). The
analysis indicated that the first two principal compo-
nents explained 73.8% of the total variance. The first
component represented 41.1% of the total variance
and the second component accounted for 32.7%. PC1
is correlated with bio16, bio12 and bio4. PC2 is cor-
related with bio14, bio16 and bio19. The first com-
ponent demonstrates positive correlations with bio16
and bio12, and negative correlations with bio4. The
second component demonstrates positive correlations
with bio14 and bio19 (Supplementary Figure S5). The occurrence of the American population of E. graminea was correlated especially with bio4, while
the Asian and Australian populations with bio16 and
bio12. To visualize changes in the distribution of suit-
able niches of the orchid, we used SDMtoolbox 2.3
for ArcGIS (Brown 2014; Brown et al. 2017). To
compare the distribution model created for current
climatic conditions with future predictions all SDMs
were converted into binary rasters and projected
using the Goode homolosine as a projection. The
presence thresholds used in the analyses equaled the
calculated minimum training presence threshold (Liu
et al. 2005). Based on obtained models the overlap of the cli-
matic niches between the native and the introduced
ranges was assessed using Schoener’s D and I statis-
tics (Warren et al. 2008). Vol : (0123456789)
Fig. 1 PCA ordination diagram of studied localities of Eulo-
phia graminea according to 21 environmental predictors (19
were related to the climate data, one described land cover and
one soil type). Legend: black dots—North American popula-
tions, red dots–Asian populations, green dots–Australian popu-
lations) Fig. 1 PCA ordination diagram of studied localities of Eulo-
phia graminea according to 21 environmental predictors (19
were related to the climate data, one described land cover and one soil type). Legend: black dots—North American popula-
tions, red dots–Asian populations, green dots–Australian popu-
lations) 1
ol.: (0 3
) 1974 M. Kolanowska et al. The sites occupied among geographical groups
did not differ significantly in annual temperature
(bio1) (Supplementary Figure S6); however, non-
native populations seemed to be adapted to slightly
higher temperatures (Asia 21.0–28.0 °C, America
23.0–35.0 °C, Australia 26.5–30.0 °C). Models evaluation and limiting factors Models evaluation and limiting factors All created models received high scores of AUC
(0.958–0.999) and mostly high scores of TSS
(0.748–0.991) tests. Both sensitivity and specificity
were also generally high indicating good reliability
of presented modelling results (Table 3). According to the jackknife test of variable impor-
tance, for both Asian and Australian populations
bio12 (annual precipitation) was the variable with
the highest gain when used in isolation, and the
same variable decreased the most when it was omit-
ted. Thus, it not only is the most informative, but
also contains information not represented by the
other variables. Models of American populations
received different scores. The variable with highest
gain when used in isolation was bio8 (mean tem-
perature of the wettest quarter) while bio1 (annual
mean temperature) decreased the gain the most
when it was omitted (Supplementary Figure S5). Differences in occupied niches between geographical
groups The mean
diurnal range of temperature (bio2) was narrower
in American group (Asia 3–15 °C, America 3–8 °C,
Australia 4–17.5 °C). Australian populations have
narrower tolerance for isothermality (bio3; Asia—
18–58, America 12–85, Australia 56–66). Consid-
ering annual precipitation (bio12) Australian popu-
lations are characterized by narrower tolerance for
rainfall (Asia – 800 to 1800 mm, America − 1000
to 2000 mm, Australia 1700–2000 mm) and precipi-
tation of the driest month (bio14; Asia 20–550 mm,
America 50–550 mm, Australia − 0.5 to 6.5 mm). The precipitation seasonality (bio15) did not dif-
fer significantly among geographical groups (Asia
5–110, America 10–100, Australia 108–137), but
precipitation of the warmest quarter was highly
variable among regions (bio18; Asia 800–5800 mm,
America 200–2000 mm, Australia 90–930 mm). Current potential orchid range Calculated niche overlap indexes (Table 2) indi-
cated low similarity of niches occupied by native
and invasive populations of E. graminea. Austral-
ian populations seem to be the most unique, while
American and Asian groups share partially similar
niches. Generally, current potential range of E. graminea is
consistent with the known distribution of the spe-
cies populations; nonetheless, Asian and Ameri-
can models indicated presence of suitable niches
in some areas not occupied by this orchid (Fig. 2). Within Asian study area, suitable niches of E. graminea are located in New Guinea which is out-
side species known native range. In America, our
model indicated Lesser Antilles, Jamaica, Domi-
nica and Haiti as additional areas suitable for E. graminea occurrence. The Australian model is con-
sistent with the distribution of known populations
of the orchid. 1
Vol:. (1234567890)
model indicated Lesser Antilles, Jamaica, Domi
nica and Haiti as additional areas suitable for E. graminea occurrence. The Australian model is con-
sistent with the distribution of known populations
of the orchid. Table 2 Niche overlap calculated using Schoener’s D and I
statistics
D\I
America
Asia
Australia
America
x
0.5919
0.0018
Asia
0.4670
x
0.0221
Australia
0.0002
0.0103
x
Table 3 Scores of model
reliability tests and value of
minimum training presence
Modelled area
AUC
TSS
Sensitivity
Specificity
Minimum training
presence logistic
threshold
America
0.986
0.959
0.977
0.982
0.0215
Asia
0.958
0.748
0.978
0.769
0.0295
Australia
0.999
0.9912
1
0.991
0.2174 Table 2 Niche overlap calculated using Schoener’s D and I
statistics Table 3 Scores of model
reliability tests and value of
minimum training presence
Modelled area
AUC
TSS
Sensitivity
Specificity
Minimum training
presence logistic
threshold
America
0.986
0.959
0.977
0.982
0.0215
Asia
0.958
0.748
0.978
0.769
0.0295
Australia
0.999
0.9912
1
0.991
0.2174 1
Vol:. ( 3
(1234567890) Climate change will likely facilitate invasion of Asian orchid Eulophia graminea into new… 1975 Fig. 2 Current distribution of suitable niches of E. graminea in Asia (A), North America (B), and northern Australia (C) t distribution of suitable niches of E. graminea in Asia (A), North America (B), and northern Australia (C) Fig. 2 Current distribution of suitable niches of E. graminea in Asia (A), North America (B), and northe Changes in the distribution of suitable niches of E.
graminea two predictions the coverage of suitable niches of E. graminea will be several times larger than currently
recorded (Table 4, Fig. 5). In these scenarios species
will expand its range in Melville and Bathusts islands,
and south from Darwin to West Daly region. In some
scenarios additional niches will also become avail-
able for E. graminea around the South Alligator River
estuary. All analysed projections indicate that E. graminea
will expand its native range in Asia (Table 4, Fig. 3);
however, niches located currently in foothills of
New Guinean Highlands, southern foothills of Mül-
ler mountains in Borneo, Indian Eastern Ghats will
become unsuitable for the orchid. CNRM projec-
tions also indicate south-eastern Himalayan foothills,
steppes and savannas of Myanmar as areas of poten-
tial range contraction for E. graminea. Expansion will
mostly occur in south-western and south-central foot-
hills of Himalayas, areas around Indian Chota Nagpur
Plateau and Vindhya Range, Chinese Sichuan Plain,
southern South Korea, and southern Honshu in Japan.i Potential versus observed geographical range Potential versus observed geographical range Every species has a fundamental niche of which only
a portion is occupied (realized niche). The elements
constituting both types of niche cannot be completely
described but can be estimated as a hypervolume of
various biophysical parameters (Blonder et al. 2014). During the expansion into non-native geographical
regions invasive species generally occupy the same
hypervolume constituted by the fundamental niche
of the species as defined by its native range (Aravind
et al. 2022). North American invasive populations will benefit
from global warming and coverage of suitable niches
of the orchid will expand for 36–193% (Table 4,
Fig. 4), generally into the Great Plains, and north-
eastern directions within US Coastal Plain. The range
contraction is expected to occur (not in all scenarios)
in Guatemala and Belize around the Belize River;
Puerto Rico along the foothills of the Sierra de Cayey
and Cordillera Central, and the Mogotes; Dominican
Republic in lowlands north of Cordillera Central,
south of the Cordillera Oriental, and the Cordillera
Septentrional; Haiti along Massif du Nord and foot-
hills of Massif de la Hotte; Jamaica primarily around
Westmoreland and Saint Catherine parishes; Cuba
along western foothills of the Sierra Cristal, western
and northern foothills of Sierra Maestra (Supplemen-
tary Figure S7). As indicated in our analyses even within Asia,
E. graminea does not occupy all climatically suit-
able niches. That may be caused either by geographi-
cal barriers preventing spread, unmeasured natural
or human-induced abiotic conditions (Wraith et al. 2020), or by constraining biotic factors in unoccu-
pied areas, e.g. lack of symbiotic mycorrhizal fungi
(Downing et al. 2020), presence of herbivores and
pathogens (Meena et al. 2018; Meena and Mani
2022). However, it should be noted that the actual
sensibility of natural orchid populations to patho-
gens is still little recognized (Melendez and Ack-
erman 1993). A similar situation of uninhabited Australian populations of E. graminea will likely
become extinct in GISS projection, but their potential
invasive range will be larger than currently observed
in both CNRM and INM simulations. In the latter 1
ol.: (01 3
123456789) 1976 M. Kolanowska et al. Table 4 Changes in the coverage (km2) of the suitable niches of E. Potential versus observed geographical range 3
(1234567890) Climate change will likely facilitate invasion of Asian orchid Eulophia graminea into new… 1977 Fig. 3 Changes in the dis-
tribution of suitable niches
of E. graminea in Asia in
various climate change
scenarios -
s (Recart et al. 2013). The invasive orchid was the
preferred host of a native, orchid specialist weevil,
Stethobaris polita, previously regarded as a relatively
rare beetle. Flower and fruit damage to S. plicata was
high with significant demographic consequences,
but population growth rates remained positive (Fal-
cón et al. 2017). Elevated beetle populations resulted
in increased weevil attack on the native orchid, sig-
nificantly reducing fruit production. Two other com-
mon, non-indigenous orchids on the island also serve
as hosts, Arundina graminifolia and Dendrobium
crumenatum, and may elevate weevil populations as
well, with possible impacts on native species (Fos-
ter and Ackerman 2021). The other orchid which is
considered by local authorities to constitute a threat
to native plants is Disa bracteata which invaded
Australia in 1944 (Wapstra et al. 2020). However, in
this case there is no evidence of negative effects on
the Australian flora. We are unaware of any studies
designed to detect the consequences of E. graminea
invasions. comparison, American and Asian groups are more
similar to each other, but still the overlap in occupied
niches between these regions is low. Impact of global warming. Climate change can accelerate non-native plant
invasions by altering environmental conditions,
which may shift the geographical distribution of suit-
able niche components and affect normal regimes
of habitat disturbance (e.g., the frequency, duration,
and severity of climatic events). Further change may
occur through human responses to these changes
(Adhikari et al. 2019; Blumenthal et al. 2013; Dukes
and Mooney 1999; Turbelin and Catford 2021). Climate change will likely be favorable for E. graminea to expand its range if the unaccounted
biotic components of its niche space (e.g., mycorrhi-
zal fungi) will respond similarly. The most significant
range expansion is predicted to occur in Australia
(in 2 of 3 projections) which is interesting consider-
ing the marginally suitable habitats that it currently
occupies. The two most obvious ecological constraints for
any orchid species persistence are (1) availability of
pollen vectors and (2) presence of microbial sym-
bionts in the environment. The former is not likely
a problem for E. Potential versus observed geographical range graminea
Geographical region
Projection
SSP scenario
Range expansion
No range change
Range contraction
Change (%)
North America
CNRM
1–2.6
503,775.4
576,167.5
27,525.28
+ 79
2–4.5
770,379.3
582,260.7
21,432.05
+ 124
3–7.0
882,609.6
476,440.1
127,252.6
+ 125
5–8.5
1,295,440
470,612.6
133,080.1
+ 193
GISS
1–2.6
444,069.4
544,644.5
59,048.22
+ 64
2–4.5
456,440.7
543,423.5
60,269.27
+ 66
3–7.0
580,439.4
528,220.5
75,472.24
+ 84
5–8.5
526,820.6
484,518
119,174.7
+ 68
INM
1–2.6
264,659.7
556,143.1
47,549.68
+ 36
2–4.5
366,845.5
546,330.8
57,361.96
+ 51
3–7.0
722,937.2
561,093.5
42,599.26
+ 113
5–8.5
929,173.8
569,635.7
34,057.05
+ 148
Asia
CNRM
1–2.6
1,591,586
5,189,896
328,331.2
+ 23
2–4.5
2,059,589
5,150,549
367,678.3
+ 31
3–7.0
2,415,786
4,936,159
582,068.7
+ 33
5–8.5
2,645,666
4,403,998
1,114,230
+ 28
GISS
1–2.6
1,286,665
5,206,398
311,829.6
+ 18
2–4.5
2,058,696
5,307,062
211,165.9
+ 33
3–7.0
2,463,861
5,351,687
166,540.4
+ 42
5–8.5
2,688,171
5,150,799
367,428.1
+ 42
INM
1–2.6
988,160.4
5,203,643
314,584.9
+ 12
2–4.5
1,479,287
5,274,574
243,653.5
+ 22
3–7.0
1,943,816
5,056,149
462,078.8
+ 27
5–8.5
2,182,075
5,137,480
380,747.9
+ 33
Australia
CNRM
1–2.6
1975.163
503.5561
564.6961
+ 132
2–4.5
13,809.15
1068.252
0
+ 1293
3–7.0
7569.475
977.3913
90.86087
+ 700
5–8.5
7622.123
701.412
366.8402
+ 679
GISS
1–2.6
0
0
1068.252
− 100
2–4.5
0
0
1068.252
− 100
3–7.0
0
0.849167
1067.403
− 100
5–8.5
0
71.33003
996.9221
− 93
INM
1–2.6
8646.219
838.977
229.2751
+ 788
2–4.5
7750.348
1068.252
0
+ 726
3–7.0
2418.428
990.1288
78.12337
+ 219
5–8.5
855.1112
723.4903
344.7618
+ 48 Table 4 Changes in the coverage (km2) of the suitable niches of E. graminea potential range is observed in North America; how-
ever, because unfilled niches are located on islands,
time and geographical barriers are most probable
reasons for unoccupied but potentially suitable areas
in this region. Time is a factor as this invasion is
relatively recent having been first noted in 2007 and
new populations are discovered every year. We have
anecdotal evidence that stratified dispersal is likely involved: long-distance dispersal to islands from
Florida (USA) via the landscape trade in topsoil and
potted plants; and local wind-dispersal of the dust-
like seeds. In Australia the species is not expected to spread
under current conditions since it already occupies
all appropriate niche space which is only marginally
suitable based on our analyses of its native range. In 1
Vol:. Fig. 3 Changes in the dis-
tribution of suitable niches
of E. graminea in Asia in
various climate change
scenarios Fig. 5 Changes in the dis-
tribution of suitable niches
of E. graminea in Australia
in various climate change
scenarios Fig. 4 Changes in the dis-
tribution of suitable niches
of E. graminea in North
America in various climate
change scenarios Potential versus observed geographical range While broad-scale analyses
across the geographical regions are needed to uncover
the importance of fungal partner(s) for orchid long-
term survival, current results suggest that availabil-
ity of OMF may not be a constraint to any of these
invasive orchids now or in the future under climate
change scenarios. Nonetheless, the orchid and its
OMF symbionts will not necessarily respond to cli-
mate change similarly (Kolanowska 2023). Although
OMF have garnered most of the attention because of
their importance in seed germination, other microbial
endophytes can have fitness consequences by affect-
ing plant growth, resistance to pathogens, and toler-
ance capacity to biotic and abiotic stresses. We know
very little about these interactions in an ecological or
biogeographical context (Bayman et al. 2002; Sar-
saiya et al. 2019). The other factor which was not included in our
analyses was geographical distribution of mating
system. We only know that flowers of native Taiwan
populations are autogamous (Chang et al. 2010),
but in other regions, native or non-native, reproduc-
tion in E. graminea may be pollinator-dependent. This may be important because orchid species and
their pollinators will not necessarily respond to cli-
mate change in the same manner so potential range
shifts of both should assessed (Kolanowska 2021;
Kolanowska et al. 2021). Some orchids are characterized by very special-
ized relationships with mycorrhizal partners and
for these species predictions of future distribu-
tion should be accompanied with the analyses of
changes in the potential ranges of their symbionts
(Kolanowska 2023). Unfortunately, little is known
of the endophytic biota of E. graminea (Downing
et al. 2020) and even less of their geographical dis-
tribution so ENM of the ecological relationships of
this species was not possible. Obviously, considering human impact on E. graminea, the most damaging is direct habitat
destruction which cannot be predicted and included
in simulations. However, the ecological ampli-
tude of this orchid is remarkable. It grows in sandy
beaches, coastal grasslands, lowland shrubs and
open forests. It also does very well in lawns, road-
sides, parks, home and hotel gardens (Ackerman
and González-Orellana 2021; Chang et al. 2010). This broad tolerance and ability to adapt to vari-
ous environments constitutes an advantage of E. graminea in human-altered ecosystems. Potential versus observed geographical range graminea since the flowers appear Orchids are generally not considered to be harm-
ful where they have invaded. However, a study con-
ducted in Puerto Rico demonstrated apparent com-
petition between mixed populations of introduced
Spathoglottis plicata on a native orchid, Bletia patula 1
ol.: (01 3
123456789) 1978 M. Kolanowska et al. 1 3
Vol:. (1234567890)
Fig. 4 Changes in the dis-
tribution of suitable niches
of E. graminea in North
America in various climate
change scenarios
Fig. 5 Changes in the dis-
tribution of suitable niches
of E. graminea in Australia
in various climate change
scenarios 1 3
Vol:. (1234567890)
Fig. 5 Changes in the dis-
tribution of suitable niches
of E. graminea in Australia
in various climate change
scenarios Fig. 5 Changes in the dis-
tribution of suitable niches
of E. graminea in Australia
in various climate change
scenarios 1
Vol:. ( 1
Vol:. ( 3
(1234567890) Climate change will likely facilitate invasion of Asian orchid Eulophia graminea into new… 1979 to be self-pollinating, perhaps by the same mecha-
nism as E. maculata (Chang et al. 2010; González-
Díaz and Ackerman 1988). The flowers contain
small quantities of nectar (0.5 μl; (Ackerman and
González-Orellana 2021) so the potential for a mutu-
alistic plant-pollinator interaction exists, but thus far
no pollinators have been reported (Ackerman et al. 2023). On the other hand, E. graminea cannot escape
the need for OMF. Based on expectations of bipartite
networks, invasive or widespread native orchids likely
specialize on a widespread OMF, or be a generalist
in the number of OMF that they can exploit (Acker-
man 2007; Bascompte et al. 2003; Vázquez and Aizen
2004). Downing et al. (Downing et al. 2020) com-
pared the OMF of E. graminea from its native range
in southwest China and the OMF of the species in its
invasive range (Florida, USA) and concluded that E. graminea is a generalist with respect to its OMF as
10 of 18 fungal strains tested had successfully germi-
nated seeds ex situ. The OMF are known for only two
other invasive orchids, Eulophia (as Oeceoclades)
maculata and Disa bracteata and both orchids asso-
ciate with widespread OMF (Bayman et al. 2016;
Bonnardeaux et al. 2007). Potential versus observed geographical range While broad-scale analyses
across the geographical regions are needed to uncover
the importance of fungal partner(s) for orchid long-
term survival, current results suggest that availabil-
ity of OMF may not be a constraint to any of these
invasive orchids now or in the future under climate
change scenarios. Nonetheless, the orchid and its
OMF symbionts will not necessarily respond to cli-
mate change similarly (Kolanowska 2023). Although
OMF have garnered most of the attention because of
their importance in seed germination, other microbial
endophytes can have fitness consequences by affect-
ing plant growth, resistance to pathogens, and toler-
ance capacity to biotic and abiotic stresses. We know
very little about these interactions in an ecological or
biogeographical context (Bayman et al. 2002; Sar-
saiya et al. 2019). However, currently it is not possible to evaluate the
possible alterations in the soil physical, chemical
or biotic properties resulting from global warming. According to our data (Supplementary Table S2)
E. graminea can grow in eleven different soil types
(acrisols, arenosols, cambisols, ferralsols, fluvisols,
gleysols, histosols, leptosols, luvisols, phaeozems,
vertisols) and apparently has rather broad tolerance
for various substrates. to be self-pollinating, perhaps by the same mecha-
nism as E. maculata (Chang et al. 2010; González-
Díaz and Ackerman 1988). The flowers contain
small quantities of nectar (0.5 μl; (Ackerman and
González-Orellana 2021) so the potential for a mutu-
alistic plant-pollinator interaction exists, but thus far
no pollinators have been reported (Ackerman et al. 2023). On the other hand, E. graminea cannot escape
the need for OMF. Based on expectations of bipartite
networks, invasive or widespread native orchids likely
specialize on a widespread OMF, or be a generalist
in the number of OMF that they can exploit (Acker-
man 2007; Bascompte et al. 2003; Vázquez and Aizen
2004). Downing et al. (Downing et al. 2020) com-
pared the OMF of E. graminea from its native range
in southwest China and the OMF of the species in its
invasive range (Florida, USA) and concluded that E. graminea is a generalist with respect to its OMF as
10 of 18 fungal strains tested had successfully germi-
nated seeds ex situ. The OMF are known for only two
other invasive orchids, Eulophia (as Oeceoclades)
maculata and Disa bracteata and both orchids asso-
ciate with widespread OMF (Bayman et al. 2016;
Bonnardeaux et al. 2007). Declarations Bayman P, Gonzalez E, Fumero J et al (2002) Are fungi nec-
essary? How fungicides affect growth and survival
of the orchid Lepanthes rupestris in the field. J Ecol
90:1002–1008 Conflict of interest The authors declare that they have no fi-
nancial or non-financial conflicts of interest. Consent for publication All authors provide their consent for
publication. Bayman P, Mosquera-Espinosa A, Saladini-Aponte C et al
(2016) Age-dependent mycorrhizal specifi city in an
invasive orchid, Oeceoclades maculata. Am J Bot
103:1880–1889 Beaury EM, Patrick M, Bradley BA (2021) Invaders for sale:
the ongoing spread of invasive species by the plant trade
industry. Front Ecol Environ 19:550–556 Bezabih Beyene B, Li J, Yuan J et al (2022) Non-native plant
invasion can accelerate global climate change by increas-
ing wetland methane and terrestrial nitrous oxide emis-
sions. Glob Change Biol 28:5453–5468 Open Access This article is licensed under a Creative Com-
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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/. Blackburn TM, Pyšek P, Bacher S et al (2011) A proposed
unified framework for biological invasions. Trends Ecol
Evol 26:333–339 Blonder B, Lamanna C, Violle C et al (2014) The n-dimen-
sional hypervolume. Glob Ecol Biogeogr 23:595–609 Blumenthal DM, Resco V, Morgan JA et al (2013) Invasive
forb benefits from water savings by native plants and car-
bon fertilization under elevated CO2 and warming. New
Phytol 200:1156–1165 Bonnardeaux Y, Brundrett M, Batty A et al (2007) Diversity
of mycorrhizal fungi of terrestrial orchids: compatibility
webs, brief encounters, lasting relationships and alien
invasions. Mycol Res 111:51–61 Brown J (2014) SDMtoolbox: a python-based GIS toolkit for
landscape genetic, biogeographic and species distribution
model analyses. Declarations Methods Ecol Evol 5:694–700 Brown JL, Bennett JR, French CM (2017) SDMtoolbox 2.0:
the next generation Python-based GIS toolkit for land-
scape genetic, biogeographic and species distribution
model analyses. PeerJ 5:e4095 Model deficiencies Author contributions All authors contributed to the study
conception and design. Material preparation, data collection
and analysis were performed by Marta Kolanowska, James
Ackerman and Agnieszka Rewicz. The first draft of the manu-
script was written by Marta Kolanowska and all authors com-
mented on previous versions of the manuscript. All authors
read and approved the final manuscript. This study, as any other simulation, has its limita-
tions. The models of future distribution of suitable
niches of E. graminea are based exclusively on
the climatic data. The grass leaved Eulophia is a
terrestrial species and depends on the soil proper-
ties which may also be altered by climate changes. 1
ol.: (01 3
123456789) 1980 M. Kolanowska et al. home” or finding a “new home”? Biodivers Conserv
31:2625–2638 Funding The authors declare that no funds, grants, or
other support were received during the preparation of this
manuscript. Arditti J, Ghani AKA (2000) Tansley Review No. 110. Numer-
ical and physical properties of orchid seeds and their bio-
logical implications. New Phytol 145:367–421 Data availability The datasets analyzed during the current
study are available as supplementary information. Bascompte J, Jordano P, Melián CJ et al (2003) The nested
assembly of plant–animal mutualistic networks. Proc
Natl Acad Sci 100:9383–9387 References Biol Cons 84:167–180 Jacquemyn H, Waud M, Merckx VS et al (2016) Habitat-driven
variation in mycorrhizal communities in the terrestrial
orchid genus Dactylorhiza. Sci Rep 6:37182 Daehler CC (2003) Performance comparisons of co-occurring
native and alien invasive plants: implications for con-
servation and restoration. Annu Rev Ecol Evol Syst
34:183–211 Juárez Gutiérrez HD, Fernández JIE, Maldonado Moreno C
et al (2023) First records of Eulophia graminea (Orchi-
daceae) naturalized in Mexico. Phytoneuron 11:1–5 Demertzis K, Iliadis L (2018) The impact of climate change
on biodiversity: the ecological consequences of invasive
species in Greece. In: Leal Filho W, Manolas E, Azul
AM, Azeiteiro UM, McGhie H (eds) Handbook of cli-
mate change communication, theory of climate change
communication, vol 1. Springer, Cham, pp 15–38 Kolanowska M (2021) The future of a montane orchid species
and the impact of climate change on the distribution of
its pollinators and magnet species. Glob Ecol Conserv
32:e01939 Kolanowska M (2023) Loss of fungal symbionts and changes
in pollinator availability caused by climate change will
affect the distribution and survival chances of myco-het-
erotrophic orchid species. Sci Rep 13:6848 Dodd AJ, Burgman MA, McCarthy MA et al (2015) The
changing patterns of plant naturalization in Australia. Divers Distrib 21:1038–1050 Downing JL, Liu H, McCormick MK et al (2020) General-
ized mycorrhizal interactions and fungal enemy release
drive range expansion of orchids in southern Florida. Ecosphere 11:e03228i Kolanowska M, Michalska E, Konowalik K (2021) The impact
of global warming on the niches and pollinator availabil-
ity of sexually deceptive orchid with a single pollen vec-
tor. Sci Total Environ 795:148850 Li J, Chen X, Kurban A et al (2021) Coupled SSPs-RCPs sce-
narios to project the future dynamic variations of water-
soil-carbon-biodiversity services in Central Asia. Ecol
Indic 129:107936 Dressler R (1981) The orchids: natural history and classifica-
tion. Harvard University Press, Cambridge Dukes JS, Mooney HA (1999) Does global change increase
the success of biological invaders? Trends Ecol Evol
14:135–139 Liu C, Berry PM, Dawson TP et al (2005) Selecting thresholds
of occurrence in the prediction of species distributions. Ecography 28:385–393 Elith J, Phillips S, Hastie T et al (2011) A statistical explana-
tion of MaxEnt for ecologists. Divers Distrib 17:43–57 Lowry E, Rollinson EJ, Laybourn AJ et al (2012) Biological
invasions: a field synopsis, systematic review, and data-
base of the literature. References Ackerman JD (2007) Invasive orchids: weeds we hate to love? Lankesteriana 7:19–21 Brzosko E, Ostrowiecka B, Kotowicz J et al (2017) Seed dis-
persal in six species of terrestrial orchids in Biebrza
National Park (NE Poland). Acta Soc Botan Polaniae
86:3557 Ackerman JD, González-Orellana N (2021) Explosive range
expansion of Eulophia graminea (Orchidaceae) in Puerto
Rico and the West Indies. Lankesteriana 21:307–312 Čengić M, Rost J, Remenska D et al (2020) On the importance
of predictor choice, modelling technique, and number of
pseudo-absences for bioclimatic envelope model perfor-
mance. Ecol Evol 10:12307–12317fi Ackerman JD, Sabat A, Zimmerman JK (1996) Seedling estab-
lishment in an epiphytic orchid: an experimental study of
seed limitation. Oecologia 106:192–198 Ackerman JD, Phillips RD, Tremblay RL et al (2023) Beyond
the various contrivances by which orchids are pollinated:
global patterns in orchid pollination biology. Bot J Linn
Soc 202:295–324 Chaffin BC, Garmestani AS, Angeler DG et al (2016) Biologi-
cal invasions, ecological resilience and adaptive govern-
ance. J Environ Manag 183:399–407l Chang C, Hu W-H, Chen Y-C et al (2010) In vitro flowering
and mating system of Eulophia graminea Lindl. Bot Stud
51:357–362fi Adhikari P, Jeon J-Y, Kim HW et al (2019) Potential impact
of climate change on plant invasion in the Republic of
Korea. J Ecol Environ 43:36 Chown SL, Hodgins KA, Griffin PC et al (2015) Biologi-
cal invasions, climate change and genomics. Evol Appl
8:23–46 Aravind NA, Shaanker MU, Bhat HNP et al (2022) Niche
shift in invasive species: is it a case of “home away from 1
Vol:. ( 3
(1234567890) Climate change will likely facilitate invasion of Asian orchid Eulophia graminea into new… 1981 European Commission (2003) Global land cover 2000 data-
base. Joint Research Centre Hänfling B, Kollmann J (2002) An evolutionary perspective of
biological invasions. Trends Ecol Evol 17:545–546 Hengl T, Mendes de Jesus J, Heuvelink GB et al (2017) Soil-
Grids250m: global gridded soil information based on
machine learning. PLoS ONE 12:e0169748 Cunze S, Kochmann J, Klimpel S (2020) Global occurrence
data improve potential distribution models for Aedes
japonicus japonicus in non-native regions. Pest Manag
Sci 76:1814–1822 Izuddin M, Yam TW, Webb EL (2019) Germination niches and
seed persistence of tropical epiphytic orchids in an urban
landscape. J Plant Res 132:383–394 Daehler CC (1998) The taxonomic distribution of invasive
angiosperm plants: ecological insights and comparison to
agricultural weeds. References Ecol Evol 3:182–196 Falcón W, Ackerman JD, Tremblay RL (2017) Quantifying
how acquired interactions with native and invasive
insects influence population growth rates of a non-
indigenous plant. Biol Invasions 19:895–911 Lugo AE (2009) The emerging era of novel tropical forests. Biotropica 41:589–591 Fand B, Shashank P, Suroshe S et al (2020) Invasion risk
of the South American tomato pinworm Tuta absoluta
(Meyrick) (Lepidoptera: Gelechiidae) in India: predic-
tions based on MaxEnt ecological niche modelling. Int
J Trop Insect Sci 40:561–571 Macrae C (2002) New weed found in Darwin. Department of
Business, Industry and Resource Development, Primary
Industry and Fisheries, Northern Territory Government,
Australia Mashhadi HR, Radosevich SR (2004) Invasive plants. In:
Inderjit (ed) Weed biology and management. Springer,
Dordrecht, pp 1–28 Fernández M, Kaur J, Sharma J (2023) Co-occurring epi-
phytic orchids have specialized mycorrhizal fungal
niches that are also linked to ontogeny. Mycorrhiza
33:87–105 Mason S, Graham N (2002) Areas beneath the relative oper-
ating characteristics (ROC) and relative operating levels
(ROL) curves: statistical significance and interpretation. Q J R Meteorol Soc 128:2145–2166 Fick S, Hijmans R (2017) WorldClim 2: new 1-km spatial reso-
lution climate surfaces for global land areas. Int J Clima-
tol 37:4302–4315 Foster EA, Ackerman JD (2021) Present and future distribution
of two non-indigenous orchids and their acquired enemy
in Puerto Rico. Biol Invasions 23:3545–3563 McCormick MK, Whigham DF, Canchani-Viruet A (2018)
Mycorrhizal fungi affect orchid distribution and popula-
tion dynamics. New Phytol 219:1207–1215 GBIF (2022) GBIF Occurrence Download - Eulophia
graminea McGee R, Williams S, Poulton R et al (2000) A longitudinal
study of cannabis use and mental health from adoles-
cence to early adulthood. Addiction 95:491–503 González-Díaz N, Ackerman JD (1988) Pollination, fruit set,
and seed production in the orchid, Oeceoclades macu-
lata. Lindleyana 3:150–155 Meena NK, Mani M (2022) Pests and their management in
orchids. In: Mani M (ed) Trends in horticultural entomol-
ogy. Springer, Singapore, pp 1239–1254 Guo W-Y, van Kleunen M, Pierce S et al (2019) Domestic gar-
dens play a dominant role in selecting alien species with
adaptive strategies that facilitate naturalization. Glob
Ecol Biogeogr 28:628–639 Meena N, Pal R, Barman D et al (2018) Indigenous approaches
of orchid pest management in North East India. Indian J
Tradit Knowl 17:209–214 1
ol.: (0 3
123456789) 1982 M. Kolanowska et al. References Meinshausen M, Nicholls Z, Lewis J et al (2020) The shared
socio-economic pathway (SSP) greenhouse gas concen-
trations and their extensions to 2500. Geosci Model Dev
13:3571–3605 Robinson TB, Martin N, Loureiro TG et al (2020) Double trou-
ble: the implications of climate change for biological
invasions. NeoBiota 62:463–487 Rodder D, Lotters S (2009) Niche shift versus niche conserva-
tism? Climatic characteristics of the native and invasive
ranges of the Mediterranean house gecko (Hemidactylus
turcicus). Glob Ecol Biogeogr 18:674–687 Melendez EJ, Ackerman JD (1993) The effects of a rust
infection on fitness components in a natural popula-
tion of Tolumnia variegata (Orchidaceae). Oecologia
94:361–367 turcicus). Glob Ecol Biogeogr 18:674–687 Rojas-Sandoval J, Ackerman JD (2021) Ornamentals lead the
way: global influences on plant invasions in the Carib-
bean. NeoBiota 64:177–197 Meyerson LA, Mooney HA (2007) Invasive alien species in an
era of globalization. Front Ecol Environ 5:199–208 Sarsaiya S, Shi J, Chen J (2019) A comprehensive review on
fungal endophytes and its dynamics on Orchidaceae
plants: current research, challenges, and future possibili-
ties. Bioengineered 10:316–334 Mitchell CE, Agrawal AA, Bever JD et al (2006) Biotic inter-
actions and plant invasions. Ecol Lett 9:726–740f Müller-Schärer H, Schaffner U, Steinger T (2004) Evolution
in invasive plants: implications for biological control. Trends Ecol Evol 19:417–422f Seebens H, Blackburn TM, Dyer EE et al (2017) No satura-
tion in the accumulation of alien species worldwide. Nat
Commun 8:14435 O’Conner M, Govender T, Jolliffe K (2006) Eulophia
graminea—the ET orchid. Orchids S Afr 37:128–131 Shabani F, Kumar L, Ahmadi M (2016) A comparison of abso-
lute performance of different correlative and mechanistic
species distribution models in an independent area. Ecol
Evol 6:5973–5986f O’Neill BC, Kriegler E, Ebi KL et al (2017) The roads ahead:
narratives for shared socioeconomic pathways describing
world futures in the 21st century. Glob Environ Change
42:169–180 Osborne BA, Gioria M (2022) Editorial: Biological invaders:
Always the bad guys? Frontiers in Ecology and Evolu-
tion 10 Simberloff D, Martin JL, Genovesi P et al (2013) Impacts of
biological invasions: what’s what and the way forward. Trends Ecol Evol 28:58–66 Singhurst JR, Mink JN, Holmes W (2020) Eulophia graminea
(Orchidaceae) naturalized in Texas. Phytoneuron 22:1–5 Owens H, Campbell L, Dornak L et al (2013) Constraints on
interpretation of ecological niche models by limited
environmental ranges on calibration areas. References Ecol Model
263:10–18 Smith AL, Hodkinson TR, Villellas J et al (2020) Global
gene flow releases invasive plants from environmen-
tal constraints on genetic diversity. Proc Natl Acad Sci
117:4218–4227 Paclibar G, Tadiosa E (2019) Ecological niche modeling of
invasive alien plant species in a protected landscape. Glob J Environ Sci Manag GJESM 5:371–382 Sonkoly J, Vojtko A, Tokolyi J et al (2016) Higher seed num-
ber compensates for lower fruit set in deceptive orchids. J
Ecol 104:343–351 Partomihardjo T (2003) Colonisation of orchids on the
Krakatau Islands. Telopea 10:299–310 Stiels D, Schidelko K, Engler J et al (2011) Predicting the
potential distribution of the invasive Common Waxbill
Estrilda astrild (Passeriformes: Estrildidae). J Ornithol
152:769–780 Pemberton RW (2013) Rapid geographic spread of the
alien orchid Eulophia graminea in Florida. Selbyana
31:47–51 Pemberton RW, Collins TM, Koptur S (2008) An asian orchid,
Eulophia graminea (Orchidaceae: Cymbidieae), natural-
izes In Florida. Lankesteriana 8:5–14 Stiels D, Gaisser B, Schidelko K et al (2015) Niche shift in four
non-native estrildid finches and implications for species
distribution models. Ibis 157:75–90 Phillips S, Dudik M (2008) Modeling of species distributions
with Maxent: new extensions and a comprehensive eval-
uation. Ecography 31:161–175 Suarez J, Eguiguren J, Herrera P et al (2016) Do mycorrhi-
zal fungi drive speciation in Teagueia (Orchidaceae) in
the upper Pastaza watershed of Ecuador? Symbiosis
69:161–168 Phillips S, Anderson R, Schapire R (2006) Maximum entropy
modeling of species geographic distributions. Ecol
Model 190:231–259 Swift S, Munroe S, Im C et al (2019) Remote tropical island
colonization does not preclude symbiotic specialists: new
evidence of mycorrhizal specificity across the geographic
distribution of the Hawaiian endemic orchid Anoectochi-
lus sandvicensis. Ann Bot 123:657–666 Phillips RD, Reiter N, Peakall R (2020) Orchid conservation:
from theory to practice. Ann Bot 126:345–362 POWO (2023) Plants of the world online. In: Royal Botanic
Gardens K (ed) Tingley R, Vallinoto M, Sequeira F et al (2014) Realized niche
shift during a global biological invasion. Proc Natl Acad
Sci 111:10233–10238 Randall RP (2017) A global compendium of weeds. R.P. Ran-
dall, Perth Recart W, Ackerman JD, Cuevas AA (2013) There goes the
neighborhood: apparent competition between invasive
and native orchids mediated by a specialist florivorous
weevil. Biol Invasions 15:283–293 Tremblay R, Ackerman J, Zimmerman J et al (2005) Variation
in sexual reproduction in orchids and its evolutionary
consequences: a spasmodic journey to diversification. References Biol J Lin Soc 84:1–54 Riahi K, van Vuuren D, Kriegler E et al (2017) The Shared
Socioeconomic Pathways and their energy, land use, and
greenhouse gas emissions implications: an overview. Glob Environ Change Hum Policy Dimens 42:153–168 Turbelin A, Catford JA (2021) Chapter 25—invasive plants and
climate change. In: Letcher TM (ed) climate change, 3rd
edn. Elsevier, New York, pp 515–539 1
Vol:. ( 3
(1234567890) Climate change will likely facilitate invasion of Asian orchid Eulophia graminea into new… 1983 Wraith J, Norman P, Pickering C (2020) Orchid conservation
and research: an analysis of gaps and priorities for glob-
ally Red Listed species. Ambio 49:1601–1611 van Kleunen M, Essl F, Pergl J et al (2018) The changing role
of ornamental horticulture in alien plant invasions. Biol
Rev Camb Philos Soc 93:1421–1437 Vázquez DP, Aizen MA (2004) Asymmetric specialization: a
pervasive feature of plant–pollinator interactions. Ecol-
ogy 85:1251–1257 Zhang S, Yang Y, Li J et al (2018) Physiological diversity of
orchids. Plant Divers 40:196–208i Zhu G, Gariepy T, Haye T et al (2017) Patterns of niche fill-
ing and expansion across the invaded ranges of Halyo-
morpha halys in North America and Europe. J Pest Sci
90:1045–1057 Vitousek PM, Walker LR (1989) Biological invasion by Myrica
Faya in Hawai’i: plant demography, nitrogen fixation,
ecosystem effects. Ecol Monogr 59:247–265 f
Vitt P, Taylor A, Rakosy D et al (2023) Global conservation
prioritization for the Orchidaceae. Sci Rep 13:6718 Publisher’s Note Springer Nature remains neutral with regard
to jurisdictional claims in published maps and institutional
affiliations. Wapstra M, Baker ML, Daniels GD (2020) Collecting history
and distribution of the potentially invasive Disa bracte-
ata (South African Orchid) in Tasmania. In: Papers and
proceedings of the royal society of Tasmani, vol 154, pp
51–60 Warren DL, Glor RE, Turelli M (2008) Environmental
niche equivalency versus conservatism: quantitative
approaches to niche evolution. Evolution 62:2868–2883 3
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Evaluation of Volumetric Change of Intracerebral Hemorrhage in Patients Treated with Thrombolysis for Intraventricular Hemorrhage
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Abstract Background: Intraventricular hemorrhage (IVH) is often caused by irruption of intracerebral hemorrhage (ICH) of
basal ganglia or thalamus into the ventricular system. Instillation of recombinant tissue plasminogen activator (rtPA)
via an external ventricular drainage (EVD) has been shown to effectively decrease IVH volumes while the impact of
rtPA instillation on ICH volumes remains unclear. In this series, we analyzed volumetric changes of ICH in patients with
and without intrathecal lysis therapy. Methods: Between 01/2013 and 01/2019, 36 patients with IVH caused by hemorrhage of basal ganglia, thalamus or
brain stem were treated with rtPA via an EVD (Group A). Initial volumes were determined in the first available com‑
puted tomography (CT) scan, final volumes in the last CT scan before discharge. During the same period, 41 patients
with ICH without relevant IVH were treated without intrathecal lysis therapy at our neurocritical care unit (Group B). Serial CT scans were evaluated separately for changes in ICH volumes for both cohorts using OsiriX DICOM viewer. The
Wilcoxon signed-rank test was performed for statistical analysis in not normally distributed variables. Results: Median initial volume of ICH for treatment Group A was 6.5 ml and was reduced to 5.0 ml after first instilla‑
tion of rtPA (p < 0.01). Twenty-six patients received a second treatment with rtPA (ICH volume reduction 4.5 to 3.3 ml,
p < 0.01) and of this cohort further 16 patients underwent a third treatment (ICH volume reduction 3.0 ml to 1.5 ml,
p < 0.01). Comparison of first and last CT scan in Group A confirmed an overall median percentage reduction of 91.7%
(n = 36, p < 0.01) of ICH volumes and hematoma resolution in Group A was significantly more effective compared to
non-rtPA group, Group B (percentage reduction = 68%) independent of initial hematoma volume in the regression
analysis (p = 0.07, mean 11.1, 95%CI 7.7–14.5). There were no adverse events in Group A related to rtPA instillation. Conclusion: Intrathecal lysis therapy leads to a significant reduction in the intraparenchymal hematoma volume
with faster clot resolution compared to the spontaneous hematoma resorption. Furthermore, intrathecal rtPA applica‑
tion had no adverse effect on ICH volume. Keywords: Intraventricular hemorrhage, Intracerebral hematoma, Intrathecal lysis, Volumetric change Keywords: Intraventricular hemorrhage, Intracerebral hematoma, Intrathecal lysis, Volumetric chang Introduction *Correspondence: Franziska.staub‑bartelt@med.uni‑duesseldorf.de
1 Department of Neurosurgery, Medical Faculty, Heinrich-Heine University
Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany
Full list of author information is available at the end of the article *Correspondence: Franziska.staub‑bartelt@med.uni‑duesseldorf.de
1 Department of Neurosurgery, Medical Faculty, Heinrich-Heine University
Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany
Full list of author information is available at the end of the article Spontaneous intracerebral hemorrhage (ICH) is a life-
threatening event leading to high mortality rates and
permanent disability in surviving patients [1]. Intraven-
tricular hemorrhage (IVH)—an extension of the ICH Evaluation of Volumetric Change
of Intracerebral Hemorrhage in Patients
Treated with Thrombolysis for Intraventricular
Hemorrhage Franziska Staub‑Bartelt1*, Jasper Hans van Lieshout1, Thomas Beez1, Rainer Kram2, Daniel Hänggi1
and Kerim Beseoglu1 © 2020 The Author(s), corrected publication 2020 © 2020 The Author(s), corrected publication 2020 Neurocrit Care (2021) 34:529–536
https://doi.org/10.1007/s12028-020-01054-7 Neurocrit Care (2021) 34:529–536
https://doi.org/10.1007/s12028-020-01054-7 *Correspondence: Franziska.staub‑bartelt@med.uni‑duesseldorf.de
1 Department of Neurosurgery, Medical Faculty, Heinrich-Heine University
Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany
Full list of author information is available at the end of the article Methods due to irruption of the bleeding into the ventricular
system—is reported in more than 50% of patients with
ICH and its volume is among the main predictors for
poor outcome [2–5]. Due to its high impact on mor-
tality and morbidity, diverse treatment strategies for
IVH have been evaluated in the past. However, a clini-
cal benefit from reduction in IVH due to irrigation of
fibrinolytic substances through an external ventricular
drainage (EVD) has been difficult to demonstrate in
humans. Studies provide evidence for a reduction in
all-cause mortality but shows no improvement in func-
tional outcome [6–9]. We performed a retrospective analysis of a single-center
cohort to investigate the effect of intrathecal recombinant
tissue plasminogen activator (rtPA) on the volumetric
changes of ICH associated with intrathecal lysis therapy. The study was approved by the local ethical committee
(Study number: 2018-295). Reporting of this study was
according to the strengthening the reporting of observa-
tional studies in epidemiology (STROBE) guidelines for
observational studies (Supplementary Material) [11]. 530 Patients Intraparenchymal hematoma expansion following
intrathecal lysis therapy of IVH could explain these dis-
appointing results, since neurological impairment due
to ICH is directly associated with the clot size [10]. The
aim of our study was to determine the effect of intrath-
ecal lysis therapy on the volume of ICH. We here report
our findings in 36 patients with ICH and concomitant
IVH treated with intrathecal fibrinolysis and compared
ICH volumes and occurrence of adverse events with a
group of patients with ICH either with or without IVH
treated with EVD alone. We identified all consecutive patients admitted to our
neurocritical care unit at the University Hospital Düssel-
dorf with an IVH due to ICH and treated with rtPA via
an EVD, between January 2013 and January 2019. Inclu-
sion criteria were: (1) age 18 years and older, (2) origin
of hemorrhage located either at the basal ganglia, thala-
mus or brain stem with relevant extension into any ven-
tricle, (3) availability of initial and follow-up computed
tomography (CT) scans and (4) at least one documented
treatment with intrathecal lysis (Fig. 1). A relevant IVH
was defined as a blood volume impending to cause a Fig. 1 Flowchart illustrating patient screening and selection. Patients were screened regarding general criteria (age, localization of ICH, EVD, no
other surgical intervention) and then divided into two groups. Group A patients receiving intrathecal lysis, Group B patients without intrathecal lysis. Afterwards, patients with missing data (e.g., no follow-UP CT scans) in both cohorts were excluded Fig. 1 Flowchart illustrating patient screening and selection. Patients were screened regarding general criteria (age, localization of ICH, EVD, no
other surgical intervention) and then divided into two groups. Group A patients receiving intrathecal lysis, Group B patients without intrathecal lysis. Afterwards, patients with missing data (e.g., no follow-UP CT scans) in both cohorts were excluded 531 hydrocephalus as evaluated by the treating physician
with obstruction of the Foramina Monroi and/or the
aqueduct. of 2 ml saline solution in order to guarantee intrathecal
application. The EVD was then closed for 30–45 min,
depending on individual patient tolerance. Upon reopen-
ing of the EVD, the drainage system was kept at a reduced
pressure threshold (10–12 mmHg) to achieve sufficient
clearance of blood. This procedure was repeated every
12 h until a sufficient reduction in intraventricular blood
in the third and/or fourth ventricle was achieved. Patients We
performed repeat CT imaging every 24 h to determine
the treatment effect as reported in previous publications
[12, 13]. For definition of a control group (Group B), we fur-
thermore screened the patient database for patients with
(1) age 18 years and older, (2) ICH located at either basal
ganglia, thalamus or brain stem, (3) receiving an EVD
without an indication for intrathecal lysis (e.g., no or
insignificant amounts of IVH) treated during the same
period of time. Patients who underwent surgery for evacuation of the
intracerebral hematoma, patients with lobar ICH and
ICH related to aneurysm rupture, arteriovenous malfor-
mation rupture or neoplasm as well as traumatic hemor-
rhages were excluded. Outcome Primary outcome measure was the median volume (ml)
of ICH on repeat CT imaging. Hematoma volume for
every patient at every defined point of time was meas-
ured by one of the authors (FSB) using Region of Interest
(ROI) volumetry in OsirixLite (Pixmeo SARL, Switzer-
land). The hematoma outline, defined as hyperdensity
compared to brain parenchyma, was outlined manually
on axial CT slices with 2–5 mm thickness. Hematoma
volume was then calculated automatically using ROI vol-
umetry function of the software (Fig. 2).hi Indication for EVD insertion in patients with ICH with
or without IVH (Group A and Group B) were uncon-
sciousness with indication for intubation, intubation due
to other reasons (e.g., pulmonary insufficiency) and was
established to monitor ICPs during the sedation/acute
unconscious phase where patients could not undergo
adequate neurological assessments. Additionally, for
patients with ICH and IVH (Group A) indication for
intrathecal lysis required EVD insertion. Indication for
intrathecal lysis were: hydrocephalus/neurological dete-
rioration due to tri-or tetra ventricular blood collection,
particularly with localization in the III and IV ventricle. All patients included in Group A were treated with
rtPA (Actilyse®, Boehringer-Ingelheim, Germany) within
12 h after initial CT scan. For each application, 2.5 mg
rtPA was dissolved in 2 ml of saline solution. Actilyse was
instilled directly into the ventricle followed by instillation Indication for EVD insertion in patients with ICH with
or without IVH (Group A and Group B) were uncon-
sciousness with indication for intubation, intubation due
to other reasons (e.g., pulmonary insufficiency) and was
established to monitor ICPs during the sedation/acute
unconscious phase where patients could not undergo
adequate neurological assessments. Additionally, for
patients with ICH and IVH (Group A) indication for
intrathecal lysis required EVD insertion. Indication for
intrathecal lysis were: hydrocephalus/neurological dete-
rioration due to tri-or tetra ventricular blood collection,
particularly with localization in the III and IV ventricle. The first available CT scan showing ICH/IVH was
defined as “initial” scan irrespective whether further CT
scans without any intervention existed or not. Outcome First fol-
low-up CT after first instillation of rtPA was defined as
“first,” second available CT scan after repeated instilla-
tion was defined as “second,“ third CT scan after further
instillation of rtPA was defined as “third.“ Accordingly
last available CT scan before discharge of the patients
was defined as “final.“ CT scans were generally done All patients included in Group A were treated with
rtPA (Actilyse®, Boehringer-Ingelheim, Germany) within
12 h after initial CT scan. For each application, 2.5 mg
rtPA was dissolved in 2 ml of saline solution. Actilyse was
instilled directly into the ventricle followed by instillation Fig. 2 Example for the region of interest (ROI) volumetry using OsirixLite (Pixmeo SARL, Switzerland). Axial CT scans were checked for slides show‑
ing hyperdensity relating to ICH, and hematoma outlines were marked on corresponding slides (left). Hematoma volume was then calculated
automatically using ROI volumetry function of the software (right) Fig. 2 Example for the region of interest (ROI) volumetry using OsirixLite (Pixmeo SARL, Switzerland). Axial CT scans were checked for slides show‑
ing hyperdensity relating to ICH, and hematoma outlines were marked on corresponding slides (left). Hematoma volume was then calculated
automatically using ROI volumetry function of the software (right) 532 Patients from Group A showed a significantly higher
clot resolution rate as compared to Group B (p < 0.02,
Fig. 4). In a regression analysis with hematoma volume
reduction in percent (mean 72.1%; 95%CI 65.3–78.8%)
as the dependent variable and initial hematoma volume
and intraventricular rtPA application as independent
variables only rtPA application (proportion 0.46; 95%CI
0.35–0.57) contributed significantly (p < 0.01) to the
model. Initial hematoma volume (mean 11.1 ml, 95%CI
7.7–14.5 ml) was excluded (p = 0.07).i within 24 h after rtPA application. The number of CT
scans were not representative for the number of intrathe-
cal lysis administration. For the 41 patients from Group B
only analyses of volume for the time periods “initial” and
“final” were conducted. Statistical Analysis Categorical data are presented as counts and percentages
and continuous variables as means with standard error
of the mean (SEM) or medians with interquartile ranges
(IQRs), depending on the normality of the data. Median time between initial CT and first administra-
tion of rtPA was 10.0 h (IQR 6.8–14.0 h). For the primary outcome measure, we calculated
median and interquartile ranges (IQR) instead of mean
values and standard deviation in order to avoid distor-
tion by outliers and extremities due to the small sample
size. The Shapiro–Wilk test was used to test for normal
distribution. Hereafter, non-parametric testing was used
for related samples to determine changes in hematoma
volumes over the treatment period (Wilcoxon signed-
rank test). Group differences between Group A and B
were evaluated using Mann–Whitney U test. Addition-
ally, we performed a regression analysis with hematoma
volume reduction (in %) as dependent variable and rtPA
application and initial hematoma volume as contributing
independent variables. The Type I error was set at 0.05
and the tests were 2-tailed. Statistical analyses were per-
formed using IBM SPSS Statistics Version 26 (IBM Cor-
poration, USA). 34 patients received repeated CT scans every 24 h after
instillation of either one or two doses of rtPA per day. In
2 patients (subject 1 and 2), the interval between initial
CT scan, start of lysis and first control CT was doubled
to 48 h. Both patients’ treatment were finalized after the
first CT scan. The median time period between the ini-
tial and final CT scan for Group A was 10.0 days (IQR
7.0–14.0). The median period between initial and final
CT scan for Group B was 11 days (IQR 9.0–17.0). No patient experienced an increase in hematoma
volume after application of rtPA. However, 6 patients
showed a hematoma progression between initial CT
scan and start of intrathecal lysis (Table 2). All of these
patients were treated with rtPA after hematoma progres-
sion and showed a significant hematoma reduction by
the time of discharge (p = 0.03). Furthermore, 3 patients
of Group B showed higher hematoma volume at final CT
scan compared to initial CT scan (subject 10, 21, 34, for
detailed volumes please refer to Table 2 of supplement
material). As these patients received no intervention,
hematoma growth was due to natural progression and
not related to any intervention. Results We included 36 patients with IVH due to ICH treated
with rtPA via an EVD (Group A, Supplement Material
Table 1) and 41 patients with ICH with or without IVH
without intrathecal rtPA application (Group B, Supple-
ment Material Table 2). All epidemiological data includ-
ing main risk factors for ICH are summarized in Table 1. Median initial ICH volume in cohort A (n = 36) was
6.5 ml (IQR 5.3–12.6 ml) and was reduced to 5.0 ml (IQR
3.0–9.9 ml) after first treatment with intrathecal rtPA
(p < 0.01). Overall, we did not see any infection related to intrath-
ecal lysis therapy. Permanent shunting was performed in
55% of patients in Group A and 34% of patients in Group
B during hospitalization. Median time from initial CT
to permanent shunting for Group A was 9.5 days (IQR
7–12) and for Group B 11 days (IQR 9–12) (Table 1). 26 out of 36 patients received a second instillation of
rtPA. Follow-up CT scan showed a decrease in median
hematoma volume for this subgroup [4.5 ml (IQR 3.1–
9.5 ml) to 3.3 ml (IQR 1.6–8.4 ml); p < 0.01]. A further 16
out of 26 patients received a third intrathecal application
of rtPA, which further decreased the mean hematoma
volume from 3.0 ml (IQR 1.4–8.5 ml) to 1.5 ml (IQR 0.7–
6.5 ml); (p < 0.01, Fig. 3).i Discussion Our analysis reveals three relevant aspects. First, intra-
ventricular application of rtPA in patients with ICH and
concomitant IVH significantly decreases parenchymal
hematoma volume without direct application into the
hematoma. Second, rtPA application accelerates intra-
parenchymatous hematoma reduction compared to
rtPA-untreated patients. Third, the intrathecal applica-
tion of rtPA does not increase the risk of parenchymal
hematoma expansion or hemorrhagic complications.hfi Comparison of initial and final hematoma volume in
Group A showed an absolute median decrease from
6.5 ml (IQR 5.3–12.6 ml) to 0.5 ml (IQR 0.0–3.9 ml). In
Group B, median hematoma volume decreased from ini-
tially 8.9 ml (IQR4.3–13.1 ml) to 3.1 ml (IQR 0.6–7.1 ml). The efficacy of intraventricular clot resolution by appli-
cation of rtPA via an EVD has been demonstrated before 533 Table 1 Details and comparison of patient Group A and B
Bold values represent significant values (p < 0.05)
Details of all patients from Group A (intrathecal lysis) and Group B (no intrathecal lysis). Location thalamus and brainstem have been grouped due to the small group
size. Discussion IQR interquartile range, CT scan computed tomography scan
Intrathecal lysis
Group A
No intrathecal lysis
Group B
p
N
36
41
Female
Absolute (%)
23 (64%)
14 (34%)
< 0.01
Age
Median (IQR)
67 (59–74)
63 (52–70)
0.32
Location absolute (%)
Basal Ganglia
26 (72%)
30 (73%)
0.93
Thalamus or Brainstem
10 (28%)
11 (27%)
Clot volume on CT scan (ml)
Median (IQR)
Initial
6.5 (5.3–12.6)
8.9 (4.3–13.1)
0.74
After first lysis
5.0 (3.0–9.9)
N/A
After second lysis
3.3 (1.6–8.4)
N/A
After third lysis
1.5 (0.7–6.5)
N/A
Final
0.5 (0–3.9)
3.1 (0.6–7.1)
0.02
Reduction in clot volume in percent initial to final CT scan
Mean (95% confidence interval)
81.8 (73.5–90.2)
63.4 (53.4–73.4)
< 0.01
Time initial CT scan—1st lysis (h)
Median (IQR)
10.0 (6.8–14.0)
N/A
Days (initial–final CT scan)
Median (IQR)
10 (7–14)
11 (9–17)
0.20
Risk factors for IVH
Absolute (%)
Arterial hypertension
33 (92%)
32 (78%)
0.10
Diabetes
4 (11%)
6 (15%)
0.65
Coronary artery disease
7 (19%)
3 (7%)
0.12
Alcohol
5 (14%)
3 (7%)
0.35
Nicotine
11 (31%)
5 (12%)
0.05
Antiplatelet
7 (19%)
4 (10%)
0.23
Anticoagulation
6 (17%)
4 (10%)
0.37
EVD-related infections (%)
0
0
–
CSF shunt rate (%)
20 (55%)
14 (34%)
0.24
Timing of permanent shunting in days
Median (IQR)
9.5 (7–12)
11 (9–12)
0.26 Table 1 Details and comparison of patient Group A and B Details and comparison of patient Group A and B [13]; however, the focus was placed on IVH and to our
knowledge data on the effect on the intraparenchymatous
hematoma has not been published before.i hydrocephalus and allow for ICP monitoring. This
simultaneously provides clinicians with a route for
application of rtPA without the need for an additional
intervention. Our analysis demonstrates that the intra-
ventricular rtPa application leads to significant reduc-
tion in ICH volume. It appears likely that rtPA diffuses
into the parenchyma via the breach in the ventricle wall
and the local effects are comparable to direct appli-
cation of fibrinolytic agents into the intraparenchy-
mal hematoma without the necessity of an additional Direct application of fibrinolytic agents into an
intracerebral hematoma via image-guided catheter
placement significantly reduces clot volume but inves-
tigated patients had no IVH and required surgery to
place the catheter [10]. In patients with deep-seated
hematomas extending into the ventricles, insertion of
an EVD is necessary in most cases to treat concomitant 534 Fig. Discussion 3 Visualization of hematoma volumes in Group A. Hematoma volume (ml) in the initial CT scan and after first, second and third application of
recombinant tissue plasminogen activator (rtPA) as well as in the final CT scan before discharge. Circles signify patients receiving a repeated rtPA
dose after CT scan, dots signify patients who did not receive further rtPA application. Patient #14 from Group A was omitted in this figure for illustra‑
tive reasons due to a very high hematoma volume; however, the value was included when calculating the medians Fig. 3 Visualization of hematoma volumes in Group A. Hematoma volume (ml) in the initial CT scan and after first, second and third application of
recombinant tissue plasminogen activator (rtPA) as well as in the final CT scan before discharge. Circles signify patients receiving a repeated rtPA
dose after CT scan, dots signify patients who did not receive further rtPA application. Patient #14 from Group A was omitted in this figure for illustra‑
tive reasons due to a very high hematoma volume; however, the value was included when calculating the medians catheter [10]. However, the underlying biophysiological
processes remain obscure in detail. suffered from symptomatic hemorrhages, only three of
them (1.2%) during the dosing phase [14]. Hematoma
expansion in 6 patients from the treated cohort occurred
before intrathecal therapy and was thus unrelated to rtPA
application and in line with previously reported hema-
toma progression during the first 24 h after hemorrhage
[15]. Compared to the spontaneous hematoma resorp-
tion rate, treated patients showed a significantly greater
reduction in ICH hematoma volume over time. Comparable to other reports, we did not experience
rebleeding complications attributable to rtPA applica-
tion. In the MISTIE III trial, no significant difference
in the prevalence of adverse events like symptomatic
bleeding between the standard medical care and inter-
ventional group was observed, even if rtPA was instilled
directly into the residual hematoma after surgical clot
removal [10]. Similarly, the CLEAR III trial showed a very
low risk for new bleeding events under rtPA instillation
therapy. It was reported that 2.4% (n = 6) of the patients Study limitations include the retrospective design and
particularly the small number of patients in both cohorts. Regarding the retrospective character of the study, we
would like to underline the following limitation. Fig. 4 Comparison of initial and final hematoma volume in Group
A and B. Initial and final hematoma volume for patients receiving
intrathecal lysis therapy (Group A, circles) compared to patients
without intrathecal lysis therapy (Group B, dots). Comparison of
hematoma volume in the final CT scan demonstrated a significant
difference between both groups (p < 0.02). Patient #14 from Group A
was omitted in this figure for illustrative reasons due to a very high
hematoma volume; however, the value was included when calculat‑
ing the medians for any reliable assessment. Still, the procedure described
was safe with regard to hematoma progression and seems
to have no negative consequences on neurological out-
come, but we cannot provide evidence for the treatment
protocol with regard to improved functional outcome. for any reliable assessment. Still, the procedure described
was safe with regard to hematoma progression and seems
to have no negative consequences on neurological out-
come, but we cannot provide evidence for the treatment
protocol with regard to improved functional outcome. In addition to the small sample size, the median ICH
size was considerably smaller compared to larger cohort
studies as the MISTIE III trial. In our analysis, we
focused on the effect of intrathecal lysis on ICH volumes;
therefore, patients with larger ICH clot sizes with indica-
tion for any surgical intervention (e.g., minimal invasive
treatments, evacuation of ICH) addressing the ICH were
excluded as hematoma volumes would not have been
comparable regarding the specific aim of the present
study. According to its aim and study design, this study con-
firmed feasibility, safety and efficacy in clot resolution
and our findings provide a basis for further prospective
data collections. A controlled prospective study would be
required to further elucidate, if the effect demonstrated is
transferable to larger patient cohorts. om cohort A with a spontaneous hematoma expansion before initiation of intrathecal rtPA (n = 6) ble 2 Patients from cohort A with a spontaneous hematoma expansion before initiation of intrathec Table 2 Patients from cohort A with a spontaneous hematoma expansion before initiation of intrathecal rtPA (n = 6)
A subgroup of patients in Group A showing details of hematoma progression between initial CT scan and last CT before intrathecal lysis. As not all patients received
repeated CT scans before treatment, CTs on admission were used for calculation of initial hematoma volume. Discussion The rel-
evance of faster ICH clot resolution for neurological out-
come was not evaluated in the present data analysis. This
was due to missing neurological outcome parameters in
a large number of patients which excluded possibilities 535 Fig. 4 Comparison of initial and final hematoma volume in Group
A and B. Initial and final hematoma volume for patients receiving
intrathecal lysis therapy (Group A, circles) compared to patients
without intrathecal lysis therapy (Group B, dots). Comparison of
hematoma volume in the final CT scan demonstrated a significant
difference between both groups (p < 0.02). Patient #14 from Group A
was omitted in this figure for illustrative reasons due to a very high
hematoma volume; however, the value was included when calculat‑
ing the medians At last, limitations of CT evaluation need to be dis-
cussed. First, hematoma evaluation was not blinded to
the groups. Secondly, the software that was used (Osi-
rix Lite) is a free software version of Osirix MD, a widely
used medical image viewer enabling various tools for
processing of MRI/CT scans and other medical imag-
ing. The full version is FDA cleared and CE IIa labelled
and might be used for radiological diagnosis. We decided
to use Osirix Lite as this is a freely available high-qual-
ity software version even though some tools are missing
compared to Osirix MD. For CT evaluation, ROI volume-
try was needed and still available through Osirix Lite. In
order to verify the results, we randomly evaluated hema-
toma volumes by ABC/2 score and could not find any
major discrepancy. A last potential confounder might be
the decrease in hematoma density over time due to deg-
radation of hemoglobin. Approximately 3–20 days after
acute onset of ICH, the appearance of intraparenchymal
hematoma in CT scans becomes less intense [16, 17]. The
median time between initial CT and initiation of therapy
in the intervention Group A was 10 h followed by repeti-
tive CT scans every 24 h (except in 2 subjects). Thus, the
treatment group received the majority of CT scans in the
hyperacute and acute ICH stages, where the clot appears
to be clearly hyperdense allowing a clear identification
of parenchyma clot borders in the ROI volumetry. For
comparison of initial and last CT in both cohorts, the
decrease in hematoma density might have had an influ-
ence on evaluation. Group values are stated as median values with
interquartile ranges
Patient
Location
CT scan admission
CT scan pre-lysis
1st CT scan post-lysis
CT scan discharge
Volume (ml)
Volume (ml)
Volume (ml)
Volume (ml)
8
Basal ganglia
22.9
33.1
24.4
3.4
11
Basal ganglia
5.9
23.3
15.3
9.1
13
Basal ganglia
30.7
34.0
32.1
18.5
18
Basal ganglia
0.1
3.8
2.7
0.0
28
Basal ganglia
25.1
33.0
32.8
32.6
29
Basal ganglia
5.3
5.9
5.7
3.7
14.4
[5.3–25.1]
28.1
[10.3–33.1]
19.8
[5.7–32.1]
6.4
[3.4–18.5] A subgroup of patients in Group A showing details of hematoma progression between initial CT scan and last CT before intrathecal lysis. As not all patients received
repeated CT scans before treatment, CTs on admission were used for calculation of initial hematoma volume. Group values are stated as median values with
interquartile ranges 536 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/. Conclusion We could demonstrate that intraventricular rtPA applica-
tion significantly reduces ICH volumes in patients with
IVH secondary to ICH in a small patient cohort. Fur-
thermore, intrathecal thrombolysis leads to accelerated
reduction in ICH volume compared to patients treated
with EVD alone. The risk of procedure-related complica-
tions was not increased, especially with regard to expan-
sion of intraparenchymatous hematoma. Nevertheless, a
prospective randomized trial will be mandatory to define
new treatment strategies. Conflict of interest 10. Hanley DF, et al. Efficacy and safety of minimally invasive surgery with
thrombolysis in intracerebral haemorrhage evacuation (MISTIE III): a ran‑
domised, controlled, open-label, blinded endpoint phase 3 trial. Lancet. 2019;393(10175):1021–32. The authors declare that they have no competing interests and all authors
have nothing to disclose. The authors declare that they have no competing interests and all authors
have nothing to disclose. Ethics Approval and Consent to Participate 11. von Elm E, et al. The strengthening the reporting of observational studies
in epidemiology (STROBE) statement: guidelines for reporting observa‑
tional studies. Lancet. 2007;370(9596):1453–7. The present retrospective study only reports data that were collected at our
local neurosurgical intensive care ward. According to local legislation research,
associates are allowed to use data that is directly accessible for research
without previous informed consent of the patient when used in scientific
context according. Due to anonymization, disclosure of identity of individual
persons is impossible. The study was approved by the local ethical committee,
Heinrich-Heine University, Faculty of Medicine, Düsseldorf, Germany (Study
Number: 2018-295-RetroDeuA). 12. Webb AJ, et al. Resolution of intraventricular hemorrhage varies by
ventricular region and dose of intraventricular thrombolytic: the clot lysis:
evaluating accelerated resolution of IVH (CLEAR IVH) program. Stroke. 2012;43(6):1666–8. 12. Webb AJ, et al. Resolution of intraventricular hemorrhage varies by
ventricular region and dose of intraventricular thrombolytic: the clot lysis:
evaluating accelerated resolution of IVH (CLEAR IVH) program. Stroke. 2012;43(6):1666–8. 13. Naff N, et al. Low-dose recombinant tissue-type plasminogen activa‑
tor enhances clot resolution in brain hemorrhage: the intraventricular
hemorrhage thrombolysis trial. Stroke. 2011;42(11):3009–16. 13. Naff N, et al. Low-dose recombinant tissue-type plasminogen activa‑
tor enhances clot resolution in brain hemorrhage: the intraventricular
hemorrhage thrombolysis trial. Stroke. 2011;42(11):3009–16. Source of Support
No funding was received. Source of Support
No funding was received. References 1. Delcourt C, et al. Intracerebral hemorrhage location and outcome among
INTERACT2 participants. Neurology. 2017;88(15):1408–14. 1. Delcourt C, et al. Intracerebral hemorrhage location and outcome among
INTERACT2 participants. Neurology. 2017;88(15):1408–14. Abbreviations IVH: Intraventricular hemorrhage; ICH: Intracerebral hemorrhage; rtPA:
Recombinant tissue plasminogen activator; EVD: External ventricular drainage;
STROBE: Strengthening the Reporting of Observational Studies in Epidemiol‑
ogy; CT: Computed tomography. 4. Young WB, et al. Prognostic significance of ventricular blood in supraten‑
torial hemorrhage: a volumetric study. Neurology. 1990;40(4):616–9. 5. Nieuwkamp DJ, et al. Treatment and outcome of severe intraventricular
extension in patients with subarachnoid or intracerebral hemorrhage: a
systematic review of the literature. J Neurol. 2000;247(2):117–21. Availability of Data and Materials 14. Dey M, et al. Bleeding and infection with external ventricular drainage: a
systematic review in comparison with adjudicated adverse events in the
ongoing clot lysis evaluating accelerated resolution of intraventricular
hemorrhage phase III (CLEAR-III IHV) trial. Neurosurgery. 2015;76(3):291–
300 di
i
301 14. Dey M, et al. Bleeding and infection with external ventricular drainage: a
systematic review in comparison with adjudicated adverse events in the
ongoing clot lysis evaluating accelerated resolution of intraventricular
hemorrhage phase III (CLEAR-III IHV) trial. Neurosurgery. 2015;76(3):291–
300 di
i
301 All data generated or analyzed during this study are included in this published
article and its supplementary information files. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12028-020-01054-7)
contains supplementary material, which is available to authorized users. 2. Trifan G, Arshi B, Testai FD. Intraventricular hemorrhage severity as
a predictor of outcome in intracerebral hemorrhage. Front Neurol. 2019;10:217. 3. Tuhrim S, et al. Volume of ventricular blood is an important determinant
of outcome in supratentorial intracerebral hemorrhage. Crit Care Med. 1999;27(3):617–21. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations. Received: 18 January 2020 Accepted: 9 July 2020
Published online: 31 July 2020 Author details
1
f 6. Dey M, et al. External ventricular drainage for intraventricular hemor‑
rhage. Curr Neurol Neurosci Rep. 2012;12(1):24–33. 1 Department of Neurosurgery, Medical Faculty, Heinrich-Heine University
Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany. 2 Department
of Anesthesiology, Medical Faculty, Heinrich-Heine University Düsseldorf,
Düsseldorf, Germany. 7. Wang D, et al. Local fibrinolytic therapy for intraventricular hemor‑
rhage: a meta-analysis of randomized controlled trials. World Neurosurg. 2017;107:1016–1024 e1. 8. Gaberel T, et al. Intraventricular fibrinolysis versus external ventricular
drainage alone in intraventricular hemorrhage: a meta-analysis. Stroke. 2011;42(10):2776–81. Source of Support Source of Support
No funding was received. Acknowledgements Acknowledgements
Open Access funding provided by Projekt DEAL. Open Access funding provided by Projekt DEAL. 9. Hanley DF, et al. Thrombolytic removal of intraventricular haemor‑
rhage in treatment of severe stroke: results of the randomised,
multicentre, multiregion, placebo-controlled CLEAR III trial. Lancet. 2017;389(10069):603–11. g p
300 ; discussion 301. 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 15. Brott T, et al. Early hemorrhage growth in patients with intracerebral
hemorrhage. Stroke. 1997;28(1):1–5. 15. Brott T, et al. Early hemorrhage growth in patients with intracerebral
hemorrhage. Stroke. 1997;28(1):1–5. 16. Macellari F, et al. Neuroimaging in intracerebral hemorrhage. Stroke. 2014;45(3):903–8. 17. Osborn AG. Osborn’s brain imaging, pathology, and anatomy. Salt Lake
City: Amirsys Publication; 2018.
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7,200 Chapter 7 Additional information is available at the end of the chapter http://dx.doi.org/10.5772/60698 © 2015 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. Autophagy, a Highly Regulated Intracellular System
Essential to Skeletal Muscle Homeostasis — Role in
Disease, Exercise and Altitude Exposure Anthony M.J. Sanchez, Robin Candau,
Audrey Raibon and Henri Bernardi Anthony M.J. Sanchez, Robin Candau,
Audrey Raibon and Henri Bernardi Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/60698 Additional information is available at the end of the chapter 1. Introduction Skeletal muscle exhibits remarkable adaptive capabilities in response to various stimuli such
as loading conditions (resistance training, microgravity), contractile activity (electrical
stimulations, endurance exercise), environmental factors (altitude exposure), or nutritional
interventions. To access this great capacity, a plethora of quantitative and functional
adaptations are involved. Changes in the size of adult muscle, in response to these external
stimuli, are mainly due to the growth of individual muscle fibers rather than an increase
in fiber number [1]. The control of muscle mass is dependent upon a balance between anabolic and catabolic
processes. Hypertrophy is associated with increased protein synthesis, while atrophy is
characterized by increased degradation of muscle proteins and/or a decrease in protein
translation. The initiation of protein synthesis is mainly mediated by a signaling pathway in
which the mammalian/mechanistic target of rapamycin complex 1 (MTORC1), a multiprotein
complex composed of MTOR (mammalian/mechanistic target of rapamycin), RPTOR (regu‐
latory-associated protein of MTOR), mLST8/GβL (MTOR-associated protein LST8 homolog),
DEPTOR (DEP domain containing MTOR-interacting protein), and PRAS40 (proline-rich Akt
substrate of 40 kDa) [2,3]. MTORC1 by phosphorylating its substrates S6K1 (ribosomal protein
p70S6 kinase 1) and 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1)
controls skeletal muscle protein translation and hypertrophy [4-7]. The Insulin signaling
pathway leads to the activation of MTORC1 through the activation of the kinases PI3K
(phosphatidylinositol 3-kinase), PDK1 (phosphoinositide-dependent kinase-1), and Akt. Akt,
also known as protein kinase B (PKB), inactivates tuberous sclerosis complex 1/2 (TSC1/2),
promoting MTOR activation by Rheb-GTP [8-10]. Akt also phosphorylates and inactivates the
glycogen synthase kinase 3β (GSK3β), resulting in the activation of the eukaryotic translation
initiation factor 2B (eIF2B) and increased protein synthesis [5,11]. Muscle atrophy leads to a state of weakness and emaciation of the body, which is encountered,
for example, in the terminal phase of certain diseases or chronic infections such as cancer, AIDS,
diabetes, bacterial infections, and nerve degeneration [12]. Muscle atrophy is also observed
during aging, immobilization, and stress or trauma to the muscle and is associated with
increased proteolysis. Protein degradation is essentially modulated by two conserved path‐
ways: the ATP-dependent ubiquitin-proteasome system and the autophagy pathways. The first one has been particularly involved in the degradation process after the discovery of
two E3 ubiquitin ligases (E3 ligases), MAFbx/atrogin-1 (muscle atrophy F-box) and MuRF1
(muscle RING finger-1), which are both overexpressed in various models of atrophy (fasting,
cancer, diabetes, immobilization, and other stresses) [13,14]. Abstract Autophagy is an evolutionarily conserved intracellular system that selectively
eliminates protein aggregates, damaged organelles, and other cellular debris. It is a
self-cleaning process critical for cell homeostasis in conditions of energy stress. Autophagy has been until now relatively overlooked in skeletal muscle, but recent
data highlight its vital role in this tissue in response to several stress conditions. The
most recognized sensors for autophagy modulation are the adenosine monophos‐
phate (AMP)-activated protein kinase (AMPK) and the mechanistic target of
rapamycin (MTOR). AMPK acts as a sensor of cellular energy status by regulating
several intracellular systems including glucose and lipid metabolisms and mitochon‐
drial biogenesis. Recently, AMPK has been involved in the control of protein synthesis
by decreasing MTOR activity and in the control of protein breakdown programs. Concerning proteolysis, AMPK notably regulates autophagy through FoxO transcrip‐
tion factors and Ulk1 complex. In this chapter, we describe the functioning of the
different autophagy pathways (macroautophagy, microautophagy, and chaperone-
mediated autophagy) in skeletal muscle and define the role of macroautophagy in
response to physical exercise, a stress that is well assumed to be a key strategy to
counteract metabolic and muscle diseases. The effects of dietary factors and altitude
exposure are also discussed in the context of exercise. Keywords: Cachexia, Endurance exercise, Hypoxia, Proteolysis, Sarcopenia 172 Muscle Cell and Tissue 1. Introduction The invalidation of these proteins
confers a resistance to certain types of induced atrophy, suggesting a critical role in the
catabolism for the ubiquitin-proteasome pathway [13]. The function of E3 ligases is to ubiq‐
uitinate specific proteins to target them for recognition by the 26S proteasome where they are
eliminated. Other E3 ligases like zinc-finger protein 216 (ZNF216), the mitochondrial E3
ubiquitin protein ligase 1 (Mul1), and the tripartite motif-containing protein 32 (Trim32) have
been shown to play an important role in skeletal muscle atrophy [15-17]. Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease…
http://dx.doi.org/10.5772/60698 173 The autophagy signaling, which constitutes the second pathway, is important for maintain‐
ing cell metabolism and organelle turnover. It involves the degradation of substrates by
hydrolases into a vesicle called lysosome [18]. Recent evidence demonstrates cross talk and
cooperation between the ubiquitin-proteasome system and autophagy [19,20]. The impor‐
tance of this pathway in skeletal muscle was long much neglected, and autophagy was thought
to be a nonselective degradation system. However, it is now well recognized that autophagy
machinery is critical for muscle homeostasis and organelle turnover in response to cellular stress
like physical exercise or hypoxia [21]. Importantly, the energy sensor AMPK (5'-adenosine
monophosphate-activated protein kinase) has been involved in the regulation of both protein
translation pathway and protein degradation systems, with a particular interest in the regula‐
tion of autophagy program for the last few years. The present chapter focuses on the role of
autophagy in skeletal muscle homeostasis. We will describe the functioning of the autophagy
signaling pathways (i.e., macroautophagy, microautophagy, and chaperone-mediated
autophagy) and detail the regulation of macroautophagy by both AMPK and MTOR, with the
final goal to discuss the potential applications of recent discoveries concerning autophagy-
related pathologies. The involvement of autophagy in response to physical exercise (acute and
chronic exercise) and altitude exposure is thereafter discussed. 2.1. The autophagy pathways 2.1.1. Description of the system 2.1.1. Description of the system Autophagy has been discovered during nutrient privation and can be referred to as “self-
eating” as cells degrade their own constituents to maintain cellular homeostasis in response to
various injuries like starvation and hypoxia and in pathological conditions, including cancer,
muscular dystrophy, and neurodegenerative diseases. One purpose of the starvation-in‐
duced autophagy is to degrade materials to provide amino acids and free fatty acids in order
to preserve metabolism and ATP levels when extracellular nutrients reach hazardous low levels
[22]. Moreover, autophagy also eliminates protein aggregates as well as unwanted and
dysfunctional organelles. The term autophagy embraces macroautophagy, microautophagy,
and chaperone-mediated autophagy that we will describe hereafter. Macroautophagy and microautophagy are conserved from yeast to humans, and these processes
were originally described as bulk degradation mechanisms. However, these two processes can
be selective for targeting different organelles, and we distinguish mitophagy selective for
degradation of mitochondria; pexophagy, selective for degradation of peroxisomes; xenopha‐
gy, selective for degradation of intracellular bacteria and virus; reticulophagy, selective for
endoplasmic reticulum; heterophagy, selective for substances taken in by phagocytosis;
golgiphagy, selective for Golgi apparatus; ribophagy, selective for ribosomes; crinophagy,
specific for the contents (proteins, peptides) of secretory granules; glycophagy, selective for
glycogen; and lipophagy, selective for lipid droplets [23-29]. Among these different varieties of Muscle Cell and Tissue 174 autophagy, mitophagy has been the most studied in the last decade, and this process involves
notably two Parkinson’s disease factors, the RING-between-RING E3 ligase Parkin and the
mitochondrial kinase PINK1 (PTEN-induced putative kinase protein 1), PTEN being “phospha‐
tase and tensin homolog”
[30,31]. After mitochondrial potential depolarization, PINK1
promotes Parkin activation through phosphorylation of its ubiquitin-like domain [32]. In
addition to these factors, the mitochondrial E3 ligase Mul1 can also be involved in mitophagy
by the degradation of the mitochondrial fusion protein Mfn2 (mitofusin-2) and the stabiliza‐
tion of the dynamin-related protein 1 (DRP1), resulting in mitochondrial fragmentation [33]. Autophagy by providing a turnover of the cellular components plays a central role in the
homeostasis of the cell. It is a key mechanism by which a starving cell reallocates nutrients from
unnecessary to more-essential processes. Macroautophagy, microautophagy, and chaperone-
mediated autophagy lead cytoplasmic substrates inside lysosomes in which their contents are
digested by a battery of acidic hydrolases [34]. Four essential ubiquitous proteases have been
identified: the cathepsins B, D, H, and L [35,36]. 2.1.2. Microautophagy Microautophagy is localized directly at the level of the lysosome which directly engulfs cytosol
components by invagination, protrusion, and/or elimination of the lysosomal limiting mem‐
brane. It is implicated in the degradation of long half-life proteins in numerous cell types and
does not respond to classical stimuli inducing chaperone-mediated autophagy and macroau‐
tophagy [51]. In contrast to macroautophagy, microautophagy has not been extensively studied
in skeletal muscle, and its functions in muscle proteolysis have to be more characterized [35]. 2.1.1. Description of the system High levels of these cathepsins are expressed
in tissues exhibiting high rates of protein turnover like the kidney, spleen, liver, or placenta,
while low concentrations of cathepsins are found in tissues with lower protein turnover as
skeletal muscles [37-40]. Similar enzymatic properties were reported for different muscles,
independent of their metabolic and contractile type. However, their concentrations differ
according to the fiber type. Indeed, slow-twitch oxidative muscles exhibit higher levels of
cathepsins than the fast-twitch glycolytic muscles [35], suggesting a more important activity of
this system in oxidative muscle. This data is in agreement with the fact that oxidative muscles
present a more important protein turnover and a greater translational activity than glycolytic
muscles. Although skeletal muscle expresses cathepsins B, D, H, and L, they play distinctive roles. During
fusion of myoblasts into myotubes, several groups have reported an increase in the expres‐
sion and activity of lysosomal cathepsins, in particular cathepsin B [41-46]. Several studies
showed that the expression of cathepsin L is induced during various forms of skeletal muscle
atrophy including starvation [12,36,47-49]. Increase in cathepsin D activity has been reported
in muscles of dystrophic mice and chicken and in muscles of patients with Duchenne muscu‐
lar dystrophy [35,50]. 2.1.4. Macroautophagy Macroautophagy, often referred to as autophagy or autophagosome-lysosome system, is an
evolutionarily conserved intracellular system that coordinates and oversees the degradation
of damaged organelles as mitochondria, peroxisomes, or ribosomes, intracellular pathogens,
and unused long-lived proteins [61]. More than 30 autophagy-specific genes (Atgs) have been
identified and are known to facilitate the sequestration of cytoplasmic substrates into a double-
membrane vesicle called autophagosome or autophagic vacuole. Atgs are essential mediators
of autophagy, by controlling the formation of the autophagosome. Autophagosome fuses with
lysosome to form an autolysosome (also called autophagolysosome). 2.1.3. Chaperone-mediated autophagy Chaperone-mediated autophagy (CMA) is a selective form of autophagy that has only been
described in mammalian cells to date [52,53]. In this form of autophagy, only cytosolic proteins
that possess the consensus pentapeptide Lys-Phe-Glu-Arg-Gln (KFERQ) are recognized. The Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease…
http://dx.doi.org/10.5772/60698
1 175 KFERQ-like sequence is recognized by the heat-shock cognate protein of 73 kDa (hsc73) then
targeted to lysosomes for degradation. This targeting needs the binding of the complex protein
substrates hsc73 to the lysosome-associated membrane protein type 2A (LAMP-2A) and to a
multi-molecular chaperone complex including hsp40, hsp70, and hsp90 at the cytosolic side of
the lysosomal membrane [54]. LAMP-2A is a glycoprotein present at the lysosomal mem‐
brane which acts as a CMA receptor. The substrates are then unfolded and translocated across
the lysosomal membrane with the hsc73 protein. Unlike other forms of autophagy, CMA is very
selective in substrate degradation and cannot eliminate organelles [55-57]. The use of an antibody directed to the KFERQ amino acid sequence substrates showed that
proteins containing this sequence are conserved in skeletal muscle during starvation, while
they are degraded in the liver and heart [58]. Moreover, the absence of consensus sequence in
most myofibrillar proteins indicates that this degradation pathway is not implicated in their
degradation. However, Nishino and colleagues showed that LAMP-2 deficiency is the primary
defect in human Danon disease, a pathology characterized by myopathy and cardiomyopathy
with massive accumulation of autophagic vacuoles [59,60].Thus, as microautophagy, CMA
has to be more characterized in the context of muscle atrophy. 2.2.1. Initiation Macroautophagy (autophagy) is initiated in response to a multitude of factors including
nutrient deprivation and oxidative stress. The activation of autophagy during muscle wasting
was shown by the accumulation of autophagosomes in muscles of fasted transgenic GFP-LC3
mice [62]. Studies showed that during starvation-induced atrophy, FoxO3a (forkhead box class
O3a) regulates the transcription of several Atgs, including Atg4B, LC3B (microtubule-
associated protein 1A/1B-light chain 3B), Beclin-1, Vps34 (vacuolar protein sorting 34)/PI3K
class III, Gabarapl1 (GABAA receptor-associated protein-like 1), Atg12, and Ulk2 (unc-51-like
kinase 2) [63,64]. Initiation of the autophagy processes involves the activation of the unc-51-
like kinase 1 (Ulk1, also called Atg1 in yeast)/Atg13/FIP200/Atg101 and the Beclin-1/Vps34
complexes. These proteins operate in conjunction with several Atgs to mediate the assembly
of the autophagosomal membrane [65-68]. Ulk1 also phosphorylates Beclin-1 at Ser-14
following amino acid withdrawal, and this stage is necessary for the Vps34 lipid kinase Muscle Cell and Tissue 176 activation and full autophagy induction [69]. In yeast and mammalian cells, Atg1 or Ulk1
(respectively) activity is suppressed under nutrient-rich conditions by MTORC1 (phosphory‐
lation of Ulk1 at Ser-757) [65,70-73]. In addition, MTOR inhibition and its subsequent dissoci‐
ation from Ulk1 are critical for Beclin-1/Vps34 complex activation by Ulk1 [69]. 2.3. Implication of autophagy in cell homeostasis and disease Autophagy plays a prominent role in the maintaining of cell homeostasis by selectively
eliminating protein aggregates, damaged organelles, and other nonactive cellular debris
[79,80]. This process is required for normal cellular function, but it is increasingly apparent
that it can have both beneficial and detrimental effects on cells and tissues, depending on the
origin of its activation [81]. Physiological function of basal autophagy in maintaining tissue
homeostasis has been demonstrated in several tissues as the brain, liver, heart, striated muscle,
intestine, pancreas, and adipose tissue [82-89]. Furthermore, exciting reports suggest that
autophagy may contribute to counteract the deleterious effects of aging by limiting the
deposition of aggregated proteins and damaged mitochondria [90-92]. By blocking apoptosis,
autophagy preserves cell survival by providing endogenous metabolites when exogenous
substrates are lacking [93]. Thus, at regular levels of activation, autophagy may represent the
first step to restore homeostasis. However, when the autophagic capacity is submerged,
apoptosis takes over [94]. Nevertheless, the relationship between autophagy and apoptosis
appears to be extremely complex, and additional data are necessary to clarify the situation,
especially in the context of disease. In numerous pathologies as diabetes, obesity, cancer, heart failure, and neurodegenerative,
infectious, and inflammatory disease, autophagy activity is affected [34,95,96]. For instance, it
has been reported that the lack in Beclin-1 expression decreases autophagy flux and leads to
increased risk of breast and prostate cancers [97]. However, the systematic beneficial effect of
autophagy should be tempered. Thus, the role of autophagy in cancer is ambivalent, and this
process can be involved in both the promotion and the prevention of this disease. In the first
stage of the malignancy (i.e., tumor initiation), inhibition of autophagy may allow the growth
of initial cancerous cells, and thus autophagy can act as a suppressor of cancer [98,99]. When
cancer is established, transformed cells may need autophagy to survive, especially in nutrient-
limiting condition [100]. In addition, for patient undergoing treatment such as chemotherapy,
cancer cells could use autophagy to protect themselves from the stress induced by the therapy. Other reports indicate also that glycogen storage disease type II (also called Pompe disease) –
an autosomal recessive metabolic disorder – is a pathology attributable in part to mutations
of Atgs. In this disease, muscle and nerve cells are damaged by an accumulation of glycogen
into the lysosomes caused by a deficiency of the lysosomal acid alpha-glucosidase enzyme
[101,102]. 2.2.2. Maturation of autophagosomes The maturation and completion of the autophagic vacuole is facilitated by a ubiquitin-like
conjugation-signaling cascade that culminates with the binding of phosphatidylethanolamine
(PE) to the cytosolic form of LC3 (LC3-I) to form LC3-II. LC3, a mammalian homolog of yeast
Atg8, is a protein with a molecular mass of 17 kDa that is distributed ubiquitously in mam‐
malian tissues. Two other LC3 homologs are GABAA receptor-associated protein (Gabarap)
and Golgi-associated ATPase enhancer (GATE-16). LC3-phosphatidylethanolamine conjugate
(LC3-II) is recruited to the autophagosomes [74,75]. LC3-II plays here a structural role that
allows the elongation and the formation of the mature autophagosome. The mature autopha‐
gosome fuses with the lysosome to form an autolysosome (Fig.1). Concomitantly, LC3-II in
autolysosomal lumen is degraded by lysosomal hydrolases; thus, turnover of the autophago‐
somal protein LC3-II reflects autophagic activity [76]. LC3 also interacts with the autophagy
adapter p62/SQSTM1 (sequestosome 1), which contains multiple apparent protein-protein
interaction domains. p62 binding to LC3 permits the entry of ubiquitinated cargo into the
autophagosome [77]. In addition, the Atg12-Atg5-Atg16 complex plays an important role in
the maximal promotion of LC3 lipidation and autophagy induction [78]. Figure 1. Processing of the macroautophagy system Figure 1. Processing of the macroautophagy system Figure 1. Processing of the macroautophagy system Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease…
http://dx.doi.org/10.5772/60698
1 Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease…
http://dx.doi.org/10.5772/60698 177 2.4. The roles of autophagy in skeletal muscle Compared to other tissues like the liver or pancreas, autophagy in skeletal muscle exhibits a
low protein turnover and a small size of autophagosomes. These peculiar characteristics have
probably constituted a brake for the detection of autophagy in this tissue for a long time. Associated to the use of transgenic mice expressing LC3 fused with GFP, autophagy process
can be now easily visualized [62]. Conversely to liver or pancreas in which autophagy is
activated transiently for a few hours, in skeletal muscle, autophagy can be activated for several
days [62]. As it was shown in other tissues, muscular autophagy is activated by nutrient
deprivation or by the absence of growth factors [108]. Although it was reported that the mRNAs coding for Atgs are present in abundance in skeletal
muscle [109], the role and the regulation of basal autophagy have been poorly characterized
in this tissue until recently. In order to assess the function of autophagy in skeletal muscle,
Masiero and colleagues performed experiments on mice deprived of the Atg7 gene, a gene
necessary to the unfolding of the autophagy program [86,110]. Importantly, mice showed
obvious signs of muscular weakness and atrophy exacerbated during ageing. Mice presented
an accumulation of degraded proteins and free radicals, a deterioration of the internal cellular
structures, and an activation of the apoptotic program. The authors clearly defined that
inhibition of basal autophagy does not protect from skeletal muscle atrophy induced by
denervation or starvation, but on the contrary, contributes to its degeneration. Similar muscle
alterations have been obtained in muscle-specific Atg5-/- mice [87], confirming the necessity to
have regular autophagic flux in the cells, even during atrophy. In many conditions varying from fasting, denervation, inactivity, microgravity, various
pathologies as cancer, AIDS, sepsis, diabetes, cardiac failure, and myopathies, autophagy is
overactive and pathologic, thus leading altered metabolism and muscle loss [81,108,111]. Contribution of autophagy to muscle loss begins to be clarified with the use of different animal
models and innovative techniques. Inactivation of autophagic flux by LC3 silencing partially
prevents FoxO3-mediated muscle atrophy [63] and atrophy caused by the expression of
mutant SOD1 G93A in skeletal muscle [112]. In another model, atrophy induced by L-type
calcium channel (DHPR) inactivation is linked to the expression of autophagic genes including
LC3, Vps34, BNIP3 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 3), and cathepsin
L (for the lysosome) [113]. 2.3. Implication of autophagy in cell homeostasis and disease In many neurodegenerative disorders such as Parkinson’s, Huntington’s, and
Alzheimer’s diseases, accumulation of autophagic vesicles has been also observed [103-105]. Regarding Alzheimer’s disease, it has been reported that Atg7 influences the accumulation of
amyloid β (Aβ) peptides, resulting in aggregation into plaques in the brain [106]. In this model,
autophagy seems to participate to the disease progression since it is involved in the generation
of Aβ peptides. In summary, by removing misfolded proteins and abnormal organelles, autophagy can be
considered as a critical mechanism for cell protection. On the contrary, by destroying excessive
fraction of cytosol and organelles, too high levels of autophagy represent a side mechanism
responsible of the initiation of pathologies [94,107]. Muscle Cell and Tissue 178 2.4. The roles of autophagy in skeletal muscle During sepsis, an upregulation of autophagy was found in parallel
to mitochondrial injury and decreased biogenesis, especially in locomotor muscles [114]. Reactive oxygen species overproduction by altered mitochondria is now considered as a
critical signal for the promotion of skeletal muscle autophagy, thus finding an opening to
practical prospects for treatment of disease [115]. Accordingly, antioxidant supplement can
lead to an inhibition of skeletal muscle autophagy through a reduction of oxidative stress and
an increase in antioxidant capacity [116]. Taken together, these recent results confirm that
excessive activation of autophagy is a critical factor for muscle wasting determinism, and
strategies attempted to control autophagy level will be promising. Data concerning sarcopenia are also specific. Sarcopenia is an age-related loss of muscle mass
and strength, which is associated with increased autophagy, apoptosis, and exacerbated
proteolysis [117]. Elevated peroxisome proliferator-activated receptor-coactivator α (PGC-1α)
expression in muscle during aging contributes to reduce the proteolytic activity associated Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease…
http://dx.doi.org/10.5772/60698 179 with atrophy. In sarcopenia, attenuation of the degradative processes and the maintenance of
mitochondrial function contribute to the preservation of muscle integrity [118]. In summary, as already described for cancer cells, autophagy in muscle is a complex process
that can be, according to its activation level, beneficial or deleterious. During disease, the
systematic question is to determine whether autophagy plays a protective function, has a
causative role, or is a result of the disease process itself. In summary, as already described for cancer cells, autophagy in muscle is a complex process
that can be, according to its activation level, beneficial or deleterious. During disease, the
systematic question is to determine whether autophagy plays a protective function, has a
causative role, or is a result of the disease process itself. 3.1. The AMP-activated protein kinase AMPK is a serine/threonine protein kinase highly conserved through evolution. AMPK is a
heterotrimeric complex composed of a catalytic subunit (AMPK-α) and two regulatory
subunits (AMPK-β and AMPK-γ). Humans have seven genes encoding AMPK subunits (α1,
α2, β1, β2, γ1, γ2, γ3) that can form at least 12 αβγ heterotrimers, increasing the diversity of
its functions [119]. The catalytic α subunit contains the threonine phosphorylation site that
upon phosphorylation leads to AMPK activation [120]. AMPK acts as a sensor of cellular
energy status by regulating several intracellular systems including glucose and lipid metab‐
olisms and mitochondrial biogenesis [121]. Thus, AMPK activation leads to increased glycol‐
ysis flux [122] and fatty acid oxidation [123-126] and on the contrary, to an inhibition of
glycogenogenesis [127,128] and cholesterol and fatty acid biosynthesis [129-131]. The enzyme
also increases the expression of PPARα (peroxisome proliferator-activated receptor α) target
genes and PGC-1 leading to mitochondrial biogenesis and enhanced oxidative metabolism of
muscle cells [132,133]. AMPK is activated by a large variety of cellular signals that decrease
cellular ATP levels and increase AMP in response to different kinds of stress like electrical-
stimulated muscle contraction, exercise, hypoxia, and heat shock or under conditions of
nutrient deprivation [124,134,135]. The recognized enzymes in the regulation of AMPK under
energy stress conditions are LKB1 (liver kinase B1), CaMKK (calmodulin-dependent protein
kinase kinase), and TAK-1 (transforming growth factor beta-activated kinase 1). 3.2. Regulation of skeletal muscle autophagy by AMPK The role of AMPK in protein turnover has been clearly defined in recent years. AMPK has been
involved in the control of protein synthesis and the repression of skeletal muscle mass by
inhibiting MTOR activity [136,137] in two ways: AMPK phosphorylates TSC2 (tuberous
sclerosis complex 2) at Thr-1227 and Ser-1345 and RPTOR at Ser-722 and Ser-792, leading to a
reduction of MTORC1 activity. Several studies also showed that AMPK increases protein
degradation through the modulation of the ubiquitin-proteasome and the autophagosome-
lysosome pathways [138,139]. AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribonucleo‐
side, an AMPK activator) treatment increases the expression of the E3 ligases MAFbx/
Atrogin-1 and MuRF1 in muscles cells. In addition, increase of autophagic flux by AMPK has
been reported in several muscle models as C2C12 myoblasts, C2C12 myotubes, and primary
myotubes [138,139]. Two major signaling pathways were characterized in AMPK-induced
muscular autophagy (Fig.2). Muscle Cell and Tissue
180 Figure 2. Role of AMPK in skeletal muscle protein turnover
The first one concerns the activation of the forkhead box class O proteins (FoxO), notably
involved in the regulation of protein breakdown, energy metabolism, and mitochondrial
turnover [140]. FoxO factors also play an important role in exercise-induced angiogenesis by
limiting it during the first days of training program [141,142]. Activation of FoxO3a by AMPK
leads to an increase in several Atgs expression, including LC3-II and Gabarapl1 that act as
promoters of autophagosome fabrication [139]. AMPK directly interacts with FoxO3a and
phosphorylates it on Ser-588, a residue known to lead to FoxO3a activation [139,143]. The
upregulation of several Atgs by FoxO factors have been described in Drosophila larval fat body
[144], mammalian cardiomyocytes [145], hepatocytes [146], and colorectal cancer cells [147]. Regarding the regulation of FoxO3a subcellular localization in muscle cells, while long
treatments (i.e., 24 h) with AMPK activators do not change FoxO3a nuclear content, an increase
in the total protein level is notable after 30 min. With a short time course (30 min–6 h), the
activation of AMPK by AICAR leads to a relocalization of FoxO3a into the nucleus [139]. Tong
and colleagues reported that AICAR treatment causes FoxO3a nuclear relocation through a
decrease in FoxO3a phosphorylation at Thr-318/321 [148]. However, Greer and colleagues have
reported an increase of FoxO3a transcriptional activity without any change in the nuclear
content of the factor after AMPK activation by 2-deoxyglucose (2DG) in HEK293T cells [143]. 3.2. Regulation of skeletal muscle autophagy by AMPK These data strongly suggest that FoxO3a relocalization into the nucleus is not necessarily
required to increase its transcriptional activity. A possibility is that AMPK may also control
FoxO3a protein stability. The second pathway involves modulation of the Ulk1 complex A multiprotein complex Figure 2. Role of AMPK in skeletal muscle protein turnover Figure 2. Role of AMPK in skeletal muscle protein turnover The first one concerns the activation of the forkhead box class O proteins (FoxO), notably
involved in the regulation of protein breakdown, energy metabolism, and mitochondrial
turnover [140]. FoxO factors also play an important role in exercise-induced angiogenesis by
limiting it during the first days of training program [141,142]. Activation of FoxO3a by AMPK
leads to an increase in several Atgs expression, including LC3-II and Gabarapl1 that act as
promoters of autophagosome fabrication [139]. AMPK directly interacts with FoxO3a and
phosphorylates it on Ser-588, a residue known to lead to FoxO3a activation [139,143]. The
upregulation of several Atgs by FoxO factors have been described in Drosophila larval fat body
[144], mammalian cardiomyocytes [145], hepatocytes [146], and colorectal cancer cells [147]. Regarding the regulation of FoxO3a subcellular localization in muscle cells, while long
treatments (i.e., 24 h) with AMPK activators do not change FoxO3a nuclear content, an increase
in the total protein level is notable after 30 min. With a short time course (30 min–6 h), the
activation of AMPK by AICAR leads to a relocalization of FoxO3a into the nucleus [139]. Tong
and colleagues reported that AICAR treatment causes FoxO3a nuclear relocation through a
decrease in FoxO3a phosphorylation at Thr-318/321 [148]. However, Greer and colleagues have
reported an increase of FoxO3a transcriptional activity without any change in the nuclear
content of the factor after AMPK activation by 2-deoxyglucose (2DG) in HEK293T cells [143]. These data strongly suggest that FoxO3a relocalization into the nucleus is not necessarily
required to increase its transcriptional activity. A possibility is that AMPK may also control
FoxO3a protein stability. The second pathway involves modulation of the Ulk1 complex. A multiprotein complex
composed of AMPK, MTORC1, Ulk1, FIP200, and Atg13 has been identified in muscle cells
(Fig.3) [139]. These data fit with the model found in other cell types showing that, under basal Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease…
http://dx.doi.org/10.5772/60698
1 181 conditions, MTORC1 prevents autophagy by interacting with Ulk1 [72]. 3.2. Regulation of skeletal muscle autophagy by AMPK Under nutrient-rich
conditions, phosphorylation of Ulk1 by MTORC1 represses Ulk1 kinase activity and its ability
to interact with Atg13 or FIP200; thereby, it coordinates the autophagy response [65,149]. In
muscle cells, activation of AMPK (by AICAR treatment) or inhibition of MTORC1 (by Torin1
treatment or amino acid privation) removes AMPK, MTOR, and RPTOR from Ulk1 [139]. These
events are known to induce the Ulk1-dependent phosphorylation of Atg13 and FIP200, leading
to the initiation of autophagy [70]. Proteomics screens of autophagy [150] and a co-immunoprecipitation study performed in
HEK293T cells [151] showed that AMPK interacts with both Ulk1 and Ulk2. In muscle cells,
Ulk1 also acts as an interacting partner of AMPK, and Ser-467 site identified by Egan and
colleagues is also phosphorylated by AMPK [139]. Ulk1 phosphorylation by AMPK may
participate to conformational changes and thus disrupts the interaction between Ulk1 and
MTORC1, in agreement with the suppression of MTORC1 anti-autophagy activity in the Ulk1
complex [72]. Moreover, Ulk1 phosphorylation by AMPK may directly activate Ulk1 kinase
activity. Indeed, in vitro studies showed that Ulk1 is highly phosphorylated and that purified
Ulk1 can phosphorylate itself and requires autophosphorylation for stability [152]. In mam‐
mals, Ulk1 phosphorylation by AMPK is critical for mitochondrial homeostasis and cell
survival during starvation [153]. In summary, AMPK regulates Ulk1 activity by decreasing
MTORC1 activity and by phosphorylating Ulk1 [121]. Figure 3. The Ulk1/Atg13/FIP200/MTORC1/AMPK complex
Time-course studies have been performed in muscle cells in order to better understand the
dynamics of UlK1 complex following autophagy induction [139]. Interestingly, AMPK
dissociates from Ulk1 3 h after AICAR treatment [139]. In agreement with these observations,
in HeLa cells, AMPK is associated with Ulk1 only under nutrient-rich condition, and it
dissociates from Ulk1 5 min after starvation [154]. Thus, in normal condition, Ulk1 is associated Figure 3. The Ulk1/Atg13/FIP200/MTORC1/AMPK complex Figure 3. The Ulk1/Atg13/FIP200/MTORC1/AMPK complex Figure 3. The Ulk1/Atg13/FIP200/MTORC1/AMPK complex Time-course studies have been performed in muscle cells in order to better understand the
dynamics of UlK1 complex following autophagy induction [139]. Interestingly, AMPK
dissociates from Ulk1 3 h after AICAR treatment [139]. In agreement with these observations,
in HeLa cells, AMPK is associated with Ulk1 only under nutrient-rich condition, and it
dissociates from Ulk1 5 min after starvation [154]. Thus, in normal condition, Ulk1 is associated Muscle Cell and Tissue 182 with AMPK; upon AICAR treatment, the complex remains stable for 3 h and then dissociates. As suggested by another group, Ulk1 dissociation from AMPK could permit to Ulk1 complex
to be more active [154]. Conversely, this dissociation can constitute a negative regulatory
feedback as proposed by Loffler et al. [155]. The authors showed that Ulk1 could mediate
phosphorylation of AMPK on the regulatory subunits, constituting an inhibitory feedback
control. Further works have to define the molecular mechanisms for these events, especially
in skeletal muscle. 4. Autophagy, exercise and altitude exposure However, Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease…
http://dx.doi.org/10.5772/60698 183 reticulophagy has not been studied yet in skeletal muscle, especially in response to exercise. Further works are needed to clarify the possible clearance of important organelles, such as
ribosomes or endoplasmic reticulum, during physical exercise. Exercise promotes better “cell
health”; it would be not surprising to discover that exercise increases the turnover of such
organelles like it is strongly suggested for mitochondria [21]. The rise of autophagy is essential to prevent mitochondrial damage during endurance exercise. Although acute inhibition of autophagy prior to exercise seems to not significantly affect
performance, it leads to accumulation of dysfunctional mitochondria and augmentation of
oxidative stress especially during eccentric contraction [164]. Thus, autophagy has a critical
role in mitochondria quality control during acute exercise. In addition, autophagy is highly
involved in exercise training-induced adaptations. Autophagy-deficient mice present attenu‐
ated improvement of endurance capacity in response to endurance training. In parallel to lower
increases of basal autophagy flux, these mice show attenuated raises of mitochondrial content
and angiogenesis [165], explaining the poor response to the training program. The dietary factors have to be considered in autophagy response to acute exercise since
essential amino acids (EAA) or carbohydrate (CHO) intake modulate protein turnover. Jamart
et al. (2014) found that exercise performed in the fasted state permits a higher raise in auto‐
phagic flux indexes compared with the fed state. Concerning resistance exercise, few works
showed a depression of autophagy markers after such an exercise like the study by Fry et al. conducted in humans [166]. In addition, autophagic flux markers can be depressed following
EAA and carbohydrate (CHO) ingestion after resistance exercise [167]. To date, autophagy
seems non-critical for muscle adaptations to resistance training [161]. However, an exception
occurs during aging in which both endurance and resistance training are able to reverse the
drop of autophagy regulatory proteins that occurs [168,169] (Fig.4). Little is known regarding protein turnover pathways, especially autophagy, in response to
exercise performed during altitude exposure in humans. Such an environment can induce a
state of hypoxia that is exacerbated according to the level of altitude considered. Hypoxia
results in decreased oxygen availability and leads to several hormonal, cardiorespiratory, and
muscular adjustments in order to preserve cell homeostasis. Long-lasting hypoxia can cause
a diminution of skeletal muscle mass and a reduction of muscle oxidative capacity. 4. Autophagy, exercise and altitude exposure Attractive data concerning the role of autophagy during exercise are emerging. Autophagic
vacuole formation during physical exercise was observed for the first time by Salminen and
coworkers in 1984 with electron microscopy [156]. Nevertheless, there were no further studies
on the topic until recently. In the last decade, data supporting the importance of autophagy in
muscle homeostasis in response to exercise have been numerous, starting with a study from
Bonaldo’s team that showed that mice presenting impaired autophagy develop severe muscle
weakness (i.e., accumulation of defective mitochondria, exacerbated apoptosis, muscle
degeneration, and atrophy) [157]. Thereafter, other studies highlight that chronic inactivation
of autophagy leads to a loss of metabolic effects related to exercise and drastic decreases in
endurance performance [158]. Concerning autophagy modulation in response to acute exercise in humans, studies by Jamart
and colleagues [159] were the first to demonstrate a raise of autophagy-regulatory genes and
autophagic flux markers after ultraendurance exercise. By showing that AMPK and FoxO3a
regulate in a coordinated way autophagy and ubiquitin-proteasome pathways during
ultraendurance exercise, the authors gave an important picture of the cross-regulation of both
degradation pathways in response to long-lasting endurance exercise [160]. Regarding more
common endurance exercises, the modulation of muscle protein turnover, autophagy, and
mitochondrial dynamics markers has been investigated thereafter in mice in response to
different exercises conducted or not until exhaustion. Endurance exercise quickly initiates the
autophagy pathway through Ulk1 activation resulting in an increase of autophagic flux,
especially near exhaustion [161]. A rise in the phosphorylation of DRP1, a GTPase essential for
mitochondrial fission, quickly occurred during exercise without any change in the expression
of fusion markers (OPA1 and Mfn2). These data are consistent with an increase in mitophagy
(i.e., the degradation of mitochondria by autophagy) since exacerbated fission can lead to
mitochondrial fragmentation. Noteworthy, exercise decreases the activity of the main protein
synthesis pathway (i.e., Akt/MTOR signaling pathway), from 90 min of moderate exercise (40–
50 % of VO2max), concomitantly to an increase in the phosphorylation of a marker of endoplas‐
mic reticulum stress (eiF2α Ser-51) [161]. Others studies reported an increase of endoplasmic
reticulum stress by the evaluation of the content of the double-stranded RNA-activated protein
kinase R (PKR)-like endoplasmic reticulum kinase (PERK), the ER stress-induced transcription
factor C/EBP homologous protein (CHOP), and the X-box binding protein 1 (XBP1s), in
response to both ultraendurance [162] and moderate-intensity exercise [163]. 4. Autophagy, exercise and altitude exposure In agree‐
ment with an alteration of protein synthesis flux, hypoxia impairs the overload-induced
increase of the PI3K/Akt/MTOR signaling pathway in rats [170]. Regarding degradation
pathways in humans, while ubiquitin-proteasome system seems not positively modulated by
environmental hypoxia, an upregulation of skeletal muscle autophagic flux markers has been
found during acute normobaric hypoxia (10.7 % O2) and after exercise conducted in such an
environment [171,172]. Another study investigated the effects of acute high-altitude exposure
(at 5,300 m altitude) in the course of the Caudwell Research Expedition to Mt. Everest [173]. The authors notably found an upregulation of heat shock cognate 71 kDa protein involved in
chaperone-mediated autophagy and a reduction of protein translation markers. Taken
together, these studies seem to highlight a preventive role of autophagy for energy expenditure
and an activation of chaperone-mediated autophagy during acute high-altitude exposure. However, the effects of chronic altitude exposure on autophagy and its combination with
training remain to be characterized to date. Chronic hypoxia leads to a change in oxidative Muscle Cell and Tissue
184 Figure 4. Exercise and autophagy in skeletal muscle. Adapted from Sanchez AMJ et al. 2014. Autophagy is essential to
support skeletal muscle plasticity in response to endurance exercise. American Journal of Physiology – Regulatory, In‐
tegrative and Comparative Physiology 307(8): R956-R969 [21]. Essential amino acids (EAA); carbohydrate (CHO) Figure 4. Exercise and autophagy in skeletal muscle. Adapted from Sanchez AMJ et al. 2014. Autophagy is essential to
support skeletal muscle plasticity in response to endurance exercise. American Journal of Physiology – Regulatory, In‐
tegrative and Comparative Physiology 307(8): R956-R969 [21]. Essential amino acids (EAA); carbohydrate (CHO) metabolism [174,175]; it is likely that autophagy, especially mitochondrial autophagy, be
amended during such an exposure. Consistent with this, experiments performed in cells
showed that mitochondrial autophagy is induced by chronic hypoxia through HIF-1 (hypoxia-
dependent factor-1) and BNIP3, and this regulation constitutes a preventive response that is
necessary to avoid accumulation of reactive oxygen species and cell death [176]. The effects of
exposition to moderate altitude (i.e., 1,500–3,000 m) have also to be definite since it concerns
a larger population (athletes, general tourist population, and highlanders) compared to high
altitude. Research of this type is leading to a better understanding of the autophagy-mediated turnover
of organelles like mitochondria in response to exercise and altitude exposure. 4. Autophagy, exercise and altitude exposure Finding optimal
training strategies represents an important objective to enhance exercise adaptations in both
athletes and patients with metabolic or muscle diseases, including COPD (chronic obstructive
pulmonary disease) that can cause systemic hypoxia and loss in muscle capability. 5. Conclusions In summary, autophagy represents a crucial mechanism for cell homeostasis and cell survival. Autophagy constitutes a recycling process that degrades used or flawed internal structures Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease…
http://dx.doi.org/10.5772/60698
1 185 into amino acid, enabling cells to survive in difficult circumstances. The identification of Ulk1
as a direct target of AMPK represents a significant step toward understanding how cellular
energy stress regulates autophagy machinery in muscle cells. Based on the data presented here,
it is clearly conceivable that the autophagy pathway must be considered in muscle pathologies
such as sarcopenia and myopathies. Fine molecular studies on AMPK/FoxO3a and AMPK/
Ulk1 axes will offer new and promising strategies in the treatment of muscular loss associated
not only to several pathologies like cancer, AIDS, and neuromuscular diseases but also to age-
related disorders. Further investigations must be conducted in order to better understand
whether exercise-induced autophagy can also prevent skeletal muscle diseases. In addition,
autophagy is involved in skeletal muscle adaptation to endurance exercise in a healthy
population. While autophagy is important to prevent mitochondria alteration and exacerbated
oxidative stress in response to severe acute exercise, its role in the improvement of endurance
capacity has also been demonstrated in response to endurance training. In addition, the feeding
pattern and the environment, especially altitude exposure, have to be considered since they
represent additional modulators of protein turnover and autophagy pathway. Regular exercise
is associated with an enhanced quality of life and represents the most profitable way to limit
metabolic disorders and the loss of muscle mass. As a result, these research directions are of
major interest in the battle against a wide range of diseases and have to be sustained. Acknowledgements Dr AMJ Sanchez thanks the “Cité de l’excellence sportive Sud de France” of Font-Romeu
(France). This work was supported by the department PHASE from INRA and by the Uni‐
versité de Montpellier 1, Faculté des Sciences du Sport. Miss A. Raibon holds a graduate
fellowship from INRA, from the region Languedoc-Roussillon, and from INCA. The authors
also thank Pr Raviola for the helpful discussions. Author details Anthony M.J. Sanchez1*, Robin Candau2, Audrey Raibon2 and Henri Bernardi2 *Address all correspondence to: anthony.sanchez@univ-perp.fr 1 Laboratoire Européen Performance Santé Altitude, EA4604, Université de Perpignan Via
Domitia, Département STAPS Font-Romeu, France 1 Laboratoire Européen Performance Santé Altitude, EA4604, Université de Perpignan Via
Domitia, Département STAPS Font-Romeu, France 2 INRA, UMR866, Dynamique Musculaire et Métabolisme, Université de Montpellier
Montpellier, France 2 INRA, UMR866, Dynamique Musculaire et Métabolisme, Université de Montpellier 2 INRA, UMR866, Dynamique Musculaire et Métabolisme, Université de Montpellier,
Montpellier, France Montpellier, France List of abbreviations 4E-BP1, eukaryotic translation initiation factor 4E-binding protein 1; Aβ, amyloid β; AIDS,
acquired immune deficiency syndrome; AMPK, 5'-adenosine monophosphate-activated
protein kinase; Atg, autophagy-specific gene; BCL2, B-cell lymphoma 2; BNIP3, BCL2/
adenovirus E1B 19 kDa-interacting protein-3; CaMKK, Ca2+/calmodulin-dependent protein
kinase; CHO, carbohydrate; CHOP, C/EBP homologous protein; CMA, chaperone-mediated
autophagy; COPD, chronic obstructive pulmonary disease; DEPTOR, DEP domain-containing
MTOR-interacting protein; EAA, essential amino acids; eIF2B, eukaryotic translation initiation
factor 2B; eIF2α, eukaryotic initiation factor 2α; FIP200, focal adhesion kinase (FAK) family-
interacting protein of 200 kDa; FoxO, forkhead box class “other” O; Gabarapl1, GABAA
receptor-associated protein-like 1; GATE-16, Golgi-associated ATPase enhancer; GSK3β,
glycogen synthase kinase 3β; hsc73, heat-shock cognate protein of 73 kDa; HIF-1, hypoxia-
dependent factor-1; IGF-1, insulin-like growth factor-1; LAMP-2A, lysosome-associated
membrane protein 2A; LC3, microtubule-associated protein 1A/ 1B light chain 3; LKB1, liver
kinase B1; MAFbx/atrogin-1, muscle atrophy F-box; Mfn2, mitofusin-2; mLST8/GβL, MTOR-
associated protein LST8 homolog; MTOR, mechanistic/mammalian target of rapamycin;
MTORC1, mechanistic/mammalian target of rapamycin complex 1; Mul1, mitochondrial E3
ubiquitin protein ligase 1; MuRF1, muscle RING-finger protein-1; OPA1, optic atrophy 1; p62/
SQSTM1, sequestosome 1; PDK1, phosphoinositide-dependent kinase-1; PE, phosphatidyle‐
thanolamine; PERK, double-stranded RNA-activated protein kinase R (PKR)-like endoplasmic
reticulum kinase; PGC-1, peroxisome proliferator activator receptor γ coactivator-1; PI3K,
phosphatidylinositol 3-kinase; PINK1, PTEN-induced putative kinase protein 1; PKB/Akt, Muscle Cell and Tissue 186 protein kinase B; PPARα, peroxisome proliferator-activated receptor α; PRAS40, proline-rich
Akt substrate of 40 kDa; PTEN, phosphatase and tensin homolog; Rheb, ras homologous
enriched in brain; RPTOR, regulatory-associated protein of MTOR, complex 1; ROS, reactive
oxygen species; S6K1, p70S6 kinase 1; TAK-1, transforming growth factor β-activated kinase
1; Trim32, tripartite motif-containing protein 32; TSC21/2, tuberous sclerosis complex 1/2; Ulk,
unc-51-like kinase; Vps34, vacuolar protein sorting 34; XBP1s, X-box binding protein 1;
ZNF216, zinc-finger protein 216 References [1] Glass DJ (2005) Skeletal muscle hypertrophy and atrophy signaling pathways. Int J
Biochem Cell Biol 37: 1974-1984. [2] Ma XM, Blenis J (2009) Molecular mechanisms of mTOR-mediated translational con‐
trol. Nat Rev Mol Cell Biol 10: 307-318. [2] Ma XM, Blenis J (2009) Molecular mechanisms of mTOR-mediated translational con‐
trol. Nat Rev Mol Cell Biol 10: 307-318. [3] Wullschleger S, Loewith R, Hall MN (2006) TOR signaling in growth and metabo‐
lism. Cell 124: 471-484. [3] Wullschleger S, Loewith R, Hall MN (2006) TOR signaling in growth and metabo‐
lism. Cell 124: 471-484. Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease…
http://dx.doi.org/10.5772/60698
1 187 [4] Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, et al. (2001) Akt/mTOR path‐
way is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle
atrophy in vivo. Nat Cell Biol 3: 1014-1019. [5] Rommel C, Bodine SC, Clarke BA, Rossman R, Nunez L, et al. (2001) Mediation of
IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/
GSK3 pathways. Nat Cell Biol 3: 1009-1013. [6] Sanchez AM, Csibi A, Raibon A, Docquier A, Lagirand-Cantaloube J, et al. (2013)
eIF3f: a central regulator of the antagonism atrophy/hypertrophy in skeletal muscle. Int J Biochem Cell Biol 45: 2158-2162. [7] Csibi A, Cornille K, Leibovitch MP, Poupon A, Tintignac LA, et al. (2010) The transla‐
tion regulatory subunit eIF3f controls the kinase-dependent mTOR signaling re‐
quired for muscle differentiation and hypertrophy in mouse. PLoS One 5: e8994. [8] Castro AF, Rebhun JF, Clark GJ, Quilliam LA (2003) Rheb binds tuberous sclerosis
complex 2 (TSC2) and promotes S6 kinase activation in a rapamycin- and farnesyla‐
tion-dependent manner. J Biol Chem 278: 32493-32496. [9] Garami A, Zwartkruis FJ, Nobukuni T, Joaquin M, Roccio M, et al. (2003) Insulin acti‐
vation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and
2. Mol Cell 11: 1457-1466. [10] Inoki K, Zhu T, Guan KL (2003) TSC2 mediates cellular energy response to control
cell growth and survival. Cell 115: 577-590. [11] Jefferson LS, Fabian JR, Kimball SR (1999) Glycogen synthase kinase-3 is the predom‐
inant insulin-regulated eukaryotic initiation factor 2B kinase in skeletal muscle. Int J
Biochem Cell Biol 31: 191-200. [12] Sacheck JM, Hyatt JP, Raffaello A, Jagoe RT, Roy RR, et al. References (2007) Rapid disuse and
denervation atrophy involve transcriptional changes similar to those of muscle wast‐
ing during systemic diseases. FASEB J 21: 140-155. [13] Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, et al. (2001) Identification of
ubiquitin ligases required for skeletal muscle atrophy. Science 294: 1704-1708. [14] Gomes MD, Lecker SH, Jagoe RT, Navon A, Goldberg AL (2001) Atrogin-1, a muscle-
specific F-box protein highly expressed during muscle atrophy. Proc Natl Acad Sci
USA 98: 14440-14445. [15] Hishiya A, Iemura S, Natsume T, Takayama S, Ikeda K, et al. (2006) A novel ubiqui‐
tin-binding protein ZNF216 functioning in muscle atrophy. EMBO J 25: 554-564. [16] Lokireddy S, Wijesoma IW, Teng S, Bonala S, Gluckman PD, et al. (2012) The ubiqui‐
tin ligase Mul1 induces mitophagy in skeletal muscle in response to muscle-wasting
stimuli. Cell Metab 16: 613-624. Muscle Cell and Tissue 188 [17] Cohen S, Zhai B, Gygi SP, Goldberg AL (2012) Ubiquitylation by Trim32 causes cou‐
pled loss of desmin, Z-bands, and thin filaments in muscle atrophy. J Cell Biol 198:
575-589. [18] Codogno P, Meijer AJ (2005) Autophagy and signaling: their role in cell survival and
cell death. Cell Death Differ 12 Suppl 2: 1509-1518. [19] Korolchuk VI, Mansilla A, Menzies FM, Rubinsztein DC (2009) Autophagy inhibition
compromises degradation of ubiquitin-proteasome pathway substrates. Mol Cell 33:
517-527. [20] Lamark T, Johansen T (2010) Autophagy: links with the proteasome. Curr Opin Cell
Biol 22: 192-198. [21] Sanchez AM, Bernardi H, Py G, Candau RB (2014) Autophagy is essential to support
skeletal muscle plasticity in response to endurance exercise. Am J Physiol Regul In‐
tegr Comp Physiol 307: R956-969. [22] Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, et al. (2004) The role of au‐
tophagy during the early neonatal starvation period. Nature 432: 1032-1036. [23] Tolkovsky AM (2009) Mitophagy. Biochim Biophys Acta 1793: 1508-1515. [24] Dunn WA, Jr., Cregg JM, Kiel JA, Van der Klei IJ, Oku M, et al. (2005) Pexophagy: the
selective autophagy of peroxisomes. Autophagy 1: 75-83. [25] Knodler LA, Celli J (2011) Eating the strangers within: host control of intracellular
bacteria via xenophagy. Cell Microbiol 13: 1319-1327. [26] Tasdemir E, Maiuri MC, Tajeddine N, Vitale I, Criollo A, et al. (2007) Cell cycle-de‐
pendent induction of autophagy, mitophagy and reticulophagy. Cell Cycle 6:
2263-2267. [27] MacIntosh GC, Bassham DC (2011) The connection between ribophagy, autophagy
and ribosomal RNA decay. References Autophagy 7: 662-663. [28] Marzella L, Ahlberg J, Glaumann H (1981) Autophagy, heterophagy, microautopha‐
gy and crinophagy as the means for intracellular degradation. Virchows Arch B Cell
Pathol Incl Mol Pathol 36: 219-234. [29] Weidberg H, Shvets E, Elazar Z (2009) Lipophagy: selective catabolism designed for
lipids. Dev Cell 16: 628-630. [30] Matsuda N, Sato S, Shiba K, Okatsu K, Saisho K, et al. (2010) PINK1 stabilized by mi‐
tochondrial depolarization recruits Parkin to damaged mitochondria and activates
latent Parkin for mitophagy. J Cell Biol 189: 211-221. [31] Vives-Bauza C, Zhou C, Huang Y, Cui M, De Vries RL, et al. (2010) PINK1-depend‐
ent recruitment of Parkin to mitochondria in mitophagy. Proc Natl Acad Sci USA
107: 378-383. Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease…
http://dx.doi.org/10.5772/60698
1 189 [32] Kondapalli C, Kazlauskaite A, Zhang N, Woodroof HI, Campbell DG, et al. (2012)
PINK1 is activated by mitochondrial membrane potential depolarization and stimu‐
lates Parkin E3 ligase activity by phosphorylating Serine 65. Open Biol 2: 120080. [33] Braschi E, Zunino R, McBride HM (2009) MAPL is a new mitochondrial SUMO E3
ligase that regulates mitochondrial fission. EMBO Rep 10: 748-754. [34] Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008) Autophagy fights disease
through cellular self-digestion. Nature 451: 1069-1075. [35] Bechet D, Tassa A, Taillandier D, Combaret L, Attaix D (2005) Lysosomal proteolysis
in skeletal muscle. Int J Biochem Cell Biol 37: 2098-2114. [36] Taillandier D, Aurousseau E, Meynial-Denis D, Bechet D, Ferrara M, et al. (1996) Co‐
ordinate activation of lysosomal, Ca 2+-activated and ATP-ubiquitin-dependent pro‐
teinases in the unweighted rat soleus muscle. Biochem J 316 (Pt 1): 65-72. [37] Bechet DM, Deval C, Robelin J, Ferrara MJ, Obled A (1996) Developmental control of
cathepsin B expression in bovine fetal muscles. Arch Biochem Biophys 334: 362-368. [38] Belkhou R, Bechet D, Cherel Y, Galluser M, Ferrara M, et al. (1994) Effect of fasting
and thyroidectomy on cysteine proteinase activities in liver and muscle. Biochim Bio‐
phys Acta 1199: 195-201. [39] Kominami E, Tsukahara T, Bando Y, Katunuma N (1985) Distribution of cathepsins B
and H in rat tissues and peripheral blood cells. J Biochem 98: 87-93. [40] Kirschke H, Wood L, Roisen FJ, Bird JW (1983) Activity of lysosomal cysteine protei‐
nase during differentiation of rat skeletal muscle. Biochem J 214: 871-877. References [41] Bechet DM, Ferrara MJ, Mordier SB, Roux MP, Deval CD, et al. (1991) Expression of
lysosomal cathepsin B during calf myoblast-myotube differentiation. Characteriza‐
tion of a cDNA encoding bovine cathepsin B. J Biol Chem 266: 14104-14112. [42] Ferrara M, Wojcik F, Rhaissi H, Mordier S, Roux MP, et al. (1990) Gene structure of
mouse cathepsin B. FEBS Lett 273: 195-199. [43] Gogos JA, Thompson R, Lowry W, Sloane BF, Weintraub H, et al. (1996) Gene trap‐
ping in differentiating cell lines: regulation of the lysosomal protease cathepsin B in
skeletal myoblast growth and fusion. J Cell Biol 134: 837-847. [44] Jane DT, DaSilva L, Koblinski J, Horwitz M, Sloane BF, et al. (2002) Evidence for the
involvement of cathepsin B in skeletal myoblast differentiation. J Cell Biochem 84:
520-531. [45] Mordier S, Bechet D, Roux MP, Obled A, Ferrara M (1993) Nucleotide sequence of
bovine preprocathepsin B. A study of polymorphism in the protein coding region. Bi‐
ochim Biophys Acta 1174: 305-311. Muscle Cell and Tissue 190 [46] Rhaissi H, Bechet D, Ferrara M (1993) Multiple leader sequences for mouse cathepsin
B mRNA? Biochimie 75: 899-904. [47] Jagoe RT, Lecker SH, Gomes M, Goldberg AL (2002) Patterns of gene expression in
atrophying skeletal muscles: response to food deprivation. FASEB J 16: 1697-1712. [48] Deval C, Mordier S, Obled C, Bechet D, Combaret L, et al. (2001) Identification of
cathepsin L as a differentially expressed message associated with skeletal muscle
wasting. Biochem J 360: 143-150. [49] Lecker SH, Jagoe RT, Gilbert A, Gomes M, Baracos V, et al. (2004) Multiple types of
skeletal muscle atrophy involve a common program of changes in gene expression. FASEB J 18: 39-51. [50] Whitaker JN, Bertorini TE, Mendell JR (1983) Immunocytochemical studies of cathe‐
psin D in human skeletal muscle. Ann Neurol 13: 133-142. [51] Li W, Yang Q, Mao Z (2011) Chaperone-mediated autophagy: machinery, regulation
and biological consequences. Cell Mol Life Sci 68: 749-763. [52] Kon M, Cuervo AM (2010) Chaperone-mediated autophagy in health and disease. FEBS Lett 584: 1399-1404. [53] Cuervo AM (2010) Chaperone-mediated autophagy: selectivity pays off. Trends En‐
docrinol Metab 21: 142-150. [54] Agarraberes FA, Dice JF (2001) A molecular chaperone complex at the lysosomal
membrane is required for protein translocation. J Cell Sci 114: 2491-2499. [55] Kaushik S, Cuervo AM (2009) Methods to monitor chaperone-mediated autophagy. Methods Enzymol 452: 297-324. [56] Kaushik S, Cuervo AM (2008) Chaperone-mediated autophagy. References Methods Mol Biol
445: 227-244. [57] Kaushik S, Massey AC, Mizushima N, Cuervo AM (2008) Constitutive activation of
chaperone-mediated autophagy in cells with impaired macroautophagy. Mol Biol
Cell 19: 2179-2192. [58] Wing SS, Chiang HL, Goldberg AL, Dice JF (1991) Proteins containing peptide se‐
quences related to Lys-Phe-Glu-Arg-Gln are selectively depleted in liver and heart,
but not skeletal muscle, of fasted rats. Biochem J 275 (Pt 1): 165-169. [59] Danon MJ, Oh SJ, DiMauro S, Manaligod JR, Eastwood A, et al. (1981) Lysosomal
glycogen storage disease with normal acid maltase. Neurology 31: 51-57. [60] Nishino I, Fu J, Tanji K, Yamada T, Shimojo S, et al. (2000) Primary LAMP-2 deficien‐
cy causes X-linked vacuolar cardiomyopathy and myopathy (Danon disease). Nature
406: 906-910. Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease…
http://dx.doi.org/10.5772/60698
1 191 [61] Yen WL, Klionsky DJ (2008) How to live long and prosper: autophagy, mitochondria,
and aging. Physiology (Bethesda) 23: 248-262. [62] Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y (2004) In vivo analy‐
sis of autophagy in response to nutrient starvation using transgenic mice expressing
a fluorescent autophagosome marker. Mol Biol Cell 15: 1101-1111. [63] Mammucari C, Milan G, Romanello V, Masiero E, Rudolf R, et al. (2007) FoxO3 con‐
trols autophagy in skeletal muscle in vivo. Cell Metab 6: 458-471. [64] Zhao J, Brault JJ, Schild A, Cao P, Sandri M, et al. (2007) FoxO3 coordinately activates
protein degradation by the autophagic/lysosomal and proteasomal pathways in atro‐
phying muscle cells. Cell Metab 6: 472-483. [65] Ganley IG, Lam du H, Wang J, Ding X, Chen S, et al. (2009) ULK1.ATG13.FIP200
complex mediates mTOR signaling and is essential for autophagy. J Biol Chem 284:
12297-12305. [66] Mizushima N (2010) The role of the Atg1/ULK1 complex in autophagy regulation. Curr Opin Cell Biol 22: 132-139. [67] Kihara A, Noda T, Ishihara N, Ohsumi Y (2001) Two distinct Vps34 phosphatidylino‐
sitol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in
Saccharomyces cerevisiae. J Cell Biol 152: 519-530. [68] Tassa A, Roux MP, Attaix D, Bechet DM (2003) Class III phosphoinositide 3-kinase--
Beclin1 complex mediates the amino acid-dependent regulation of autophagy in
C2C12 myotubes. Biochem J 376: 577-586. [69] Russell RC, Tian Y, Yuan H, Park HW, Chang YY, et al. (2013) ULK1 induces autoph‐
agy by phosphorylating Beclin-1 and activating VPS34 lipid kinase. Nat Cell Biol 15:
741-750. References [70] Jung CH, Jun CB, Ro SH, Kim YM, Otto NM, et al. (2009) ULK-Atg13-FIP200 com‐
plexes mediate mTOR signaling to the autophagy machinery. Mol Biol Cell 20:
1992-2003. [71] Jung CH, Ro SH, Cao J, Otto NM, Kim DH (2010) mTOR regulation of autophagy. FEBS Lett 584: 1287-1295. [72] Hosokawa N, Hara T, Kaizuka T, Kishi C, Takamura A, et al. (2009) Nutrient-de‐
pendent mTORC1 association with the ULK1-Atg13-FIP200 complex required for au‐
tophagy. Mol Biol Cell 20: 1981-1991. [73] Kim J, Kundu M, Viollet B, Guan KL (2011) AMPK and mTOR regulate autophagy
through direct phosphorylation of Ulk1. Nat Cell Biol 13: 132-141. [74] Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, et al. (2000) LC3, a mam‐
malian homologue of yeast Apg8p, is localized in autophagosome membranes after
processing. EMBO J 19: 5720-5728. Muscle Cell and Tissue 192 [75] Tanida I, Ueno T, Kominami E (2008) LC3 and Autophagy. Methods Mol Biol 445:
77-88. [76] Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, et al. (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8: 445-544. [77] Bjorkoy G, Lamark T, Brech A, Outzen H, Perander M, et al. (2005) p62/SQSTM1
forms protein aggregates degraded by autophagy and has a protective effect on hun‐
tingtin-induced cell death. J Cell Biol 171: 603-614. [78] Walczak M, Martens S (2013) Dissecting the role of the Atg12-Atg5-Atg16 complex
during autophagosome formation. Autophagy 9: 424-425. [79] Mizushima N (2005) The pleiotropic role of autophagy: from protein metabolism to
bactericide. Cell Death Differ 12 Suppl 2: 1535-1541. [80] Komatsu M, Ueno T, Waguri S, Uchiyama Y, Kominami E, et al. (2007) Constitutive
autophagy: vital role in clearance of unfavorable proteins in neurons. Cell Death Dif‐
fer 14: 887-894. [81] Vainshtein A, Grumati P, Sandri M, Bonaldo P (2014) Skeletal muscle, autophagy,
and physical activity: the menage a trois of metabolic regulation in health and dis‐
ease. J Mol Med (Berl) 92: 127-137. [82] Cadwell K, Liu JY, Brown SL, Miyoshi H, Loh J, et al. (2008) A key role for autophagy
and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells. Nature
456: 259-263. [83] Ebato C, Uchida T, Arakawa M, Komatsu M, Ueno T, et al. (2008) Autophagy is im‐
portant in islet homeostasis and compensatory increase of beta cell mass in response
to high-fat diet. Cell Metab 8: 325-332. References [84] Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, et al. (2006) Suppression
of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature
441: 885-889. [85] Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, et al. (2005) Impairment of starva‐
tion-induced and constitutive autophagy in Atg7-deficient mice. J Cell Biol 169:
425-434. [86] Masiero E, Agatea L, Mammucari C, Blaauw B, Loro E, et al. (2009) Autophagy is re‐
quired to maintain muscle mass. Cell Metab 10: 507-515. [87] Raben N, Hill V, Shea L, Takikita S, Baum R, et al. (2008) Suppression of autophagy
in skeletal muscle uncovers the accumulation of ubiquitinated proteins and their po‐
tential role in muscle damage in Pompe disease. Hum Mol Genet 17: 3897-3908. [88] Singh R, Xiang Y, Wang Y, Baikati K, Cuervo AM, et al. (2009) Autophagy regulates
adipose mass and differentiation in mice. J Clin Invest 119: 3329-3339. Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease…
http://dx.doi.org/10.5772/60698 193 [89] Mehrpour M, Esclatine A, Beau I, Codogno P (2010) Autophagy in health and dis‐
ease. 1. Regulation and significance of autophagy: an overview. Am J Physiol Cell
Physiol 298: C776-C785. [90] Cuervo AM, Bergamini E, Brunk UT, Droge W, Ffrench M, et al. (2005) Autophagy
and aging: the importance of maintaining "clean" cells. Autophagy 1: 131-140. [91] Harrison DE, Strong R, Sharp ZD, Nelson JF, Astle CM, et al. (2009) Rapamycin fed
late in life extends lifespan in genetically heterogeneous mice. Nature 460: 392-395. [92] Hashimoto Y, Ookuma S, Nishida E (2009) Lifespan extension by suppression of au‐
tophagy genes in Caenorhabditis elegans. Genes Cells 14: 717-726. [93] Boya P, Gonzalez-Polo RA, Casares N, Perfettini JL, Dessen P, et al. (2005) Inhibition
of macroautophagy triggers apoptosis. Mol Cell Biol 25: 1025-1040. [94] Shintani T, Klionsky DJ (2004) Autophagy in health and disease: a double-edged
sword. Science 306: 990-995. [95] Levine B, Kroemer G (2008) Autophagy in the pathogenesis of disease. Cell 132:
27-42. [96] Tzelepis F, Verway M, Daoud J, Gillard J, Hassani-Ardakani K, et al. (2015) Annexin1
regulates DC efferocytosis and cross-presentation during Mycobacterium tuberculo‐
sis infection. J Clin Invest 125: 752-768. [97] Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, et al. (1999) Induction of au‐
tophagy and inhibition of tumorigenesis by beclin 1. Nature 402: 672-676. References [98] Gozuacik D, Kimchi A (2004) Autophagy as a cell death and tumor suppressor mech‐
anism. Oncogene 23: 2891-2906. [99] Ogier-Denis E, Codogno P (2003) Autophagy: a barrier or an adaptive response to
cancer. Biochim Biophys Acta 1603: 113-128. [100] Cuervo AM (2004) Autophagy: in sickness and in health. Trends Cell Biol 14: 70-77. [101] Fukuda T, Ahearn M, Roberts A, Mattaliano RJ, Zaal K, et al. (2006) Autophagy and
mistargeting of therapeutic enzyme in skeletal muscle in Pompe disease. Mol Ther
14: 831-839. [102] Fukuda T, Roberts A, Ahearn M, Zaal K, Ralston E, et al. (2006) Autophagy and lyso‐
somes in Pompe disease. Autophagy 2: 318-320. [103] Cheung ZH, Ip NY (2009) The emerging role of autophagy in Parkinson's disease. Mol Brain 2: 29. [104] Williams A, Sarkar S, Cuddon P, Ttofi EK, Saiki S, et al. (2008) Novel targets for Hun‐
tington's disease in an mTOR-independent autophagy pathway. Nat Chem Biol 4:
295-305. Muscle Cell and Tissue 194 [105] Moreira PI, Santos RX, Zhu X, Lee HG, Smith MA, et al. (2010) Autophagy in Alz‐
heimer's disease. Expert Rev Neurother 10: 1209-1218. [106] Nilsson P, Sekiguchi M, Akagi T, Izumi S, Komori T, et al. (2015) Autophagy-related
protein 7 deficiency in amyloid beta (Abeta) precursor protein transgenic mice de‐
creases abeta in the multivesicular bodies and induces abeta accumulation in the gol‐
gi. Am J Pathol 185: 305-313. [107] Fortun J, Dunn WA, Jr., Joy S, Li J, Notterpek L (2003) Emerging role for autophagy
in the removal of aggresomes in Schwann cells. J Neurosci 23: 10672-10680. [108] Sandri M (2010) Autophagy in skeletal muscle. FEBS Lett 584: 1411-1416. [109] Shea L, Raben N (2009) Autophagy in skeletal muscle: implications for Pompe dis‐
ease. Int J Clin Pharmacol Ther 47 Suppl 1: S42-S47. [110] Masiero E, Sandri M (2010) Autophagy inhibition induces atrophy and myopathy in
adult skeletal muscles. Autophagy 6: 307-309. [111] Sandri M (2010) Autophagy in health and disease. 3. Involvement of autophagy in
muscle atrophy. Am J Physiol Cell Physiol 298: C1291-C1297. [112] Dobrowolny G, Aucello M, Rizzuto E, Beccafico S, Mammucari C, et al. (2008) Skele‐
tal muscle is a primary target of SOD1G93A-mediated toxicity. Cell Metab 8: 425-436. [113] Pietri-Rouxel F, Gentil C, Vassilopoulos S, Baas D, Mouisel E, et al. (2010) DHPR al‐
pha1S subunit controls skeletal muscle mass and morphogenesis. EMBO J 29:
643-654. References [114] Mofarrahi M, Sigala I, Guo Y, Godin R, Davis EC, et al. (2012) Autophagy and skele‐
tal muscles in sepsis. PLoS One 7: e47265. [115] Rahman M, Mofarrahi M, Kristof AS, Nkengfac B, Harel S, et al. (2014) Reactive oxy‐
gen species regulation of autophagy in skeletal muscles. Antioxid Redox Signal 20:
443-459. [116] Qi Z, He Q, Ji L, Ding S (2014) Antioxidant supplement inhibits skeletal muscle con‐
stitutive autophagy rather than fasting-induced autophagy in mice. Oxid Med Cell
Longev 2014: 315896. [117] Marzetti E, Lees HA, Wohlgemuth SE, Leeuwenburgh C (2009) Sarcopenia of aging:
underlying cellular mechanisms and protection by calorie restriction. Biofactors 35:
28-35. [118] Wenz T, Rossi SG, Rotundo RL, Spiegelman BM, Moraes CT (2009) Increased muscle
PGC-1alpha expression protects from sarcopenia and metabolic disease during ag‐
ing. Proc Natl Acad Sci USA 106: 20405-20410. [119] Jorgensen SB, Viollet B, Andreelli F, Frosig C, Birk JB, et al. (2004) Knockout of the
alpha2 but not alpha1 5'-AMP-activated protein kinase isoform abolishes 5-aminoi‐ Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease…
http://dx.doi.org/10.5772/60698
1 195 midazole-4-carboxamide-1-beta-4-ribofuranosidebut not contraction-induced glucose
uptake in skeletal muscle. J Biol Chem 279: 1070-1079. midazole-4-carboxamide-1-beta-4-ribofuranosidebut not contraction-induced glucose
uptake in skeletal muscle. J Biol Chem 279: 1070-1079. [120] Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, et al. (2003) Complexes between
the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream
kinases in the AMP-activated protein kinase cascade. J Biol 2: 28. [121] Sanchez AM, Candau RB, Csibi A, Pagano AF, Raibon A, et al. (2012) The role of
AMP-activated protein kinase in the coordination of skeletal muscle turnover and en‐
ergy homeostasis. Am J Physiol Cell Physiol 303: C475-C485. [122] Marsin AS, Bertrand L, Rider MH, Deprez J, Beauloye C, et al. (2000) Phosphoryla‐
tion and activation of heart PFK-2 by AMPK has a role in the stimulation of glycoly‐
sis during ischaemia. Curr Biol 10: 1247-1255. [123] Merrill GF, Kurth EJ, Hardie DG, Winder WW (1997) AICA riboside increases AMP-
activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle. Am J
Physiol 273: E1107-1112. [124] Vavvas D, Apazidis A, Saha AK, Gamble J, Patel A, et al. (1997) Contraction-induced
changes in acetyl-CoA carboxylase and 5'-AMP-activated kinase in skeletal muscle. J
Biol Chem 272: 13255-13261. References [125] Winder WW, Hardie DG (1996) Inactivation of acetyl-CoA carboxylase and activa‐
tion of AMP-activated protein kinase in muscle during exercise. Am J Physiol 270:
E299-E304. [126] Winder WW, Wilson HA, Hardie DG, Rasmussen BB, Hutber CA, et al. (1997) Phos‐
phorylation of rat muscle acetyl-CoA carboxylase by AMP-activated protein kinase
and protein kinase A. J Appl Physiol (1985) 82: 219-225. [127] Aschenbach WG, Hirshman MF, Fujii N, Sakamoto K, Howlett KF, et al. (2002) Effect
of AICAR treatment on glycogen metabolism in skeletal muscle. Diabetes 51:
567-573. [128] Carling D, Hardie DG (1989)The substrate and sequence specificity of the AMP-acti‐
vated protein kinase. Phosphorylation of glycogen synthase and phosphorylase kin‐
ase. Biochim Biophys Acta 1012: 81-86. [129] Carling D, Clarke PR, Zammit VA, Hardie DG (1989) Purification and characteriza‐
tion of the AMP-activated protein kinase. Copurification of acetyl-CoA carboxylase
kinase and 3-hydroxy-3-methylglutaryl-CoA reductase kinase activities. Eur J Bio‐
chem 186: 129-136. [130] Carling D, Zammit VA, Hardie DG (1987) A common bicyclic protein kinase cascade
inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis. FEBS
Lett 223: 217-222. Muscle Cell and Tissue 196 [131] Garton AJ, Campbell DG, Carling D, Hardie DG, Colbran RJ, et al. (1989) Phosphory‐
lation of bovine hormone-sensitive lipase by the AMP-activated protein kinase. A
possible antilipolytic mechanism. Eur J Biochem 179: 249-254. [132] Lee WJ, Kim M, Park HS, Kim HS, Jeon MJ, et al. (2006) AMPK activation increases
fatty acid oxidation in skeletal muscle by activating PPARalpha and PGC-1. Biochem
Biophys Res Commun 340: 291-295. [133] Liang H, Ward WF (2006) PGC-1alpha: a key regulator of energy metabolism. Adv
Physiol Educ 30: 145-151. [134] Derave W, Ai H, Ihlemann J, Witters LA, Kristiansen S, et al. (2000) Dissociation of
AMP-activated protein kinase activation and glucose transport in contracting slow-
twitch muscle. Diabetes 49: 1281-1287. [135] Inoki K, Kim J, Guan KL (2012) AMPK and mTOR in cellular energy homeostasis
and drug targets. Annu Rev Pharmacol Toxicol 52: 381-400. [136] Mounier R, Lantier L, Leclerc J, Sotiropoulos A, Pende M, et al. (2009) Important role
for AMPKalpha1 in limiting skeletal muscle cell hypertrophy. FASEB J 23: 2264-2273. [137] Lantier L, Mounier R, Leclerc J, Pende M, Foretz M, et al. (2010) Coordinated mainte‐
nance of muscle cell size control by AMP-activated protein kinase. FASEB J 24:
3555-3561. References [138] Nakashima K, Yakabe Y (2007) AMPK activation stimulates myofibrillar protein deg‐
radation and expression of atrophy-related ubiquitin ligases by increasing FOXO
transcription factors in C2C12 myotubes. Biosci Biotechnol Biochem 71: 1650-1656. [139] Sanchez AM, Csibi A, Raibon A, Cornille K, Gay S, et al. (2012) AMPK promotes
skeletal muscle autophagy through activation of forkhead FoxO3a and interaction
with Ulk1. J Cell Biochem 113: 695-710. [140] Sanchez AM, Candau RB, Bernardi H (2014) FoxO transcription factors: their roles in
the maintenance of skeletal muscle homeostasis. Cell Mol Life Sci 71: 1657-1671. [141] Slopack D, Roudier E, Liu ST, Nwadozi E, Birot O, et al. (2014) Forkhead BoxO tran‐
scription factors restrain exercise-induced angiogenesis. J Physiol 592: 4069-4082. [142] Sanchez AM (2015) FoxO transcription factors and endurance training: a role for
FoxO1 and FoxO3 in exercise-induced angiogenesis. J Physiol 593: 363-364. [143] Greer EL, Oskoui PR, Banko MR, Maniar JM, Gygi MP, et al. (2007) The energy sen‐
sor AMP-activated protein kinase directly regulates the mammalian FOXO3 tran‐
scription factor. J Biol Chem 282: 30107-30119. [144] Juhasz G, Puskas LG, Komonyi O, Erdi B, Maroy P, et al. (2007) Gene expression
profiling identifies FKBP39 as an inhibitor of autophagy in larval Drosophila fat
body. Cell Death Differ 14: 1181-1190. Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease…
http://dx.doi.org/10.5772/60698
1 197 [145] Sengupta A, Molkentin JD, Yutzey KE (2009) FoxO transcription factors promote au‐
tophagy in cardiomyocytes. J Biol Chem 284: 28319-28331. [146] Liu HY, Han J, Cao SY, Hong T, Zhuo D, et al. (2009) Hepatic autophagy is sup‐
pressed in the presence of insulin resistance and hyperinsulinemia: inhibition of
FoxO1-dependent expression of key autophagy genes by insulin. J Biol Chem 284:
31484-31492. [147] Chiacchiera F, Matrone A, Ferrari E, Ingravallo G, Lo Sasso G, et al. (2009) p38alpha
blockade inhibits colorectal cancer growth in vivo by inducing a switch from HIF1al‐
pha- to FoxO-dependent transcription. Cell Death Differ 16: 1203-1214. [148] Tong JF, Yan X, Zhu MJ, Du M (2009) AMP-activated protein kinase enhances the ex‐
pression of muscle-specific ubiquitin ligases despite its activation of IGF-1/Akt sig‐
naling in C2C12 myotubes. J Cell Biochem 108: 458-468. [149] Hara T, Mizushima N (2009) Role of ULK-FIP200 complex in mammalian autophagy:
FIP200, a counterpart of yeast Atg17? Autophagy 5: 85-87. References [150] Behrends C, Sowa ME, Gygi SP, Harper JW (2010) Network organization of the hu‐
man autophagy system. Nature 466: 68-76. [151] Lee JW, Park S, Takahashi Y, Wang HG (2010) The association of AMPK with ULK1
regulates autophagy. PLoS One 5: e15394. [152] Dorsey FC, Rose KL, Coenen S, Prater SM, Cavett V, et al. (2009) Mapping the phos‐
phorylation sites of Ulk1. J Proteome Res 8: 5253-5263. [153] Egan DF, Shackelford DB, Mihaylova MM, Gelino S, Kohnz RA, et al. (2011) Phos‐
phorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy
sensing to mitophagy. Science 331: 456-461. [154] Shang L, Chen S, Du F, Li S, Zhao L, et al. (2011) Nutrient starvation elicits an acute
autophagic response mediated by Ulk1 dephosphorylation and its subsequent disso‐
ciation from AMPK. Proc Natl Acad Sci USA 108: 4788-4793. [155] Loffler AS, Alers S, Dieterle AM, Keppeler H, Franz-Wachtel M, et al. (2011) Ulk1-
mediated phosphorylation of AMPK constitutes a negative regulatory feedback loop. Autophagy 7: 696-706. [156] Salminen A, Vihko V (1984) Autophagic response to strenuous exercise in mouse
skeletal muscle fibers. Virchows Arch B Cell Pathol Incl Mol Pathol 45: 97-106. [157] Grumati P, Coletto L, Schiavinato A, Castagnaro S, Bertaggia E, et al. (2011) Physical
exercise stimulates autophagy in normal skeletal muscles but is detrimental for colla‐
gen VI-deficient muscles. Autophagy 7: 1415-1423. [158] He C, Bassik MC, Moresi V, Sun K, Wei Y, et al. (2012) Exercise-induced BCL2-regu‐
lated autophagy is required for muscle glucose homeostasis. Nature 481: 511-515. Muscle Cell and Tissue 198 [159] Jamart C, Benoit N, Raymackers JM, Kim HJ, Kim CK, et al. (2012) Autophagy-relat‐
ed and autophagy-regulatory genes are induced in human muscle after ultraendur‐
ance exercise. Eur J Appl Physiol 112: 3173-3177. [160] Jamart C, Francaux M, Millet GY, Deldicque L, Frere D, et al. (2012) Modulation of
autophagy and ubiquitin-proteasome pathways during ultra-endurance running. J
Appl Physiol. [161] Pagano AF, Py G, Bernardi H, Candau RB, Sanchez AM (2014) Autophagy and pro‐
tein turnover signaling in slow-twitch muscle during exercise. Med Sci Sports Exerc
46: 1314-1325. [162] Kim HJ, Jamart C, Deldicque L, An GL, Lee YH, et al. (2011) Endoplasmic reticulum
stress markers and ubiquitin-proteasome pathway activity in response to a 200-km
run. Med Sci Sports Exerc 43: 18-25. References [163] Jamart C, Naslain D, Gilson H, Francaux M (2013) Higher activation of autophagy in
skeletal muscle of mice during endurance exercise in the fasted state. Am J Physiol
Endocrinol Metab 305: E964-E974. [164] LoVerso F, Carnio S, Vainshtein A, Sandri M (2014) Autophagy is not required to
sustain exercise and PRKAA1/AMPK activity but is important to prevent mitochon‐
drial damage during physical activity. Autophagy 10. [165] Lira VA, Okutsu M, Zhang M, Greene NP, Laker RC, et al. (2013) Autophagy is re‐
quired for exercise training-induced skeletal muscle adaptation and improvement of
physical performance. FASEB J 27: 4184-4193. [166] Fry CS, Drummond MJ, Glynn EL, Dickinson JM, Gundermann DM, et al. (2013)
Skeletal muscle autophagy and protein breakdown following resistance exercise are
similar in younger and older adults. J Gerontol A Biol Sci Med Sci 68: 599-607. [167] Glynn EL, Fry CS, Drummond MJ, Dreyer HC, Dhanani S, et al. (2010) Muscle pro‐
tein breakdown has a minor role in the protein anabolic response to essential amino
acid and carbohydrate intake following resistance exercise. Am J Physiol Regul In‐
tegr Comp Physiol 299: R533-R540. [168] Kim YA, Kim YS, Oh SL, Kim HJ, Song W (2013) Autophagic response to exercise
training in skeletal muscle with age. J Physiol Biochem 69: 697-705. [169] Luo L, Lu AM, Wang Y, Hong A, Chen Y, et al. (2013) Chronic resistance training ac‐
tivates autophagy and reduces apoptosis of muscle cells by modulating IGF-1 and its
receptors, Akt/mTOR and Akt/FOXO3a signaling in aged rats. Exp Gerontol 48:
427-436. [170] Chaillou T, Koulmann N, Simler N, Meunier A, Serrurier B, et al. (2012) Hypoxia
transiently affects skeletal muscle hypertrophy in a functional overload model. Am J
Physiol Regul Integr Comp Physiol 302: R643-654. Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease…
http://dx.doi.org/10.5772/60698
1 199 [171] Masschelein E, Van Thienen R, D'Hulst G, Hespel P, Thomis M, et al. (2014) Acute
environmental hypoxia induces LC3 lipidation in a genotype-dependent manner. FASEB J 28: 1022-1034. [172] D'Hulst G, Jamart C, Van Thienen R, Hespel P, Francaux M, et al. (2013) Effect of
acute environmental hypoxia on protein metabolism in human skeletal muscle. Acta
Physiol (Oxf) 208: 251-264. [173] Levett DZ, Vigano A, Capitanio D, Vasso M, De Palma S, et al. (2015) Changes in
muscle proteomics in the course of the Caudwell Research Expedition to Mt. Everest. References Proteomics 15: 160-171. [174] Ferretti G (2003) Limiting factors to oxygen transport on Mount Everest 30 years af‐
ter: a critique of Paolo Cerretelli's contribution to the study of altitude physiology. Eur J Appl Physiol 90: 344-350. [175] Howald H, Hoppeler H (2003) Performing at extreme altitude: muscle cellular and
subcellular adaptations. Eur J Appl Physiol 90: 360-364. [176] Zhang H, Bosch-Marce M, Shimoda LA, Tan YS, Baek JH, et al. (2008) Mitochondrial
autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia. J Biol
Chem 283: 10892-10903.
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A System for Converting and Recovering Texts Managed as Structured Information
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Scientific reports
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A System for Converting
and Recovering Texts Managed
as Structured Information
OPEN Edgardo Samuel Barraza Verdesoto 1,3,4*, Marlly Yaneth Rojas Ortiz 3 &
Richard de Jesus Gil Herrera 2 Edgardo Samuel Barraza Verdesoto 1,3,4*, Marlly Yaneth Rojas Ortiz 3 &
Richard de Jesus Gil Herrera 2 This paper introduces a system that incorporates several strategies based on scientific models of how
the brain records and recovers memories. Methodologically, an incremental prototyping approach
has been applied to develop a satisfactory architecture that can be adapted to any language. A special
case is studied and tested regarding the Spanish language. The applications of this proposal are vast
because, in general, information such as text way, reports, emails, and web content, among others,
is considered unstructured and, hence, the repositories based on SQL databases usually do not
handle this kind of data correctly and efficiently. The conversion of unstructured textual information
to structured one can be useful in contexts such as Natural Language Generation, Data Mining, and
dynamic generation of theories, among others. Written communication is a type of information that has a basic structure well defined in each language which
is useful in information processing1. Additionally, some other types of communication can be totally or partially
converted into text whereupon the final processing is carried out2,3. Written communication is a type of information that has a basic structure well defined in each language which
is useful in information processing1. Additionally, some other types of communication can be totally or partially
converted into text whereupon the final processing is carried out2,3. i
Several applications have been developed in information processing following principles or attributes of the
text, for instance, to build ontologies or micro-theories4–7 which are convenient for automatic decision-making
tasks8. Furthermore, In process automation, the speed at which the information is produced reduces human
performance and delays the decision-making processes, this has generated the urgent need to delegate some
decisions to machines and create applications for resolving these problems9–11.h pp
g
p
The information retrieval and the generation of natural language promote the generation of sentences/phrases
with meaning from a large amount of data generated by interactivity, shared repositories, and homogeneous or
heterogeneous data sources, one of these types of application is presented in12 where is exposed a framework
able to generate three classes of question and answers from corpora: fill in the gaps, multiple choice, and shuffled
sentences. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports 1Universidad Americana de Europa (UNADE), Cancún, México. 2Universidad Internacional de la Rioja, Logroño,
Spain. 3Research Department, Tecnológica Autónoma de Bogotá (FABA), Bogotá, Colombia. 4Universidad de
Santander (UDES), Bogotá, Colombia. *email: edgardo.barraza@correo.faba.edu.co Scientific Reports | (2022) 12:22249 Conceptual framework p
Object‑action dissociation/integration. These studies and approaches suggest that the information
held in the brain is a set of clusters (cores) that could be affected by the ambiguity and the context in both, the
dissociation and integration. Likewise, according to some theories, the brain saved our memories in two ways:
semantically and the episodic way13,14, this latter manner is very important to explain the development of the
strategy followed in this paper. Historically, the dissociation of the information by the human brain was observed when comparing Broca’s
aphasic agrammatical patients, whose speech involves the use of very few verbs in contrast with other anomic
patients that had great difficulty finding concrete nouns15. Initially, the major difficulty with verbs for Broca’s
patients was interpreted based on the highest syntactical complexity of verbs compared to nouns16–18. However,
the idea that verbs are, in general, harder to produce has been undermined in other studies where it is indicated
that patients with anomic difficulties produce verbs more easily than nouns19,20. From a neurophysiological point
of view, there are different opinions and theoretical proposals21, of which three hypotheses have been put for-
ward regarding verb-noun storage issues within neural networks: partial separation of verbs and nouns22,23, word
separation based on morphosyntax24, and separation between actions and objects25,26. Psycholinguistics also agree
that exist, in the brain, the distinction between various grammatical categories, particularly between verbs and
nouns, and propose three possible starting points or context to access the information: availability of information
related to the grammatical class, a required grammatical knowledge, and the independence between the definition
of grammatical class and the semantic differences27–30. A considerable number of studies have dealt with aspects
associated with the dissociation of the information within the human mind and the conclusions are similar31,
there are a dissociation between verbs(actions) and nouns (objects).h j
The counterpart of the dissociation process is the integration process. According to32, grammatical informa-
tion is relevant to understanding and producing sentences, but a plausible conclusion suggests that the gram-
matical class information is not a lexical property that can be retrieved automatically; instead, this property is
likely to play an important role in the context of a sentence. Fundamentally, the role of the grammatical class in
sentence processing is modulated by the linguistic differences regarding the way as words of certain grammati-
cal classes are used within sentences. A System for Converting
and Recovering Texts Managed
as Structured Information
OPEN The framework aims to create a pedagogical tool able to automatically generate tests in the context of
a topic, the parser divides texts according to the processed language and prepares the type of question selected. These approaches that allow recording and recovering of fragments or whole texts from a repository, conceiv-
ing and improving strategies applied in recovering unstructured information are very important in the current
Computer Science.h This article introduces an architecture that allows building applications capable of dissociating texts/sentences
in subsets of cores with properties and simple operations such as those that the algebraic groups incorporate. These operations are preferable because they have properties that promote a straightforward and reliable manner
to retrieve a whole text or part of it by keeping the structure of the language. Additionally, relationships between
subsets are incorporated because they play an essential role in maintaining the meaning of the recovered text/
sentences.h The main objective of this manuscript is to show the design of a system with the capability of processing
sentences (part of the text), storing them in databases, and finally, recovering them while keeping the original
text’s basic meaning. To reach that, it is reviewing some previous concepts and experiences about linguistic
computational to support the architectural design; it is described and justified how the algebraic groups help in
the organization of the components of sentences for storage and recovering them while keeping the meaning and
structure; Also, it is treated how to design an architecture for a processing system the objects/data as structured
information (into structured databases), and finally, it is shown the functionality of a system for some illustrative
instances and test cases for the Spanish language. 1Universidad Americana de Europa (UNADE), Cancún, México. 2Universidad Internacional de la Rioja, Logroño,
Spain. 3Research Department, Tecnológica Autónoma de Bogotá (FABA), Bogotá, Colombia. 4Universidad de
Santander (UDES), Bogotá, Colombia. *email: edgardo.barraza@correo.faba.edu.co | https://doi.org/10.1038/s41598-022-26304-w www.nature.com/scientificreports/ This paper is organized as follows. Firstly, a theoretical framework under which the proposal is based will be
explored. Secondly, a general architecture will be proposed that incorporates each one of the elements exposed
in the theoretical framework. Thirdly, an approach, based on the architecture presented, applied to the Spanish
Language will be analyzed. Finally, the findings and future works will be exhibited. Conceptual framework In all languages, verbs commonly require higher processing than nouns at
various levels, firstly, because the processing of verbs is about events and could exist many elements that will have
to be integrated. Secondly, the verb syntax also demands more processing because verbs should be connected
to other words to convey their meaning. Lastly, nouns are linked to objects, but they might refer to events too,
and it is necessary its disambiguation. In conclusion, the effects of the grammatical class in the retrieval and
representation of simple words are more productive when the context is present33–35. In addition, Neuroscience states that there are two kind of memory for storing and remembering facts and
events consciously; such events are stored in the episodic memory such as a storyteller whereas the semantic
memory records the same event as part of our overall knowledge (dictionary). In specific, the episodic memory
is intended as a repository in our brain where is recorded an event similar to a text well-written13,14. p
y
In summary, there are two processes well conceived in our brain, dissociation and integration of an event
These tasks are the fundamentals of the proposal in this paper. From sentences to clusters of words. The word classification has been a normal practice in linguistics,
computer sciences, and education, among others (see Fig. 1); this practice normally has different targets and
results. Furthermore, as an instance, ConceptNet is a project based on the sense common concept that was
conceived as a semantic network containing lots of things that the computers should know about the world36–38. Another example is the WordNet project which resembles a thesaurus in that words are grouped based on their
meanings, the result is a network that can be browseable easily39–42.i g
y
A text is more complex than simple words, it is a texture that relates, firstly, words to create sentences, sec-
ondly paragraphs, and ignoring other structures, finally several paragraphs directly or indirectly (e.g. using the
anaphor) linked between one another result in a text. Each language has rules to build sentences and paragraphs. According to43, there are various ways to classify and describe the languages, but a very common is the order of
each one of their main components (Subject, Object, and Verb) in the sentence: • Subject-Object-Verb (SOV). This is the most frequent type of word order in spoken languages. • Subject-Verb-Object (SVO). Conceptual framework It is a relevant type of word order because of its speakers worldwide. • Verb-Subject-Object (VSO): It represents a relatively small set of languages. • Verb-Object-Subject (VOS): Very few languages use this kind of order. • Subject-Object-Verb (SOV). This is the most frequent type of word order in spoken languages. j
j
(
)h
q
yp
p
g
g
Subject-Verb-Object (SVO). It is a relevant type of word order because of its speakers worldwide. j
j
p
y
g
g
• Verb-Object-Subject (VOS): Very few languages use this kind of order. Some approaches use such classifications to divide sentences, expressions, paragraphs, and texts, and, ultimately,
to generate categories that are used in specific applications44,45. Additionally, other applications use these charac-
teristics in a reverse way, for instance, to build sentences and paragraphs, or concatenate textual expressions from
the same or different sources for generating new expressions; this is being applied in Human Machine Interfaces
(HMI) development46. On the other hand, a text not only has nouns and verbs, else other types of words with
different purposes, e.g., emphasizing words, which to join small sentences to produce effects like generalization
or itemization, etc. These words play an important role to decide how the relations between words, sentences, https://doi.org/10.1038/s41598-022-26304-w Scientific Reports | (2022) 12:22249 | www.nature.com/scientificreports/ Figure 1. Some word classifications techniques. Source: Extracted and adapted from37,42. Figure 1. Some word classifications techniques. Source: Extracted and adapted from37,42. and paragraphs are. They can be linked to verbs or nouns, e.g. the determinants which comply with the function
of generalization or quantification of nouns47.h and paragraphs are. They can be linked to verbs or nouns, e.g. the determinants which comply with the function
of generalization or quantification of nouns47.h i
In48 was analyzed the preliminary results focused on the dissociation of sentences in clusters. The sentences
studied were in the Spanish Language. Section 2 of this reference exposes why it is necessary to migrate from
String-set dependence to another algebraic structure for modeling a sentence, and why this algebraic structure
must be an Abelian group, it also supplied the proof. In summary, the dissociation between verbs and nouns,
mainly, is a convenient strategy to generate new sentences, also, it is important to create an adequate environ-
ment for it. Algebraic environment. Conceptual framework Modern Algebra is a discipline that deals with the properties of the sets and their
elements, and the operations that can be executed within them. Modern algebra classifies the sets as semigroups, https://doi.org/10.1038/s41598-022-26304-w Scientific Reports | (2022) 12:22249 | www.nature.com/scientificreports/ Figure 2. Dissociation of a sentence. Source: Own elaboration. Figure 2. Dissociation of a sentence. Source: Own elaboration. monoids, groups, rings, and fields; all of these are named algebraic structures. These classifications depend on
the number and type of properties that the operation fulfills. i
If the elements of sentences are treated like components of an algebraic set, then such components could be
used to build phrases and new sentences easily by applying an operation that complies with certain properties. This section shows that converting the conventional algebraic structure of the set of strings (sentences) to a
structure more adequate allows for reaching this purpose.i q
g
p
p
A class very important for this approach is the groups, specifically, the Abelian groups49 these last ones have
significant properties that guarantee that by operating elements of a dissociated sentence, the original sentence
can be rebuilt; A key property is to be commutative because it allows that the result of an operation among ele-
ments will be the same, although the operands change their place in the operation. g
p
g
p
p
A sentence could be treated as an ordered set of strings which implies an algebraic structure very simple, but
this structure does not is adequate because each string in the sentence complies with a function depending on
its position in it, if the sentence is dissociated and then it is reassembled, this last process must guarantee that
the product is at least coherent with the structure of the language. p
g
g
A sentence could be treated as an ordered set of strings which implies an algebraic structure very simple, it
is ordered because each string in the sentence complies with a function depending on its position in it, if the
sentence is dissociated into strings and later is required its reassembling, this last process must guarantee that
the final sentence keeps the structure of the language and its meaning. These conditions comply if the set gener-
ated in the dissociation has associated an operation with certain properties which will be shown in this section. Conceptual framework Components
Description
[v0 : v1 : · · · : vk], [n0 : n1 : · · · : nk] or X
Components: Each component is a vector of vectors. First-internal-vector only contains
verbal clusters (v-elements). Second-internal-vector only nominal clusters (n-elements). X generalizes the internal vectors. k
−element : Represents a null string. The subscript is the position in the internal vector. [0/∞], [0/∞
Ŵ −element A component with only −elements in all internal vectors. Figure 3. Dissociation of a Spanish sentence. Mapping from Pvn to Ovn . Source: Own elaboration. Table 1. Set Ovn and description of its components. Source: Own elaboration. Components
Description
[v0 : v1 : · · · : vk], [n0 : n1 : · · · : nk] or X
Components: Each component is a vector of vectors. First-internal-vector only contains
verbal clusters (v-elements). Second-internal-vector only nominal clusters (n-elements). X generalizes the internal vectors. k
−element : Represents a null string. The subscript is the position in the internal vector. [0/∞], [0/∞
Ŵ −element A component with only −elements in all internal vectors. n1 : · · · : nk] or X
Components: Each component is a vector of vectors. First-internal-vector only contains
verbal clusters (v-elements). Second-internal-vector only nominal clusters (n-elements). X generalizes the internal vectors. Table 1. Set Ovn and description of its components. Source: Own elaboration. able 1. Set Ovn and description of its components. Source: Own elaboration. Figure 3. Dissociation of a Spanish sentence. Mapping from Pvn to Ovn . Source: Own elaboration. Figure 3. Dissociation of a Spanish sentence. Mapping from Pvn to Ovn . Source: Own elaboration. E0 + E3 =null + “Fred′′ +′′ quiere ir a′′ + “Hong Kong′′ =“Fred quiere ir a Hong Kong′′(correct) E2 + E4 =null + “sitios turisticos′′ +′′ quiere ir′′ +′′ a Hong Kong′′ =“sitios turisticos quiere ir a Hong Kong′′(no meaning) So far, this strategy revolves around the closure property and other properties such as associative, and the
neutral element; but this is not enough to guarantee structure and meaning, at least compared to the source text. Conceptual framework To improve this proposal is necessary to include more properties to the set along with the operation, this is only
possible by exploring other possible set types that can build up an algebraic structure more useful, and thus,
it is decisive to map Pvn to another set that will be named Ovn . Table 1 shows the new set and its components. Mapping F
: P
→O : Let us define F
as: • Pairs < vi, nj > belong Pvn with different index ( i = j ) will be mapped to Ŵ in Ovn. • Pairs < vi, nj > belong Pvn with different index ( i = j ) will be mapped to Ŵ in Ovn. j
gf
j
pp
• All pairs mapped must contain at least a n-element, then, pairs such as < vi, > will be mapped to Ŵ in j
gf
j
pp
• All pairs mapped must contain at least a n-element, then, pairs such as < vi, > will be mapped to Ŵ in Ovn. • Additionally, if couples such as < vi, ni > exist in Pvn , then, elements such as < , ni > will be mapped to Ŵ
in Ovn. • All pairs mapped must contain at least a n-element, then, pairs such as < vi, > will be mapped to Ŵ in Ovn. • Additionally, if couples such as < vi, ni > exist in Pvn , then, elements such as < , ni > will be mapped to Ŵ
in Ovn. • Additionally, if couples such as < vi, ni > exist in Pvn , then, elements such as < , ni > will be mapped to Ŵ
in Ovn. Figure 3 show the mapping made from Pvn to Ovn for the example. In O
the operation used also change and it is defined as follows Figure 3 show the mapping made from Pvn to Ovn for the example. In Ovn the operation used, also, change, and it is defined as follows: In Ovn the operation used, also, change, and it is defined as follows: • Dual. It is Dual because of whether two components are operated, then the operation takes place indepen-
dently in each internal vector. This property allows to separate completely verbs and nouns. • Positional. It is Positional because the operation is carried out by two elements with the same subscript. Conceptual framework p
p
p
Supposing the following sentence in the Spanish language: ”Fred quiere ir a Hong Kong y visitar sitios turísticos”
(English meaning: ”Fred wants to go to Hong Kong and visit tourist places”), and it is dissociated in strings with a
word each one. One scenario for creating Natural Language from this dissociation will be to use the conventional
algebraic structure of strings which is composed of the set of strings, and an operator able to join the strings
and generate others (closure property). In this algebraic structure the closure property functions as follows: String-set = {“Fred′′, “Hong Kong′′, “quiere′′, “ir′′, “a′′,′′ y′′,′′ visitar′′,′′ sitios′′, turisticos}
A new string = “Fred′′ + “quiere′′ + “ir′′ + “a′′ + “Hong Kong′′
= “Fred quiere ir a Hong Kong′′ String-set = {“Fred′′, “Hong Kong′′, “quiere′′, “ir′′, “a′′,′′ y′′,′′ visitar′′,′′ sitios′′, turisticos}
A new string = “Fred′′ + “quiere′′ + “ir′′ + “a′′ + “Hong Kong′′
′′ ng = “Fred′′ + “quiere′′ + “ir′′ + “a′′ + “Hong Kong′′ = “Fred quiere ir a Hong Kong′′ But the closure property is not enough, because the generation of a new string in natural language must
ensure structure and meaning, and this is not completely possible in this set with this operation, for example: A new string = “ir′′ + “quiere′′ + “a′′ + “Fred′′ + “Hong Kong′′ = “ir quiere a Fred Hong Kong′′(no meaning) = “ir quiere a Fred Hong Kong′′(no meaning) = “ir quiere a Fred Hong Kong′′(no meaning) A possible solution is to divide the sentence into adequate strings forming ordered sets of clusters. The sets
generated by this process will be named Kn (noun-cores/noun-clusters) and Kv(verb-cores/verb-clusters)), but
the set used to generate phrases would be the Cartesian product of these sets ( Kv × Kn = Pvn ). In this strategy,
the verb-core must contain the null string because the SVO languages (Spanish and English, among others) allow
generating phrases without verbs. Figure 2 shows a dissociation following the heuristics in48:hh The operation should destroy the operand pairs and apply the operation concatenation or plus (+). This
method can generate several sentences, but any of them without meaning, or, at least in the context of the
original sentence: Scientific Reports | (2022) 12:22249 | https://doi.org/10.1038/s41598-022-26304-w www.nature.com/scientificreports/ Table 1. Set Ovn and description of its components. Source: Own elaboration. Conceptual framework To create the sentence starting from this new core is applied a process that states that for each position in
the vectors a verb is concatenated with the noun corresponding and the result will be added to the next result
as follows: =′′(quiere
ir′′ +′′ a
Hong
Kong)′′
=′′quiere
ir′′ +′′ a
Hong
Kong′′ +′′ (y
visitar′′ +′′ sitios
turisticos)′′
=′′quiere
ir
a
Hong
Kong
y
visitar
sitios
tursticos′′ =′′quiere
ir′′ +′′ a
Hong
Kong′′ +′′ (y
visitar′′ +′′ sitios
turisticos)′′ =′′quiere
ir
a
Hong
Kong
y
visitar
sitios
tursticos′′ It is too easy to deduct that this new operation in Ovn is commutative, i.e., the result is the same, although
the operands will change their position. This commutative structure is known as an Abelian monoid structure
and, in48, and by including the symmetrical element, is converted to an Abelian group.i An algebraic structure as has been defined is very useful because reduces the complexity in the reconstruc-
tion of phrases because the operation is easy to implement and its behavior is similar to the add operation in the
numbers by managing sentences as sets of discrete cores. In the section ”An approach” will be explained that a
sentence can generate several Abelian groups, and each one can generate sentences separately. Conceptual framework This • Dual. It is Dual because of whether two components are operated, then the operation takes place indepen-
dently in each internal vector. This property allows to separate completely verbs and nouns.h yh
p
p
y
p
p
y
Positional. It is Positional because the operation is carried out by two elements with the same subscript. This
property allows to implement commutativity. For example:
This operation is the same that:
[0 : v1 : 2][0 : n1 : 2] + [0 : 1 : v2][0 : 1 : n2]
=[0 + 0 : v1 + 1 : 2 + v2][0 + 0 : n1 + 1 : 2 + n2]
=[0 : v1 : v2][0 : n1 : n2]
=v1 + n1 + v2 + n2 same that:
[0 : v1 : 2][0 : n1 : 2] + [0 : 1 : v2][0 : 1 : n2]
=[0 + 0 : v1 + 1 : 2 + v2][0 + 0 : n1 + 1 : 2 + n2]
=[0 : v1 : v2][0 : n1 : n2]
=v1 + n1 + v2 + n2 https://doi.org/10.1038/s41598-022-26304-w www.nature.com/scientificreports/ Figure 4. Use case for the architecture of the system. Source: Own elaboration. igure 4. Use case for the architecture of the system. Source: Own elaboration. ? [0 :′′ quiere
ir′′ : 2][0 :′′ a
Hong
Kong′′ : 2] + [0 : 1 :′′ y
visitar′′][0 : 1 :′′ sitios
turisticos′′]? =? [0 + 0 :′′ quiere
ir′′ + 1 : 2 +′′ y
visitar′′][0 + 0 :′′ a
Hong
Kong′′ + 1 : 2 +′′ sitios
turisticos′′]? =? [0 :′′ quiere
ir′′ :′′ y
visitar′′][0 :′′ a
Hong
Kong′′ :′′ sitios
turisticos′′]? Methodology
Th This section proposes an architecture of a system, for dissociating and recovering texts and sentences, based on
the concepts, theories, and regulations aforementioned. Figure 4 shows a scheme of the system based on use
cases view50. The system would include three major sub-systems: dissociation, memory and recovery. The two
first sub-systems will be activated serially and immediately after a reading takes place, and the latter process is
executed when a query promotes the generation of sentences. Nevertheless, in terms of the information pro-
cessing associated with each sub-system, they operate independently. The entire system is conceptualized as a
framework that could be up-gradable and enriched with plug-in modules.h The class diagram is shown in Fig. 5. h
g
g
And the activities diagram is shown in Fig. 6, this last diagram is only for dissociating, because the recovery
depends on the implementation which is shown in Section ”An approach”. Dissociation subsystem. The function of the dissociation subsystem is to split a text/sentence into special
units. As previously mentioned in section From sentences to clusters of words, all languages share a common char-
acteristic which is the identification of three basic clusters within a sentence: Subject(S), Verb(V), and Object(O). They can occur within a sentence in a different order depending on the language. hf
In this paper, the expression SOV-trio or simply SOV will be used to represent the trio that models a sentence
or a text. Given that Subject and Object have similarities both will be treated as (S). Additionally, each of the
components of a SOV will be named a core.h The cores may contain one or more words from the sentence. For example, it is possible to have a verb fol-
lowed by another verb in the same core, as in the following sentence: ”Fred quiere ir a Hong Kong y visitar sitios
turísticos” the two verbs (”quiere ir”) constitute a V core. Once a SOV is generated, this is dispatched to the
memory subsystem. Strategies to generate SOVs
l
d
b As explained in section Object-action dissociation/integration, there is a consensus about the dissociation
between actions (verbs) and objects (nouns) inside the human mind. However,32 emphasizes the existence of
problems by establishing the grammar category that can generate confusion between verbs and nouns, this also Scientific Reports | (2022) 12:22249 | https://doi.org/10.1038/s41598-022-26304-w www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. Class diagram of the framework. Source: Own elaboration. Figure 5. Class diagram of the framework. Source: Own elaboration. Figure 6. Activities diagram of the framework. Source: Own elaboration. Figure 6. Activities diagram of the framework. Source: Own elaboration. Figure 6. Activities diagram of the framework. Source: Own elaboration. https://doi.org/10.1038/s41598-022-26304-w Scientific Reports | (2022) 12:22249 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 7. A generic UML sequence diagram for the dissociation process. Source: Own elaboration. Figure 7. A generic UML sequence diagram for the dissociation process. Source: Own elaboration. can happen in the process of dissociation in this subsystem. To dissociate the sentences correctly, the subsystem
should implement modules such as: can happen in the process of dissociation in this subsystem. To dissociate the sentences correctly, the subsystem
should implement modules such as: can happen in the process of dissociation in this subsystem. To dissociate the sentences correctly, the subsystem
should implement modules such as: • Syntactic Analysis (Parsing). An ordinary parser generates a syntax tree from which the SOVs can be rapidly
built. Although this strategy is good, it does not avoid that the syntax tree generated may require the involve-
ment of some other heuristic processes to “refine” the creation of the cores, for instance, in cases of slang
interpretation as is shown in Fig. 7, Parser module of, routine parserMethod(). d
d
d l
fi
ll • Dictionaries and conjugators. Sometimes, parsers can produce an incorrect word classification, especially
when the parser has not well-trained in a particular language, in such case it is necessary to perform an
analysis and debugging process over these words. For this purpose, software like dictionaries and conjuga-
tors modules could be useful to validate the category as is shown in Fig. 7, Parser module, routine correct- • Grouping of elements. The dissociation in cores requires identifying elements like determinants, adverbs,
prepositions, conjunctions, etc., in such a way that they will be inserted in the adequate core. This process
should be customized for each language as is shown in Fig. 7, Groups module. To summarize, some procedures, syntactic-semantic strategies, and heuristics should be implemented to help
in building the S/O/V cores correctly. Memory subsystem. An important function of the memory system is to store the information generated
by the dissociation subsystem. Hence, it is mandatory to build a structure that guarantees order and efficiency. Scientific Reports | (2022) 12:22249 | www.nature.com/scientificreports/ Therefore, the memory system should contain a repository to save the SOVs generated by each text read inter-
related between them. This storage should maintain these cores in such a way that can be retrieved in the exact
order as they were read. According to these principles, the implementation should comply with the following
conditions: • SQL-database. The type of database towards has been addressed in this research is the SQL-database because
it is the most used to store information. The main idea is to save the texts in such a way that their elements
will be organized in groups or clusters representing sets that, joining them, can reproduce the source without
losing their meaning. g
g
• Repository based on queries of cores. Firstly, a repository based on query means that uses SQL technol-
ogy to save and recover information. Secondly, the queries can be attended by modules that recover cores,
compare against the queries, determine similarity, and create sentences, paragraphs, and full texts as of the
cores chosen. g
g
• Repository based on queries of cores. Firstly, a repository based on query means that uses SQL technol-
ogy to save and recover information. Secondly, the queries can be attended by modules that recover cores,
compare against the queries, determine similarity, and create sentences, paragraphs, and full texts as of the
cores chosen. https://doi.org/10.1038/s41598-022-26304-w Scientific Reports | (2022) 12:22249 | www.nature.com/scientificreports/ Figure 8. Relationship between groups generated by the sentence: ”Fred quiere ir a Hong Kong y visitar sitios
turísticos”. Source: Own elaboration. Figure 8. Relationship between groups generated by the sentence: ”Fred quiere ir a Hong Kong y visitar sitios
turísticos”. Source: Own elaboration. • Meta-engine. Each implementation should program a meta-engine that works over the database in a superior
layer that the database engine, this should be equipped with the algebraic operation explained in subsection
Algebraic environment and mappings between groups to integrate them and build a part or whole original
text. • Meta-engine. Each implementation should program a meta-engine that works over the database in a superior
layer that the database engine, this should be equipped with the algebraic operation explained in subsection
Algebraic environment and mappings between groups to integrate them and build a part or whole original
text. The Engineh The queries are expected in natural language and it would transform into a set of SOVs. The key is to com-
pare SOVs for finding the closest results. The strategies to match the SOVs.can be wide. An example could be to
establish matches of SOVs that contain elements that could respond contextually to the query as in Fig. 9. The
degree of coincidence will be the measure.h g
This strategy could recover sentences that do not answer the query completely, hence, it would be important
to implement another stage. For instance, that compares sentences in a logical context. This can be carried out
by converting the query and the text recovered into small text-theories that can be matched logically. Dynamic structureh The dissociation in SOVs and the mapping create sets distributed and connected in terms of their original
semantic content. Figure 8 shows a scheme that illustrates the relationship between the sets of SOVs (Abelian
group).h g
p
The nodes will be related with adequate functions (mapping) to guarantee that the recovery of the part, or
the whole, of a sentence/text will be executed correctly as will be explained later. Recovery subsystem. The purpose of this subsystem is to generate, in a dynamic way, a sentence/text part
or entirely. This subsystem is closely interrelated to the dynamic structure because this subsystem is composed of
the functions that connect the nodes.h The Engineh An approachh This section has a summary of a prototype designed as a layered framework that could be used for any language
characterized as S-V-O (Spanish, English, etc.) The most relevant layers of the dissociation processes are the
following: 1. Identifying the language. This first layer has been designed to identify the language of the text and divide
it into sentences, and finally, their results will send to the next layer one at a time.h 1. Identifying the language. This first layer has been designed to identify the language of the text and divide
it into sentences, and finally, their results will send to the next layer one at a time.h i
y
y
2. Planning. This second layer chooses the modules required to dissociate the sentences based on the language
recognized. This layer makes flexible the framework because it allows changing the rules of dissociation
depending on the language to be processed.h i
2. Planning. This second layer chooses the modules required to dissociate the sentences based on the language
recognized. This layer makes flexible the framework because it allows changing the rules of dissociation
depending on the language to be processed.h p
g
g
g
p
3. Reaction layer. This layer is related to the strategies to generate SOVs which were described in the para-
graph Strategies to generate SOVs. Figure 7 is shown the execution of two modules in a pipeline way, but new
modules could be included to improve the results, this will depend on the implementation. The name of this
layer is due to the modules chosen by the plan layer being triggered dynamically and executed like a chain
reaction in a pipeline. p
g
g
g
p
3. Reaction layer. This layer is related to the strategies to generate SOVs which were described in the para-
graph Strategies to generate SOVs. Figure 7 is shown the execution of two modules in a pipeline way, but new
modules could be included to improve the results, this will depend on the implementation. The name of this
layer is due to the modules chosen by the plan layer being triggered dynamically and executed like a chain
reaction in a pipeline. An approachh In this implementation, the modules created in the reaction layer dealt with sentences in the Spanish language
(S-V-O language) and were organized in three linear phases following the guidelines described in the paragraph In this implementation, the modules created in the reaction layer dealt with sentences in the Spanish language
(S-V-O language) and were organized in three linear phases following the guidelines described in the paragraph https://doi.org/10.1038/s41598-022-26304-w Scientific Reports | (2022) 12:22249 | www.nature.com/scientificreports/ Source: Extracted from52. In this approach, there are two types of determinants p-det and a J-det; both interrelate the sets with func-
tions, but the Abelian group pointed by a J-det is considered optional in the rebuild of the sentence. All of these
properties were established empirically.h The restoring process is not the reverse operation exactly, else it is a complex process that executes tasks
from the repository trying to preserve syntax and the original semantics. This purpose is successful due to the
properties of the Abelian groups (see52) and the hierarchy of sets created by the determinants in the dissociation
process. The process is shown in Fig. 10h ph
p
g
The sets are operated and mapped in a domino way from the core where the matching occurs up until the
root of the hierarchy. For example, in Fig. 10, if the core matched is in G3 and corresponds to ” n2 = críticas” then
the recovered sentence will be: “La marcha programada, para el próximo 26 de marzo, recibía críticas”. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 9. Creating a sentence from a query. Source: Own elaboration. Figure 9. Creating a sentence from a query. Source: Own elaboration. Figure 9. Creating a sentence from a query. Source: Own elaboration. Strategies to generate SOVs and shown in Fig. 7. In the first phase, each sentence is processed by a linguistic tool,
commanded by the VISL parser51 reaction layer, in this stage, it, also, corrects possible inconsistencies generated
by the parser as the wrong classification of the words, e.g., some words classified as nouns or vice versa. The
information produced by the parser is significant, therefore, it is discriminated, and sent to the next module in
the pipeline. The second phase receives the information and classification and applies heuristics for generating,
initially, raw-clusters, then refined by another heuristic, and finally to produce the set Ov . Lastly, in the third phase,
the set Ov is saved in a standard database (SQL-style). The heuristics applied in this approach are not extendable
to other languages. However, currently, they are being tested in the English Language, also S-V-O language, to
measure their effectiveness in it. Each sentence is organized in Abelian groups hierarchically organized with a
binary operation capable of building phrases (see Fig. 3). The Abelian groups obey the specifications done in
section Algebraic environment.ih g
Table 2 shows the classification established heuristically for the Spanish language cores in this approach. This
process involves a loop where neighboring words that comply with certain conditions are packed into a single
class named: nominal core (S), determinant, and verbal core (V). A determinant is used to interrelate Abelian
groups as in Fig. 8 in the paragraph Dynamic structure. It is important note that the punctuation signs are useful
to create these categories, some are part of the determinants and other are par of the verbal cores or nominal
cores, for example, in Fig. 8 the nominal core n6 in G2 include a comma: ”, la Paz y la justicia”, similarly, the
verbal cores v5 in G3 : ”, recibía”. https://doi.org/10.1038/s41598-022-26304-w Scientific Reports | (2022) 12:22249 | www.nature.com/scientificreports/ Table 2. Final categorization scheme. Category
Description
p, J
Determinants
v, V
Verbal core
n
Noun core Table 2. Final categorization scheme. Category
Description
p, J
Determinants
v, V
Verbal core
n
Noun core Table 2. Final categorization scheme. Figure 10. Scheme to recover sentences. Source: Extracted from52. Figure 10. Scheme to recover sentences. Proposal comparison Currently, generating sentences and small texts is a task very significant in several fields of Computer Science. The approach named Rhetorical Structure Theory (RST)53 is one of the first proposals created to divide the
discourse and has been the inspiration for Natural Language Generation (NLG) schemes. Th
d
d
d
h
d
h
h
h
ll (
l
d
ll
) The strategy used in RST to divide the texts into cores and organize them hierarchically (nucleus and satellite)
requires training of neural networks54,55. The relevance of the framework presented in this document is that it
does not need training. g
With respect to the generation of sentences, the implementations and approaches are very exigent. According
to56, they should carry out several complicated tasks as below: . Determining the information relevant. This part is associated with the context and scope, basically related
to the searching57–60. 2. Determining the order as the words should appear. Some approaches try to resolve this part from texts by
collecting, recovering, and organizing sentences inside them61,62hfi 3. Determining: how should be the information aggregated? This stage is considered very difficult because the
information can be provided by several sources or it is not the correct response to any query. Some works
use the context to resolve discrepancies or the domain to explore the sources63,64. Scientific Reports | (2022) 12:22249 | https://doi.org/10.1038/s41598-022-26304-w www.nature.com/scientificreports/ 4. Determining the right words and phrases (verbs and nouns). This part contains two stages but they will be
joined in one because can be carried out jointly. In this part the sentence is organized in one of the following
structures: SVO, SOV, VSS, VOS, also, it is analyzed the verb times65.h 4. Determining the right words and phrases (verbs and nouns). This part contains two stages but they will be
joined in one because can be carried out jointly. In this part the sentence is organized in one of the following
structures: SVO, SOV, VSS, VOS, also, it is analyzed the verb times65.h y
5. Combining words and phrases to generate well-formed sentences. This phase builds the sentences, sometimes
through templates, or grammar-based techniques, among others66,67. y
5. Combining words and phrases to generate well-formed sentences. This phase builds the sentences, someti
through templates, or grammar-based techniques, among others66,67. Conclusions and future worksh The high demand for information has caused an increasingly important in the automation of processes such as
decision-making, pattern recognition, and interaction human-machine, among others. Several of these processes
require the use of the text, either to understand queries, generate reports, or answer in natural language, hence,
building applications with these functions takes a greater relevance. This paper presents an architecture for dis-
sociating the text/sentences, saving it in a SQL database, and recovering it without loss of meaning. This is highly
productive in process automation because the textual information is converted from unstructured to structured
format and the queries and other processes in natural language can be more efficient.hi g
gfi
The suggested system has been inspired and based on processes verified by scientists related to the dissociation
of the information inside the human brain, memory models in the Neuroscience field, and the structure of the
languages in Linguistic and Psycholinguistic disciplines. The proposed framework divides a sentence/text into
clusters like the brain dissociates the speech into nominal and verbal categories. The scheme will divide the text/
sentence into sets of cores named nominal cores and verbal cores, and implement an algebraic operation that can
be used to generate new sentences that keep the original meaning without loosing the structure of the language. This proposal was applied by the approach studied in the last section successfully.h h
The explored implementation resolved a great part of the challenges described in the paper by implementing
a framework with abstract modules that can be custom implemented, for instance, the processes described in
the architecture, the generic abstract modules for different languages, and the recovery modules, among others. dd
ll
h
l
l
f
h
h l
b
h
f
b
h f
Additionally, the implementation creates a solution for the Spanish language by using heuristics for both
dissociation and recovery processes. The application suggests interrelating the algebraic sets by employing func-
tions to recover the whole or part of the textual information by maintaining the meaning. The approach shows
that for the Spanish language is possible to have an implementation. In68 is exposed several proposals of NLG. A system has been proposed for converting unstructured textual information to be computationally man-
aged structured information. This proposal has been tested in an approach for the Spanish language successfully. Conclusions and future worksh Future works will be addressed to implement this framework for other languages and to generate applications
for these approaches. Proposal comparison In the framework exposed in this document, the first three steps are part of the recovery system in this frame-
work, specifically, corresponding to the engine searching. the last two steps can be resolved by responding to the
queries and executing the algebra of the groups and mapping between them which are tasks easy and efficient. All of these show a framework simple to implement. Received: 1 February 2022; Accepted: 13 December 2022 Data availabilityh The current document has been focused on the discussion about a framework able to compose strategies to
divide sentences and texts into cores to save them in a SQL-databases. Under this context the data used to verify
the effectiveness belong to other work where the purpose was to study heuristics for dividing the sentences into
these clusters, these data do not form part of the current research. Therefore, all data generated or analyzed dur-
ing this study are included in this published article. Received: 1 February 2022; Accepted: 13 December 2022 References
1. D’Souza, S. Parser extraction of triples in unstructured text. arXiv preprint arXiv:1811.05768 (2018).
2. YL. Shuea, C. V., P. Keatingb & Yub, K. Voicesauce: A program for voice analysis. In INTERNATIONAL CONGRESS OF PHONETIC
SCIENCES (ICPhS XVII) 1846 1849 (2011) Contexts: A formalization and some applications (1992). 6. Guha, R. Contexts: A formalization and some applications (1992). pp
(
)
7. Lenat, D. B. & Guha, R. V. Building Large Knowledge-Based Systems; Representation and Inference in the Cyc Project (Addison-Wesley
Longman Publishing Co., Inc, USA, 1989). 8 Herrera R J G & Martin-Bautista M J A novel process-based kms success framework empowered by ontology learning technol- 7. Lenat, D. B. & Guha, R. V. Building Large Knowledge-Based Systems; Representation and Inference in the Cyc Project (Addison-Wesley
Longman Publishing Co., Inc, USA, 1989). g
g
8. Herrera, R. J. G. & Martin-Bautista, M. J. A novel process-based kms success framework empowered by ontology learning technol-
ogy. Eng. Appl. Artif. Intell. 45, 295–312 (2015). gy
g
pp
f
(
)
9. Abbes, H. & Gargouri, F. Mongodb-based modular ontology building for big data integration. J. Data Semant. 7, 1–27 (2017). 0. Gruber, T. R. Toward principles for the design of ontologies used for knowledge sharing. Int. J. Hum.-Comput. Stud. 43, 907–928
https://doi.org/10.1006/ijhc.1995.1081 (1995). gy
g
pp
f
9. Abbes, H. & Gargouri, F. Mongodb-based modular ontology building for big data integration. J. Data Semant. 7, 1–27 (2017). 0. Gruber, T. R. Toward principles for the design of ontologies used for knowledge sharing. Int. J. Hum.-Comput. Stud. 43, 907–928 9. Abbes, H. & Gargouri, F. Mongodb-based modular ontology building for big data integration. J. Data Semant. 7, 1–27 (2017). 10. Gruber, T. R. Toward principles for the design of ontologies used for knowledge sharing. Int. J. Hum.-Comput. Stud. 43, 907–928. https://doi.org/10.1006/ijhc.1995.1081 (1995). p
g
j
(
)
11. Guo, K. & Ma, J. Semantic-based heterogeneous multimedia big data retrieval. In Li, K., Jiang, H., Yang, L. T. & Cuzzocrea, A. (eds.) Big Data - Algorithms, Analytics, and Applications, 18 (Chapman and Hall/CRC, New York, 2015). p
g
j
11. Guo, K. & Ma, J. Semantic-based heterogeneous multimedia big data retrieval. In Li, K., Jiang, H., Yang, L. T. & Cuzzocrea, A. (eds.) Big Data - Algorithms, Analytics, and Applications, 18 (Chapman and Hall/CRC, New York, 2015). g
g
y
pp
p
12. Perez, N. & Cuadros, M. Multilingual call framework for automatic language exercise generation from free text. In Proceedings of
the Software Demonstrations of the 15th Conference of the European Chapter of the Association for Computational Linguistics, 49–52
(2017). 12. Perez, N. & Cuadros, M. References
1. D’Souza, S. Parser extraction of triples in unstructured text. arXiv preprint arXiv:1811.05768 (2018).
2. YL. Shuea, C. V., P. Keatingb & Yub, K. Voicesauce: A program for voice analysis. In INTERNATIONAL CONGRESS OF PHONETIC
SCIENCES (ICPhS XVII) 1846 1849 (2011) 1. D’Souza, S. Parser extraction of triples in unstructured text. arXiv preprint arXiv:1811.05768 (2018). 2. YL. Shuea, C. V., P. Keatingb & Yub, K. Voicesauce: A program for voice analysis. In INTERNATIONAL CONGRESS OF PHONETIC
SCIENCES (ICPhS XVII) 1846 1849 (2011) 1. D’Souza, S. Parser extraction of triples in unstructured text. arXiv preprint arXiv:1811.05768 (2018). 2. YL. Shuea, C. V., P. Keatingb & Yub, K. Voicesauce: A program for voice analysis. In INTERNATIONAL CONGRESS OF PHONETIC
SCIENCES (ICPhS, XVII), 1846–1849 (2011). 2. YL. Shuea, C. V., P. Keatingb & Yub, K. Voicesauce: A program for voice analysis. In INTERNATIONAL CONGRESS OF PHONETIC
SCIENCES (ICPhS, XVII), 1846–1849 (2011). 2. YL. Shuea, C. V., P. Keatingb & Yub, K. Voicesauce: A program for voice analysis. In INTERNATIONAL CONGRESS OF PHONETIC
SCIENCES (ICPhS, XVII), 1846–1849 (2011). 3. Jain, A. K. & Yu, B. Automatic text location in images and video frames. Pattern Recogn. 31, 2055–2076 (1998). g
g
4. Cimiano, P. & Völker, J. Text2onto - a framework for ontology learning and data-driven change discovery. In Proceedings of the 10th
International Conference on Applications of Natural Language to Information Systems (NLDB), 227–238 (Alicante, Spain, 2005). 5. Ghidini, C. & Giunchiglia, F. Local models semantics, or contextual reasoning=locality+compatibility. Artif. Intell. 127, 221–259. https://doi.org/10.1016/S0004-3702(01)00064-9 (2001). 4. Cimiano, P. & Völker, J. Text2onto - a framework for ontology learning and data-driven change discovery. In Proceedings of the 10th
International Conference on Applications of Natural Language to Information Systems (NLDB), 227–238 (Alicante, Spain, 2005). 5 Ghidi i C & Gi
hi li
F L
l
d l
ti
t
t
l
i
l
lit +
tibilit
A tif I t ll 127 221 259 4. Cimiano, P. & Völker, J. Text2onto - a framework for ontology learning and data-driven change discovery. In Proceedings of the 10th
International Conference on Applications of Natural Language to Information Systems (NLDB), 227–238 (Alicante, Spain, 2005). 5. Ghidini, C. & Giunchiglia, F. Local models semantics, or contextual reasoning=locality+compatibility. Artif. Intell. 127, 221–259. htt
//d i
/10 1016/S0004 3702(01)00064 9 (2001) gy
g
g
y
g
f
International Conference on Applications of Natural Language to Information Systems (NLDB), 227–238 (Alicante, Spain, 2005). 5. Ghidini, C. & Giunchiglia, F. Local models semantics, or contextual reasoning=locality+compatibility. Artif. Intell. 127, 221–259
https://doi.org/10.1016/S0004-3702(01)00064-9 (2001). 5. Ghidini, C. & Giunchiglia, F. Local models semantics, or contextual reasoning=locality+compatibility. Artif. Intell. 127, 221–259. https://doi.org/10.1016/S0004-3702(01)00064-9 (2001). 6. Guha, R. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 13. Tulving, E. Episodic and semantic memory. In Tulving, E. & Donaldson, W. (eds.) Organization of Memory, 381–403 (Acad
Press, New York, 1972). )
14. Havel, I. M. Strategies of Remembrance: From Pindar to Hölderlin, chap. 2 (Cambridge Scholars, England, 2009).i 5. D. C. Bradley, M. F. G. & Zurif, E. B. Syntactic deficit in broca’s apahsia. In Caplan, D. (ed.) Biological studies of mental processes
vol. 14, 345–360 (MIT Press, 1988).f 6. Saffran, E. M. Neuropsychological approaches to the study of language. Br. J. Psychol. 73, 317–337. https://doi.org/10.1111/j.2044-
8295.1982.tb01815.x (1982).fh 7. E. M. Saffran, M. F. S. & Marin, O. S. M. The word order problem in agrammatism: Ii. production. Brain and Language10, 263—280
https://doi.org/10.1016/0093-934X(80)90056-5 (1980). p
g
8. Feng, S., Qi, R., Yang, J., Yu, A. & Yang, Y. Neural correlates for nouns and verbs in phrases during syntactic and semantic process-
ing: An fmri study. Journal of Neurolinguistics 53, 100860 (2020). g
y
f
g
(
)
19. Miceli, G., Silveri, M. C., Nocentini, U. & Caramazza, A. Patterns of dissociation in comprehension and production of nouns and
verbs. Aphasiology 2, 351–358. https://doi.org/10.1080/02687038808248937 (1988).fi p
gy
p
g
20. Miceli, G., Silveri, M. C., Nocentini, U. & Caramazza, A. On the basis of the agrammatics’ difficulty in producing main v
Cortex 20, 207–220. https://doi.org/10.1016/S0010-9452(84)80038-6 (1984). heng, W. et al. Chasing language through the brain: Successive par 22. Damasio, A. R. & Tranel, D. Nouns and verbs are retrieved with differently distributed neural systems. In Proceedings of the Na
Academy of Sciences U.S.A., vol. 90, 4957-4960, https://doi.org/10.1073/pnas.90.11.4957 (1993). 22. Damasio, A. R. & Tranel, D. Nouns and verbs are retrieved with differently distributed neural systems. In Proceedings of the National
Academy of Sciences U.S.A., vol. 90, 4957-4960, https://doi.org/10.1073/pnas.90.11.4957 (1993). 23. Daniele, A., Giustolisi, L., Silveri, M. C., Colosimo, C. & Gainotti, G. Evidence for a possible neuroanatomical basis for le
processing of nouns and verbs. Neuropsychologia 32, 1325–1341. https://doi.org/10.1016/0028-3932(94)00066-2 (1994). p
g
p y
g
p
g
24. K. A. Shapiro, L. R. M. & Caramazza, A. Cortical signatures of noun and verb production. In P
of Sciences U.S.A., vol. 103, 1644—1649, https://doi.org/10.1073/pnas.0504142103 (2006).hf 24. K. A. Shapiro, L. R. M. & Caramazza, A. Cortical signatures of noun and verb production. In Proceedings of the National Academy
of Sciences U.S.A., vol. 103, 1644—1649, https://doi.org/10.1073/pnas.0504142103 (2006).hf 5. Siri, S. et al. www.nature.com/scientificreports/ The neural substrate of naming events: Effects of processing demands but not of grammatical class. Cereb. Cortex 18
171–177. https://doi.org/10.1093/cercor/bhm043 (2008). p
g
6. Tyler, L. K. & Marslen-Wilson, W. Fronto-temporal brain systems supporting spoken language comprehension. Philos. Trans. R
Soc. B 363, 1037–1054. https://doi.org/10.1098/rstb.2007.2158 (2008).h p
g
27. Levelt, W. J. M. Speaking: From intention to articulation. The American Journal of Psychology (1990). M. Speaking: From intention to articulation. The American Journal Levelt, W. J. M. Speaking: From intention to articulation. The Ame J
p
gh
J
f
y
gy (
)
28. Garrett, M. F. Syntactic processes in sentence production. In R. J. Wales, E. W. (ed.) New Approaches to Language Mechan
vol. 12, 231—255 (North Holland Publishing Company, Netherlands, 1976).h h
. Syntactic processes in sentence production. In R. J. Wales, E. W —255 (North Holland Publishing Company, Netherlands, 1976).h 29. Garrett, M. F. The organization of processing structure for language production: applications to aphasic speech. In D. Caplan, A. S.,
A. R. Lecours (ed.) Biological Perspectives on Language, vol. 12, 172—193 (The MIT Press, 1984). h
0. Ullman, M. T. et al. A neural dissociation within language: Evidence that the mental dictionary is part of declarative memory and
that grammatical rules are processed by the procedural system. J. Cognitive Neurosci. 9, 266–276. https://doi.org/10.1162/jocn
1997.9.2.266 (1997). 31. Elli, G. V., Lane, C. & Bedny, M. A double dissociation in sensitivity to verb and noun semantics across cortical networks. Cereb. Cortex 29, 4803–4817 (2019). 32. Vigliocco, G., Vinson, D. P., Druks, J., Barber, H. & Cappa, S. F. Nouns and verbs in the brain: A review of behavioural, electro-
physiological, neuropsychological and imaging studies. Neurosci. Biobehav. Rev. 35, 407–426. https://doi.org/10.1016/j.neubiorev.
2010.04.007 (2011).h 33. Blything, L. P. & Cain, K. The role of memory and language ability in children’s production of two-clause sentences contai
before and after. J. Exp. Child Psychol. 182, 61–85 (2019).f t
p
y
(
)
34. Khader, P., J. S., Scherag, A. & Rösler, F. Differences between noun and verb processing in a minimal phrase context: A semantic
priming study using eventrelated brain potentials. Cognitive Brain Research17, 293—313, https://doi.org/10.1016/S0926-6410(03)
00130-7 (2003). 35. Gomes, W., Ritter, V. C., Tartter, H. G., Vaughan, J. R. & Rosen, J. J. Lexical processing of visually and auditorily presented nouns and
verbs: evidence from reaction time and n400 priming data. J. Cogn. Neurosci. 6, 121–134. References
1. D’Souza, S. Parser extraction of triples in unstructured text. arXiv preprint arXiv:1811.05768 (2018).
2. YL. Shuea, C. V., P. Keatingb & Yub, K. Voicesauce: A program for voice analysis. In INTERNATIONAL CONGRESS OF PHONETIC
SCIENCES (ICPhS XVII) 1846 1849 (2011) Multilingual call framework for automatic language exercise generation from free text. In Proceedings of
the Software Demonstrations of the 15th Conference of the European Chapter of the Association for Computational Linguistics, 49–52
(2017). Scientific Reports | (2022) 12:22249 | https://doi.org/10.1038/s41598-022-26304-w www.nature.com/scientificreports/ https://doi.org/10.1016/S0926-6410(97)
00023-2 (1997).h (
)
36. Rodosthenous, C. et al. Using crowdsourced exercises for vocabulary training to expand conceptnet. In Proceedings of The 12th
Language Resources and Evaluation Conference, 307–316 (2020). 37. Singh, H. L. P. Conceptnet - a practical commonsense reasoning tool-kit. BT Technol. J. 22, 211–226. https://doi.org/10.102
BTTJ.0000047600.45421.6d (2004). 38. Speer, R. & Havasi, C. Representing general relational knowledge in conceptnet 5. In LREC, 3679–3686 (European Lang
Resources Association (ELRA), Istanbul, Turkey, 2012). 39. Vial, L., Lecouteux, B. & Schwab, D. Sense vocabulary compression through the semantic knowledge of wordnet for neural w
sense disambiguation. arXiv preprint arXiv:1905.05677 (2019). sense disambiguation. arXiv preprint arXiv:1905.05677 (2019). g
p p
40. Miller, G. A., Beckwith, R., Fellbaum, C., Gross, D. & Miller, K. J. Introduction to wordnet: An on-line lexical database
Lexicogr. 3, 235–244. https://doi.org/10.1093/ijl/3.4.235 (1990). g
p
g
j
(
)
41. Fellbaum, C. English verbs as a semantic net. Int. J. Lexicogr. 3, 278–301. https://doi.org/10.1093/ijl/3.4.278 (1990). 41. Fellbaum, C. English verbs as a semantic net. Int. J. Lexicogr. 3, 278–301. https:/ 42. Group, P. F. Visualization of wordnet using force-directed gra 43. Boeree, G. Basic language structures. 43. Boeree, G. Basic language structures. 44. García-Méndez, S., Fernández-Gavilanes, M., Costa-Montenegro, E., Juncal-Martínez, J. & González-Castaño, F. J. A library for
automatic natural language generation of spanish texts. Expert Syst. Appl. 120, 372–386 (2019).i 44. García-Méndez, S., Fernández-Gavilanes, M., Costa-Montenegro, E., Juncal-Martínez, J. & González-Castaño, F. J. A librar
automatic natural language generation of spanish texts Expert Syst Appl 120 372 386 (2019) 44. García-Méndez, S., Fernández-Gavilanes, M., Costa-Mont automatic natural language generation of spanish texts. Expe 5. Palmirani, M., Bincoletto, G., Leone, V., Sapienza, S. & Sovrano, F. Hybrid refining approach of pronto ontology. In Internationa
Conference on Electronic Government and the Information Systems Perspective, 3–17 (Springer, 2020). 45. Palmirani, M., Bincoletto, G., Leone, V., Sapienza, S. & Sovrano, F. Hybrid refining approach of pronto ontology. In
C
f
El t
i G
t
d th I f
ti
S t
P
p ti
3 17 (S
i
2020) 45. Palmirani, M., Bincoletto, G., Leone, V., Sapienza, S. & Sovrano, F. Hybrid refining approach of pronto ontology. In International
Conference on Electronic Government and the Information Systems Perspective, 3–17 (Springer, 2020). ,
,
,
,
,
,
p
,
,
yi
g pp
p
gy
Conference on Electronic Government and the Information Systems Perspective, 3–17 (Springer, 2020). Competing interests h p
g
The authors declare no competing interests. www.nature.com/scientificreports/ In INLG’2000
Proceedings of the First International Conference on Natural Language Generation, 186–193, https://doi.org/10.3115/1118253.11182
79 (Association for Computational Linguistics, Mitzpe Ramon, Israel, 2000).i 64. Cheng, H. & Mellish, C. Capturing the interaction between aggregation and text planning in two generation systems. In INLG’2000
Proceedings of the First International Conference on Natural Language Generation, 186–193, https://doi.org/10.3115/1118253.11182
79 (Association for Computational Linguistics, Mitzpe Ramon, Israel, 2000).i p
g
p
5. Kennedy, C. & McNally, L. Scale structure, degree modification, and the semantics of gradable predicates. Language 345–381
(2005). 6. Angeli, G., Liang, P. & Klein, D. A simple domain-independent probabilistic approach to generation. In Proceedings of the 2010
Conference on Empirical Methods in Natural Language Processing, 502–512 (2010). 66. Angeli, G., Liang, P. & Klein, D. A simple domain-independent probabilistic approach to generation. In Proceedings of the 2010
Conference on Empirical Methods in Natural Language Processing, 502–512 (2010). f
p
g
g
g
67. Kondadadi, R., Howald, B. & Schilder, F. A statistical nlg framework for aggregated planning and realization. In Proceedings of the
51st Annual Meeting of the Association for Computational Linguistics (Volume 1: Long Papers), 1406–1415 (2013). 7. Kondadadi, R., Howald, B. & Schilder, F. A statistical nlg framework for aggregated planning and realization. In Proceedings of the
51st Annual Meeting of the Association for Computational Linguistics (Volume 1: Long Papers), 1406–1415 (2013). 8. Gatt, A. & Krahmer, E. Survey of the state of the art in natural language generation: Core tasks, applications and evaluation. J
Artif. Int. Res. 61, 65–170 (2018). www.nature.com/scientificreports/ www.nature.com/scientificreports/ 55. Hou, S., Zhang, S. & Fei, C. Rhetorical structure theory: A comprehensive review of theory, parsing methods and applications. Expert Syst. Appl. 157, 113421. https://doi.org/10.1016/j.eswa.2020.113421 (2020). p
y
pp
p
g
j
(
)
56. Reiter, E. & Dale, R. Building Natural Language Generation Systems (Cambridge University Press, 2000).i g
g
g
y
g
y
57. Bouayad-Agha, N., Casamayor, G., Wanner, L. & Mellish, C. Overview of the first content selection challenge from open semantic
web data. In Proceedings of the 14th European Workshop on Natural Language Generation, 98–102 (Association for Computational
Linguistics, Sofia, Bulgaria, 2013). gi
g
8. Wanner, L. et al. Getting the environmental information across: from the web to the user. Expert. Syst. 32, 405–432. https://doi
org/10.1111/exsy.12100 (2015). g
y
9. Kutlak, R., Mellish, C. & van Deemter, K. Content selection challenge - University of Aberdeen entry. In Proceedings of the 14th
European Workshop on Natural Language Generation, 208–209 (Association for Computational Linguistics, Sofia, Bulgaria, 2013) p
p
g
g
(
p
gi
g
)
60. Barzilay, R. & Lee, L. Catching the drift: Probabilistic content models, with applications to generation and summarization. In
Proceedings of the Human Language Technology Conference of the North American Chapter of the Association for Computational
Linguistics: HLT-NAACL 2004, 113–120 (Association for Computational Linguistics, Boston, Massachusetts, USA, 2004). 60. Barzilay, R. & Lee, L. Catching the drift: Probabilistic content models, with applications to generation and summarization. In
Proceedings of the Human Language Technology Conference of the North American Chapter of the Association for Computational
Linguistics: HLT-NAACL 2004, 113–120 (Association for Computational Linguistics, Boston, Massachusetts, USA, 2004). g
p
g
61. Lapata, M. Automatic evaluation of information ordering: Kendall’s tau. Comput. Linguist. 32, 471–484. https://doi.org/10.1162/
coli.2006.32.4.471 (2006). 62. Bollegala, D., Okazaki, N. & Ishizuka, M. A bottom-up approach to sentence ordering for multi-document summarization. In
Proceedings of the 21st International Conference on Computational Linguistics and 44th Annual Meeting of the Association for Com-
putational Linguistics, 385–392, https://doi.org/10.3115/1220175.1220224 (Association for Computational Linguistics, Sydney,
Australia, 2006). 63. Walker, M. A., Rambow, O. & Rogati, M. SPoT: A trainable sentence planner. In Second Meeting of the North American Chap
the Association for Computational Linguistics (2001). 64. Cheng, H. & Mellish, C. Capturing the interaction between aggregation and text planning in two generation systems. www.nature.com/scientificreports/ h
f h
f h
l l
k
l
d
l i
Conference on Electronic Government and the Information Systems Perspective, 3–17 (Springer, 2020). 6. Gatt, A. & Krahmer, E. Survey of the state of the art in natural language generation: Core tasks, applications and evaluation. J
Artif. Int. Res. 61, 65–170 (2018). 47. Martínez Jiménez, J. A., Muñoz Marquina, F. & Sarrió Mora, M. Á. Lengua Castellana y Literatura, 36 (Akal Sociedad Anónima,
Madrid, España, Madrid, 2011). p
arraza Verdesoto, E. S., Rivas Trujillo, E. & Rodríguez Molano, J. I. Model texts with svo sentences as a system composed. struc-
ure such as the spanish language. International Journal of Mechanical and Production Engineering Research and Development10,
6111 16118 htt
//d i
/10 24247/ij
dj
20201528 (2020) 48. Barraza Verdesoto, E. S., Rivas Trujillo, E. & Rodríguez Molano, J. I. Model texts with svo sentences as a system composed. struc-
ture such as the spanish language. International Journal of Mechanical and Production Engineering Research and Development10,
16111–16118, https://doi.org/10.24247/ijmperdjun20201528 (2020). h
l
b
l
( h
l
) 49. Cohn, P. M. Algebra, vol. 3 (Jhon Wiley & Sons, 1991). g
(J
y
)
50. Miles, R. & Hamilton, K. Learning UML 2.0 (O’Reilly, 2006). 50. Miles, R. & Hamilton, K. Learning UML 2.0 (O’Reilly, 2006 51. Bick, E. A constraint grammar-based parser for spanish. In TIL (2006). 52. Barraza Verdesoto, E. S., Rivas Trujillo, E., Medina García, V. H. & Cardona Sánchez, D. Algebraic model to formalize sentences
and their context: Use case scenario of the spanish language. In Applied Computer Sciences in Engineering, 182–193, https://doi.
org/10.1007/978-3-030-00350-0_16 (2018).h 53. Mann, W. C. & Thompson, S. A. Rhetorical structure theory: Toward a functional theory of text organization. Text - Interdiscip. J. Study Discourse 8, 243–281. https://doi.org/10.1515/text.1.1988.8.3.243 (1988). y
p
g
54. Mabona, A., Rimell, L., Clark, S. & Vlachos, A. Neural generative rhetorical structure parsing. arXiv preprint arXiv:1909.1
(2019). https://doi.org/10.1038/s41598-022-26304-w Scientific Reports | (2022) 12:22249 | Author contributions Edgardo Barraza is the main author, he generated the main idea of the paper and its proof previously published
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Historical contingency and spatial processes rather than ecological niche differentiation explain the distribution of invasive goldenrods (Solidago and Euthamia)
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Plant ecology
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Plant Ecol (2016) 217:565–582
DOI 10.1007/s11258-016-0601-1 Historical contingency and spatial processes rather
than ecological niche differentiation explain the distribution
of invasive goldenrods (Solidago and Euthamia) Magdalena Szymura . Tomasz H. Szymura Received: 19 January 2016 / Accepted: 5 April 2016 / Published online: 13 April 2016
The Author(s) 2016. This article is published with open access at Springerlink.com the species that was first introduced occupied available
habitats and prevented the establishment of other
species. Solidago canadensis was distributed ran-
domly throughout the entire region and did not differ
from S. altissima in its ecology. Euthamia graminifo-
lia differed from the other goldenrods in habitat
preferences and environmental requirements. Its dis-
tribution was strongly spatially structured, clumping
around initial infestation sites. The extents of the
differences resulting from ecological niche differen-
tiation and the limitation of long-range dispersal are
unclear. We argue that the under-representation of E. graminifolia in abandoned fields originates from the
inhibition of establishment in habitats already occu-
pied by other goldenrods. The results underline that
the effect of historical contingency can be more
prominent than ecological niche differentiation on
invasive
species
distribution
and
invasion
effectiveness. Abstract
The effectiveness of invasion depends on
the interacting environmental factors and biology of
the invader. The effects of these interactions are
contingent on their contexts and difficult to predict. Among the most successful plant invaders in Europe
are the goldenrods (Solidago and Euthamia genera). We assessed the roles of ecological niche differenti-
ation and historical contingency on their distribution
in Silesia (Central Europe, studied area approximately
32,000 km2). The distributions of the two most
common species (S. gigantea and S. altissima) were
clumped, and the species dominated different areas. The distribution was unrelated to ecological niche
differences but originates rather from the history of
invasion and subsequent density-dependent spread; Communicated by Scott J Meiners. Electronic supplementary material
The online version of
this article (doi:10.1007/s11258-016-0601-1) contains supple-
mentary material, which is available to authorized users. Keywords
Biological invasions Dispersal
limitation Ecological contingency Habitat
preferences Invasion effectiveness Local
distribution Priority effect Range infilling Keywords
Biological invasions Dispersal
limitation Ecological contingency Habitat
preferences Invasion effectiveness Local
distribution Priority effect Range infilling M. Szymura (&)
Department of Agroecosystems and Green Areas
Management, Wrocław University of Environmental and
Life Sciences, Grunwaldzki Sq. 24A, 50-363 Wrocław,
Poland M. Szymura (&)
Department of Agroecosystems and Green Areas
Management, Wrocław University of Environmental and
Life Sciences, Grunwaldzki Sq. 24A, 50-363 Wrocław,
Poland Introduction 2013; Vannette and
Fukami 2014; Marchante et al. 2015; Young et al. 2015). Numerous studies of species’ interactions
indicate that the order of arrival into an ecosystem,
the so-called priority effect (Shulman et al. 1983), can
influence local community assembly (Fukami 2010;
Grman and Suding 2010; Kardol et al. 2013; Putten
et al. 2013; Zefferman 2015; Fraser et al. 2015). As a
result, ecological contingencies can govern the suc-
cess of colonisation of dispersing individuals and limit
the distribution of invasive species (Ricklefs 2010;
Fraser et al. 2015). The priority effect should be
particularly strong when interacting species are sim-
ilar in resource use, or when the early-arriving species
strongly affect the environment, and the late-arriving
species have resource high requirements (Vannette
and Fukami 2014). The native ranges of these species partially overlap
but differ in extent: S. altissima has the largest range,
the ranges of S. gigantea and E. graminifolia are
similar but narrower than that of S. altissima, while the
range of S. canadensis is relatively the smallest
(Weber and Schmid 1998; Weber 2001; Semple and
Cook 2006). They are considered as broad tolerant in
terms of soil characteristics, but S. gigantea is usually
associated with moist to wet habitats, while the S. altissima is sensitive to flooding (Weber 2000; Abra-
hamson et al. 2005; Weber and Jakobs 2005). In the
native range, several goldenrod species can co-occur,
but the particular combination of the species present at
one site can vary according to site conditions (Rhoads
and Block 2000; Abrahamson et al. 2005). Observa-
tion at the local scale in Pennsylvania, USA, showed
that S. altissima and S. gigantea were associated with
neutral soils, whereas E. graminifolia achieved high
abundance on acidic soils. S. altissima occurred more
often on well-drained soils, S. gigantea on moister
soils with stable water levels over time, whereas E. graminifolia preferred clay-rich soils. In Europe, S. altissima and S. gigantea are also
considered as broad tolerant in terms of soil and
climate conditions. They primarily invade abandoned
fields and meadows as well as ruderal and disturbed
habitats (Weber 2000; Weber and Jakobs 2005;
Szymura and Szymura 2013; Bartha et al. 2014). Nonetheless, it appears that S. altissima prefers drier
sites occurring along roadsides, railway lines and
disturbed places around settlements, while S. Introduction T. H. Szymura
Department of Ecology, Biogeochemistry and
Environmental Protection, University of Wrocław, Maksa
Borna Sq. 9, 50-328 Wrocław, Poland Invasions of alien species alter biodiversity, landscape
structure, ecosystem functions and services, and the 12 3 Plant Ecol (2016) 217:565–582 566 2003). Four alien goldenrod species (Solidago and
Euthamia) invade Central Europe: S. gigantea Aiton,
S. canadensis L., S. altissima L. (S. canadensis var. scabra (Muhl.) Torr. and Gray) and E. graminifolia
(L.) Nutt. Alien Solidago species cause the decrease of
plant species richness (Hejda et al. 2009; Del Fabbro
et al. 2013; Fenesi et al. 2015a; Pal et al. 2015), alter
spontaneous succession (Bornkamm 2007; Bartha
et al. 2014) and negatively influence the diversity of
birds (Sko´rka et al. 2010) and insects (Moron´ et al. 2009). Due to their locally high abundance and
substantial environmental impact, their populations
need to be controlled (Sheppard et al. 2006; Sko´rka
et al. 2010; Fenesi et al. 2015a). local economy, as well as human health and well-
being (Chapuis-Lardy et al. 2006; Pejchar and
Mooney 2009; Hejda et al. 2009; Pysˇek and Richard-
son 2010; Vila` et al. 2011; Hulme et al. 2014, 2015). The total number of invasive species is still increasing,
and habitats may be invaded by multiple species
(Kuebbing et al. 2013; Kuebbing and Nun˜ez 2015). Thus, knowledge about the interactions between non-
native species becomes critical for understanding their
distribution and abundance, particularly from the
perspectives of nature conservation and management
(Kuebbing et al. 2013; Kuebbing and Nun˜ez 2015). Anthropogenic species movement is the primary
driver of plant invasion (Chytry´ et al. 2008; Pysˇek and
Richardson 2010; Kuebbing and Nun˜ez 2015). How-
ever, the subsequent effectiveness of invasion, con-
sidered as the efficient distribution and habitat infilling
within the new range and/or the abundance of
individuals, is driven by the interaction of environ-
mental factors (land-use system, climate, habitat
disturbances and resident vegetation) and the biology
of the invasive species (Lake and Leishman 2004;
Pysˇek and Richardson 2008; Catford et al. 2009; van
Kleunen et al. 2010; Essl et al. 2011; Johnson et al. 2012; Warren et al. 2013). The effects of these
interactions are highly contingent on their context
(Chamberlain et al. 2014), e.g. plant community
composition of the new territory, specifically the
presence of interacting species, availability of suit-
able space and resources, and the time a population has
been established (Putten et al. Introduction gigantea
is more frequent on river banks and moist areas often
connected with river networks (Guzikowa and May-
cock 1986; Weber 2001, 2011; Weber and Jakobs
2005). The presence of E. graminifolia was related to One of the most successful worldwide plant
invaders are the goldenrods (Pysˇek 1998; Weber 123 123 Plant Ecol (2016) 217:565–582 567 limitation and historical contingency in determining
the distribution of invasive goldenrods in Silesia,
Central Europe. We tested the differences among
species with respect to the following aspects: (a) habi-
tat preferences of studied species, (b) composition of
co-occurring vascular plant species, (c) climatic and
topographical variables and (d) soil properties. To
assess the potential role of priority effects and the
subsequent density-dependent processes that lead to
infilling by one species in a particular area, we
modelled the spatial structure of plot locations, effects
of distance from initial infestation sites and the
environmental heterogeneity on goldenrod distribu-
tion. Finally, the quantitative effects of each of these
factors on species distribution were separated. moist and wet habitats disturbed by humans (Guzi-
kowa and Maycock 1986). There is a lack of systematic measurements of
primary environmental gradients that can be used for
testing the hypothesis concerning the distribution of
invasive goldenrods or for modelling their distribu-
tion. This is important because an increase of abun-
dance and infilling available habitats within the
species secondary range is occurring (Szymura and
Szymura 2011; Bartha et al. 2014; Fenesi et al. 2015a,
b). The relationship of a particular goldenrod species
distribution with environmental conditions is unclear,
particularly the recently observed tendency of S. gigantea to occupy drier habitats (Gu¨sewell et al. 2005; Weber and Jakobs 2005; La´nı´kova´ et al. 2009;
Bartha et al. 2014). The establishment of goldenrods from seedlings on
abandoned lands occurs soon after the abandonment,
whereas germination of seeds in undisturbed, well-
established native vegetation is rather infrequent
(Hartnett and Bazzaz 1985; Bartha et al. 2014; Fenesi
et al. 2015b). After establishing at a new site, the
subsequent spread of goldenrods is almost exclusively
clonal via horizontal rhizomes, and the death of an
established genet is a rare event (Meyer and Schmid
1999a, b). The age of single genets of S. altissima and
S. canadensis has been reported to be 20–100 years
(Whitham 1983; Carson and Root 2000). Conse-
quently, goldenrods last for a long time during mid-
successional stages of vegetation (Cain 1990; Wise
et al. Introduction 2006; Bornkamm 2007; Bartha et al. 2014;
Fenesi et al. 2015a), and in such well-established
stands, the recruitment of new individuals from seeds
is rather unlikely (Hartnett and Bazzaz 1985; Meyer
and Schmid 1999a). Therefore, it could be hypothe-
sised that the pattern of goldenrod distribution is
influenced by propagule pressure related to the
invasion history; species that occur first may infill
the available habitats and dominate the local vegeta-
tion. Since the number of goldenrod populations is
increasing, the constant presence of their propagules
increases the probability of successful colonisation
when a gap occurs. Because goldenrods are similar
with respect to the use of environmental resources and
strongly affect the environment, the historical contin-
gency caused by priority effect should restrict their
distribution. Studied species Studied species We studied four alien goldenrod species (Solidago and
Euthamia): S. gigantea Aiton, S. canadensis L., S. altissima L. and E. graminifolia (L.) Nutt. E. gramini-
folia is most commonly recognised in Europe as
Solidago graminifolia (L.) Elliot; however, based on
anatomical and DNA studies (Semple et al. 1981,
1984), the taxon should be classified to Euthamia Nutt. genus. The taxonomical problem concerned also
considers S. canadensis and S. altissima as separate
taxa. In several articles, these taxa are reported as two
varieties of S. canadensis s.l.: var. canadensis and var. scabra (Guzikowa and Maycock 1986; Weber 1997;
Weber and Schmid 1998). Among the primary traits
that differ between S. altissima and S. canadensis, the
length and arrangement of hair covering the stem and
leaves and the shape and margins of the leaves (Weber
1997; Semple and Cook 2006), micro-morphological
characteristics of leaf epidermis (Szymura and Wolski
2011) and rhizome systems (Schmid et al. 1988) have
been described. In this study, the aforementioned
species were separated into S. canadensis and S. altissima. Goldenrods were introduced from North America
in the eighteenth century and distributed to gardens in
different parts of Europe (Hitchmough et al. 2004). After a short time, the goldenrods escaped from
cultivation. At present, S. canadensis s.l. and S. gigantea are aggressive invaders that are widespread In this study, we assessed the relative roles of
ecological niche differentiation, long-range dispersal 12 3 3 Plant Ecol (2016) 217:565–582 568 et al. 2005). Each of the plots consisted of a buffer with
a radius of 282 m (area 25 ha) within which the alien
goldenrods were sampled. The centre of the buffer and
the buffer extent were determined in the field using a
GPS receiver. The land use/land cover (LULC) in the
centre of the buffer was noted. If a population of alien
goldenrods was found, the vegetation was sampled. The sampling plot was sized 10 9 10 m (area
100 m2), and the cover of all vascular plants found
was assessed using the Braun–Blanquet abundance
scale. The nomenclature of plant species fallows to
Mirek et al. (2002). The Polish vegetation database
housed all the vegetation data (Kacki and Sliwinski
2012). Plots were established wherever Solidago first
occurred, regardless of its abundance. If a few
separated populations of a goldenrod species were
found within the buffer, the stand placed nearest the
buffer centre was sampled. Studied species If another species of alien
goldenrods, forming a separate stand, was found, an
additional sampling plot was established. By this way,
in a particular buffer, no sampling plot (absence of
goldenrods), a plot with a single goldenrod taxon, a
plot with two or more goldenrod species or separate
plots with different goldenrod taxa in each could be
established. For each sampling plot, the LULC was
noted, and the cover of trees and shrub canopy was
visually assessed (canopy). The climatic conditions in Europe (Weber and Schmid 1998; Weber 2001;
Schlaepfer et al. 2008). In contrast, E. graminifolia is
present only at a few localities in Europe but can form
dense, monospecific stands (Weber and Schmid 1998;
Kompała-Ba˛ba and Ba˛ba 2006; Dajdok and Nowak
2007; Szymura and Szymura 2013). In Poland, the
occurrence of S. gigantea was noted in 1853, S. canadensis s.l. in 1872 and E. graminifolia in 1885
(Guzikowa and
Maycock
1986; Tokarska-Guzik
2003). Studied area and sampling plot design Studied area and sampling plot design The fieldwork was performed in the Silesia region
(Poland, Central Europe, sampled area approximately
32,000 km2, Fig. 1a). The studied area was mostly
lowlands with a rather small portion of foothills and
mountains (Fig. 1b); the land was mostly used for
agriculture (approximately 64 %) and forestry (ap-
proximately 28 %). Within the study region (Fig. 1a),
we established 309 plots, placed at the nodes of
10 9 10 km regular grid. The altitudinal range of
plots was from 50 to 1100 m above sea level; however,
most of the plots were placed at an altitude below the
300 m a.s.l. The total annual precipitation varied
between 533 and 858 mm, and the average annual
temperature varied between 3.5 and 9.2 C (Hijmans Fig. 1 Studied region
(panel a, dark grey) and
pattern of historical (before
1939) infestation of
particular goldenrod species
(according to Guzikowa and
Maycock 1986) in Silesia on
the background of altitude
and main rivers (panel b) Fig. 1 Studied region
(panel a, dark grey) and
pattern of historical (before
1939) infestation of
particular goldenrod species
(according to Guzikowa and
Maycock 1986) in Silesia on
the background of altitude
and main rivers (panel b)
123 Fig. 1 Studied region
(panel a, dark grey) and
pattern of historical (before
1939) infestation of
particular goldenrod species
(according to Guzikowa and
Maycock 1986) in Silesia on
the background of altitude
and main rivers (panel b) 12 12 3 Plant Ecol (2016) 217:565–582 569 occurred. Then, the difference between the proportion
of goldenrods and those in the buffer centre with
respect to particular species and habitat type was
calculated. A negative value indicates that a particular
goldenrod taxon was under-represented in a given
habitat type, whereas a positive value indicated that it
was over-represented. The significance of these
differences compared to random was assessed with a
v2 test. [average annual temperature (tempaver), the maximal
temperature of the warmest month (tempmax), the
minimal temperature of the coldest month (tempmin)
and the annual sum of precipitation (precipitation)]
were determined from the climatic model of Hijmans
et al. (2005). Using the digital elevation model (Jarvis
et al. 2008), the altitude was defined and the
topographic wetness index (TWI) was calculated. Based on the LULC in the buffer centres and in the
sampling plots, LULC was categorised. Studied area and sampling plot design A total of 11
types of habitats were distinguished: (1) urban,
industrial and transport areas (urbanised areas); (2)
unpaved roads as footpaths, skidder trail in forests and
sporadically used agricultural roads (unpaved roads);
(3) drainage ditches and strips of land along paved
roads
(road
verges);
(4)
habitat
edges,
e.g. forest/grassland ecotones; (5) various anthropogenic
vegetation types, other than agriculture—allotments,
orchards, gardens, parks (green areas); (6) meadows
and pastures (grasslands); (7) forests and scrublands;
(8) rivers, ditches, embankments or escarpments; (9)
arable fields; (10) unmanaged strips of lands along
fencing and other types of borders (property bound-
aries); and (11) abandoned lands. Sampling of the
plots gave us knowledge about the frequency and
distribution of each goldenrod species. For 78 sam-
pling plots selected using a stratified-random method,
topsoil samples were collected. As strata, species
identity and geographical space were used. We tended
to sample as much as possible of rare species (E. graminifolia and S. canadensis) and to obtain spatially
diversified In additionsamples of common species (S. altissima and S. gigantea). The selected 78 plots were
the ‘topsoil subset’. Soil samples from the top 20 cm
were taken with a soil auger from four randomly
chosen positions and mixed into one sample repre-
senting the plot. Then the subsamples collected from
upper soil layer (approximately 20 cm) were mixed
into one sample representing the plot. In the labora-
tory, the samples were assessed for soil texture, pH of
H2O, carbon (C), total nitrogen (N), phosphorus (P),
potassium (K), magnesium (Mg) and calcium (Ca)
content. The tendency of the studied species to cluster or
occur with a regular or random distribution was tested
using Ripley’s Kd function. To verify whether the patches of vegetation invaded
by particular goldenrod taxa differed with respect to
vascular plant species composition, we analysed the
similarity (ANOSIM) and, additionally, performed an
ordination with non-metric multidimensional scaling
(NMDS) using Bray–Curtis distance. As a grouping
factor in the ANOSIM, the occurrence of particular
goldenrod taxa was used; in the case of plots invaded
by multiple goldenrod species, we classified it as a
‘mixture’. Because the results of NMDS ordination
would be under the strong influence of the presence of
particular goldenrod taxa, they were excluded from the
analysis. Studied area and sampling plot design The significance of differences in environmental
traits across species was tested using permutation and
Kruskal–Wallis analysis of variance on ranks with a
Nemenyi–Damico–Wolfe–Dunn post hoc test (Hol-
lander and Wolfe 1999; Hothorn et al. 2013). For all plots, the shortest distances to the initial
invasion location in Silesia were calculated. Data
regarding the initial invasion (distribution before WW
II) were derived from Guzikowa and Maycock (1986;
Fig. 1b). As the authors of that article did not
distinguish S. altissima, we calculated the distances
to the locations of initial infestations by E. gramini-
folia (minEut), S. gigantea (minGig) and S. canaden-
sis sensu lato (minCan). For modelling goldenrod spatial distributions, we
used the principal coordinate analysis of neighbour
matrices (PCNMs; Borcard and Legendre 2002; Dray
et al. 2006). The geographical coordinates of the plots
were used to compute PCNM, from which vectors that
represented statistically significant positive spatial cor-
relations were selected, using Moran’s I (Borcard and
Legendre 2002; Borcard et al. 2004). The PCNM vectors
were calculated independently for the entire dataset
(N = 232) as well as for the topsoil subset (N = 78). Statistical methods The percentage of different habitat types found in the
buffer centres was calculated, including the percent-
age of habitats where particular goldenrod species 12 3 3 570 Plant Ecol (2016) 217:565–582 All environmental data, distances to initial invasion
sites and the PCNM vectors were used as explanatory
variables in a canonical correspondence analysis
(CCA) of alien goldenrod distribution using pres-
ence/absence data. Two independent analyses were
performed. First, using the all predictor variables for
the entire dataset. The second analysis used the canopy,
topsoil properties, the shortest distances and PCNM
vectors for the topsoil subset. Within the analysis,
models exclusively for environmental data (environ-
ment) were separately constructed, which were used as
the explanatory variables solely for the shortest
distances (distance) and for the spatial data alone
(space) as explanatory variables. In these models, using
forward selection procedures, the variables that signif-
icantly shaped alien goldenrod distribution were
selected. Finally, combined models, including signif-
icant environmental, spatial and distance variables,
were constructed. For these combined goldenrod
distribution models, variance partitioning (Borcard
et al. 1992; Peres-Neto et al. 2006) was used to quantify
the amount of variation explained by the three different
groups of factors (space, environment and distance). In
addition, correlations between the site scores obtained
in the CCA models with space as explanatory variables
on one hand and species data, environment and the
distance group of variables on the other hand were
checked to determine which species and/or which
variables were associated with the spatial structures of
goldenrod population. The significance of the correla-
tions was checked using the Monte Carlo permutation
method (Hothorn et al. 2013). Solidago canadensis occurred most often as a few
ramets in the 10 9 10 m plots (‘?’ according to the
Braun–Blanquet scale), S. altissima and S. gigantea
mostly covered up to 5 % of the plot (‘1’ in B–B
scale), while E. graminifolia covers most often
5–25 % of the plot area (‘2’ in B–B scale). However,
S. altissima and S. gigantea in some plots can cover
more than 50 % of the plot (‘4’ and ‘5’ according to B–
B scale, Online Resource A). The distributions of S. gigantea and S. altissima
were clustered (values of Ripley’s Kd function above
random ranges, Fig. 3), and their ranges did not
overlap entirely within the studied region. S. canaden-
sis was randomly distributed throughout the region. The range of E. Statistical methods graminifolia was restricted to one area,
but within that range, its distribution was random. Goldenrods were most often found on abandoned
lands and road verges. The number of plots for
particular species and habitat types is listed in Online
Resource B. All species avoided arable fields and
urbanised areas but were over-represented on road
verges (Fig. 4). We observed differences between the
percentage of habitats invaded by E. graminifolia and
other species. E. graminifolia was over-represented on
unpaved roads and, in contrast to other species, under-
represented on abandoned lands but did not signifi-
cantly avoid forests and scrublands. The most frequent species co-occurring with the
goldenrods were Cirsium arvense (57.3 %), Artemisia
vulgaris (56.9 %), Urtica dioica (50.0 %), Achillea Fig. 2 Percentage of plots with particular goldenrod species
growing in monocultures or mixtures with other goldenrod taxa. In total, 232 plots were sampled with one or more goldenrod
species on each Statistical analyses were performed using ‘vegan’
and ‘coin’ packages in R environment (Oksanen et al. 2008; Hothorn et al. 2013) and PASSAGE (Rosenberg
and Anderson 2011) software for spatial analysis. Fig. 3 Maps of the studied
species distribution (grey
dots) and values of Ripley’s
Kd function of different
spatial extents. The grey
area on Ripley’s Kd graphs
shows ranges for random
distribution. The crosses on
the maps denote the centres
of the studied buffers Results Among the analysed 309 buffers, 232 sampling plots
were established. Monospecific stands of S. gigantea
(N = 92, 40 %) or S. altissima (N = 88, 38 %) were
found in most of the plots. In contrast, plots with a
mixture of these two species (N = 23, 10 %) rarely
occurred. The two remaining species, S. canadensis
(N = 21) and E. graminifolia (N = 9), were much
rarer and mostly occurred in stands along with other
goldenrod species (Fig. 2). Fig. 2 Percentage of plots with particular goldenrod species
growing in monocultures or mixtures with other goldenrod taxa. In total, 232 plots were sampled with one or more goldenrod
species on each 12 123 Plant Ecol (2016) 217:565–582 571 and
Dactylis
glomerata
cies found in the sample plots
Resource C. The results of
there were no significant
06, p = 0.660) between the
rent goldenrod species with
nt species composition. The
plots invaded by particular
upper panel) was consistent
with particular species composition. The exception
was the tendency of E. graminifolia to concentrate on
the right side of the graph with forest species. The
ordination of species (Fig. 5, lower panel) suggests
that the first axis is related to the gradient from
frequently disturbed habitats (left wing of the graph,
arable weeds and annual or biennial ruderal species
such
as
Tripleurospermum
inodorum,
Conyza
canadensis
and
Setaria
viridis)
through
mead- Fig. 3 Maps of the studied
species distribution (grey
dots) and values of Ripley’s
Kd function of different
spatial extents. The grey
area on Ripley’s Kd graphs
shows ranges for random
distribution. The crosses on
the maps denote the centres
of the studied buffers millefolium
(49.1 %)
and
Dactylis
glomerata
(43.1 %). All of the species found in the sample plots
are listed in Online Resource C. The results of
ANOSIM showed that there were no significant
differences (R = -0.006, p = 0.660) between the
plots invaded by different goldenrod species with
respect to vascular plant species composition. The
NMDS ordination of plots invaded by particular
goldenrod taxa (Fig. 5, upper panel) was consistent
with the results of ANOSIM; that is, the goldenrod
species did not have any tendency to form clusters
with particular species composition. The exception
was the tendency of E. graminifolia to concentrate on
the right side of the graph with forest species. The
ordination of species (Fig. Results 5, lower panel) suggests
that the first axis is related to the gradient from
frequently disturbed habitats (left wing of the graph,
arable weeds and annual or biennial ruderal species
such
as
Tripleurospermum
inodorum,
Conyza
canadensis
and
Setaria
viridis)
through
mead-
ows/grassland vegetation (centre of the graph, Ar-
rhenatherum
elatius,
D. glomerata
and
Lolium
Kd function of different
spatial extents. The grey
area on Ripley’s Kd graphs
shows ranges for random
distribution. The crosses on
the maps denote the centres
of the studied buffers with particular species composition. The exception
was the tendency of E. graminifolia to concentrate on
the right side of the graph with forest species. The
ordination of species (Fig. 5, lower panel) suggests
that the first axis is related to the gradient from
frequently disturbed habitats (left wing of the graph,
arable weeds and annual or biennial ruderal species
such
as
Tripleurospermum
inodorum,
Conyza
canadensis
and
Setaria
viridis)
through
mead-
ows/grassland vegetation (centre of the graph, Ar-
rhenatherum
elatius,
D. glomerata
and
Lolium millefolium
(49.1 %)
and
Dactylis
glomerata
(43.1 %). All of the species found in the sample plots
are listed in Online Resource C. The results of
ANOSIM showed that there were no significant
differences (R = -0.006, p = 0.660) between the
plots invaded by different goldenrod species with
respect to vascular plant species composition. The
NMDS ordination of plots invaded by particular
goldenrod taxa (Fig. 5, upper panel) was consistent
with the results of ANOSIM; that is, the goldenrod
species did not have any tendency to form clusters 12 3 3 Plant Ecol (2016) 217:565–582 572 Fig. 4 Differences between the percentages of habitat types
calculated for the goldenrods and the buffer centres. The
positive values reveal over-representation of goldenrod species
in the particular type, while the negative values show under-
representation. The asterisks show statistical significance Fig. 4 Differences between the percentages of habitat types
calculated for the goldenrods and the buffer centres. The
positive values reveal over-representation of goldenrod species
in the particular type, while the negative values show under-
representation. The asterisks show statistical significance Fig. 4 Differences between the percentages of habitat types
calculated for the goldenrods and the buffer centres. The
positive values reveal over-representation of goldenrod species Fig. 4 Differences between the percentages of habitat types
calculated for the goldenrods and the buffer centres. 123 Results The
positive values reveal over-representation of goldenrod species
in the particular type, while the negative values show under-
representation. The asterisks show statistical significance Fig. 5 Results of NMDS ordination. The upper panel shows the
ordination of the plots. Because of the low number of plots with E. graminifolia (asterisks) and S. canadensis (grey trapeze) in these
two groups, plots where these two species occur in combination
with S. altissima and/or S. gigantea were also included. The group
mixture (grey dots) consists of plots with a mixture of S. gigantea
and S. altissima, exclusively. The lower panel shows the
ordination of the species. For legibility, only the species,
distribution of which was best explained by the ordination model,
and/or the most frequent species were shown. The species labels
consist of the first three letters of genus and species Latin names. The full names are given in Online Resource C perenne) up to relatively undisturbed forest vegetation
(right wing, tree and shrub species such as Sorbus
aucuparia, Corylus avellana and Quercus robur). The
second axis can be interpreted as decreasing moisture
gradient from moderate (upper part of the graph,
Oxalis acetosella, Pinus sylvestris and Equisetum
pratense) to wet habitats (lower part, Phragmites
australis, Stellaria media and Filipendula ulmaria). Goldenrods occurred in a wide range of soils with
respect to pH, nutrient content and texture. We found
significant differences between taxa with respect to
environmental variables (Table 1). E. graminifolia
occurred in more over-shaded plots than S. altissima
and S. canadensis; however, the latter species were
also sporadically found under canopy cover. We also
found differences between E. graminifolia and the
remaining
species
with
respect
to
the
average precipitation (S. gigantea) and minimum temperature
(S. altissima and S. canadensis); nonetheless, the
ranges of these climatic variables overlapped among
species. Analysis of the topsoil subset also revealed
differences of E. Results graminifolia in lower K and Mg
contents and a tendency to occur on more sandy soils 123 123 Plant Ecol (2016) 217:565–582 573 Table 1 The average ± standard deviation and range (in parenthesis) of environmental variables for each goldenrod species, the
shortest distance to the initial infestation location (distance) and the results of statistical tests (v2, p) Table 1 The average ± standard deviation and range (in parenthesis) of environmental variables for each goldenrod species, the
shortest distance to the initial infestation location (distance) and the results of statistical tests (v2, p) Table 1 The average ± standard deviation and range (in parenthesis) of environmental variables for each goldenrod species, the
shortest distance to the initial infestation location (distance) and the results of statistical tests (v2, p)
S. altissima
S. canadensis
S. gigantea
E. Results The valu
of PCNM vectors that significantly shaped goldenr
distribution plotted against geographical coordinat
the entire dataset and topsoil subset is not equal to the num
Table 2 Results of canonical correspondence analysis (CC
(R2, F and p) of the entire dataset and topsoil subset
R2
F
p
Entire dataset
Environment
0.031
6.531
0.0
Distance
0.081
10.053
0.0
Space
0.169
6.531
0.0
Topsoil subset
Environment
0.161
4.152
0.0
Distance
0.121
3.402
0.0
Space
0.208
4.786
0.0
123 are presented in Online Resource D. It was found that
not only the distribution of all the studied goldenrod
species but also the canopy, precipitation and the
distance (minCan, minEut) were correlated with the
CCA axis calculated on the basis of PCNM vectors
(Table 3). This implies that the environmental vari-
ables were not randomly distributed throughout the
studied region but spatially structured and that the
PCNM variables also well reflected the distance. The
combined model that joins environment, distance and
space variables significantly explained approximately
19.6 % of goldenrod distribution variability (Fig. 6,
lower panel). The combined model explained the
distribution variabilities of 32.5 % of E. graminifolia,
23.5 % of S. altissima, 15.8 % of S. gigantea and
5.6 % of S. canadensis. The procedure of variation
partitioning showed that space explains far more
variation than the environment. The fraction of
variation explained by pure distance was relatively
less; however, the high amount of explained variation
was shared with space. The fraction of explained
variation shared by these three components concur-
rently was relatively small (Fig. 6, lower panel). The
first ordination axis, explaining most of the variation,
well modelled the distribution of E. graminifolia. The
second axis reflected the differences between S. altissima and S. gigantea (Fig. 6, lower panel). compared to S. altissima as well as on soils with a
higher C/N ratio compared to S. canadensis (Table 1). The results of CCA of the entire dataset and the
topsoil subset are presented in Table 2. The CCA
model results, developed for the entire dataset, using
the environmental variables showed that the distribu-
tion of goldenrods was significantly shaped by
precipitation and canopy. Their distribution was also
significantly influenced by the shortest distance to
initial infestation sites (distance) of E. graminifolia
(minEut) and S. canadensis (minCan). It was found
that the distance explained more variation than the
environment (Table 2). Results graminifolia
Entire dataset
N observations
127
21
129
9
Canopy
11.7b ± 21.4
10.2b ± 20.3
17.2a,b ± 27.7
33.3a ± 28.2
v2 = 9.166
(0.0 to 100.0)
(0.0 to 70.0)
(0.0 to 100.0)
(0.0 to 80.0)
p = 0.024
Precipitation
590.8a,b ± 38.7
592.0a ± 32.3
580.7b ± 34.8
600.7a ± 24.5
v2 = 9.736
(535.0 to 737.0)
(556.0 to 671.0)
(533.0 to 675.0)
(567.0 to 648.0)
p = 0.017
TWI
19.9 ± 2.3
19.5 ± 2.6
19.8 ± 2.0
21.3 ± 0.8
v2 = 7.611
(9.0 to 22.7)
(11.2 to 22.7)
(12.2 to 22.9)
(20.2 to 22.5)
p = 0.051
Altitude
204.9 ± 106.3
218.3 ± 95.8
185.5 ± 83.3
181.0 ± 33.6
v2 = 4.723
(53.0 to 674.0)
(98.0 to 424.0)
(86.0 to 487.0)
(147.0 to 246.0)
p = 0.198
Tempaver
8.1 ± 0.7
8.0 ± 0.6
8.1 ± 0.5
8.3 ± 0.1
v2 = 2.358
(5.4 to 9.2)
(6.7 to 8.9)
(6.4 to 8.9)
(8.1 to 8.4)
p = 0.514
Tempmax
23.1 ± 0.9
23.0 ± 0.8
23.2 ± 0.6
23.3 ± 0.2
v2 = 1.197
(19.2 to 24.5)
(21.3 to 23.8)
(20.9 to 24.2)
(23.1 to 23.5)
p = 0.760
Tempmin
-5.7a ± 1.0
-5.7a ± 0.8
-5.6a,b ± 0.7
-5.0b ± 0.2
v2 = 9.729
(-8.3 to -4.0)
(-7.2 to -4.3)
(-7.7 to -4.0)
(-5.4 to -4.8)
p = 0.019
Distance*
53.2a ± 37.2
20.8b ± 16.9
11.9b ± 6.5
v2 = 80.744
(0.2 to 152.2)
(2.2 to 108.5)
(2.2 to 21.2)
p = 0.000
Topsoil subset
N observations
46
18
43
8
Canopy
12.1 ± 21.1
10.8 ± 22.0
12.2 ± 21.4
31.9 ± 29.8
v2 = 7.145
(0.0 to 80.0)
(0.0 to 70.0)
(0.0 to 80.0)
(0.0 to 80.0)
p = 0.066
pH
5.9 ± 0.8
5.8 ± 0.9
5.8 ± 0.9
5.7 ± 1.1
v2 = 0.502
(4.3 to 7.9)
(4.3 to 8.0)
(4.3 to 8.0)
(4.4 to 7.1)
p = 0.922
C
3.3 ± 3.4
2.1 ± 1.0
3.7 ± 4.0
4.2 ± 3.9
v2 = 3.769
(0.3 to 20.0)
(0.6 to 4.1)
(0.6 to 20.3)
(1.3 to 13.6)
p = 0.296
N
1.6 ± 1.3
1.2 ± 0.4
1.5 ± 0.9
1.3 ± 0.7
v2 = 1.799
(0.3 to 9.1)
(0.7 to 1.8)
(0.6 to 4.7)
(0.6 to 2.9)
p = 0.622
C/N
2.0a,b ± 0.7
1.7b ± 0.5
2.1a,b ± 0.9
2.9a ± 1.1
v2 = 10.485
(0.9 to 4.7)
(0.9 to 2.7)
(0.9 to 5.3)
(1.5 to 4.8)
p = 0.014
P
47.7 ± 58.7
38.7 ± 26.6
47.9 ± 53.8
13.4 ± 8.4
v2 = 7.036
(3.8 to 279.5)
(6.2 to 102.8)
(3.6 to 211.2)
(5.9 to 29.5)
p = 0.069
K
155.2a ± 122.0
141.4a,b ± 96.7
120.2a,b ± 111.5
41.9b ± 26.2
v2 = 11.139
(20.0 to 545.0)
(20.0 to 355.0)
(5.0 to 520.0)
(20.0 to 95.0)
p = 0.011
Mg
98.2a ± 55.7
84.0a,b ± 47.4
80.9a,b ± 64.5
50.7b ± 32.2
v2 = 8.428
(22.2 to 234.8)
(25.4 to 170.0)
(17.1 to 380.6)
(22.2 to 115.0)
p = 0.032
Ca
710.1 ± 883.5
760.9 ± 1135.2
709.5 ± 837.1
1351.0 ± 1664.1
v2 = 0.356
(147.0 to 4206.0)
(170.0 to 4206.0)
(18.0 to 3372.0)
(18.0 to 4206.0)
p = 0.953
Sand
55.4c ± 21.2
55.2b,c ± 22.4
66.8a,b ± 18.7
81.3a ± 5.2
v2 = 14.434
(21.0 to 90.0)
(27.0 to 90.0)
(27.0 to 91.0)
(73.0 to 88.0)
p = 0.001
Silt
41.3a ± 19.4
41.8a,b ± 20.6
31.1b,c ± 17.0
17.6c ± 5.0
v2 = 14.252
(10.0 to 73.0)
(10.0 to 68.0)
(9.0 to 68.0)
(11.0 to 25.0)
p = 0.002
Clay
3.3a ± 2.3
2.9a,b ± 2.0
2.1b,c ± 1.8
1.1c ± 0.4
v2 = 13.386
(0.0 to 12.0)
(0.0 to 6.0)
(0.0 to 8.0)
(1.0 to 2.0)
p = 0.002 12 Plant Ecol (2016) 217:565–582 574 Table 1 continued
S. Results altissima
S. canadensis
S. gigantea
E. graminifolia
Precipitation
595.7 ± 41.9
596.0 ± 33.1
582.7 ± 30.9
601.5 ± 26.0
v2 = 4.484
(540.0 to 720.0)
(556.0 to 671.0)
(536.0 to 671.0)
(567.0 to 648.0)
p = 0.215
Distance*
55.2a ± 39.6
20.4b ± 17.7
13.3b ± 8.9
v2 = 31.595
(7.4 to 152.2)
(2.2 82.8)
(2.3 to 25.9)
p = 0.000
Asterisk indicates the Distance was calculated for S. canadensis s.I. Bold values indicate statistically significant results at p level 0.05
The different superscript letters denote significant differences between pairs of taxa. The sum of observations for particular taxa for
the entire dataset and topsoil subset is not equal to the number of studied plots since two or more species can occur on some plots Bold values indicate statistically significant results at p level 0.05 y
g
p
The different superscript letters denote significant differences between pairs of taxa. The sum of observations for particular taxa for
the entire dataset and topsoil subset is not equal to the number of studied plots since two or more species can occur on some plots The different superscript letters denote significant differences between pairs of taxa. The sum of observations for particular taxa for
the entire dataset and topsoil subset is not equal to the number of studied plots since two or more species can occur on some plots compared to S. altissima as well as on soils with
higher C/N ratio compared to S. canadensis (Table
The results of CCA of the entire dataset and t
topsoil subset are presented in Table 2. The CC
model results, developed for the entire dataset, usi
the environmental variables showed that the distrib
tion of goldenrods was significantly shaped
precipitation and canopy. Their distribution was al
significantly influenced by the shortest distance
initial infestation sites (distance) of E. graminifo
(minEut) and S. canadensis (minCan). It was fou
that the distance explained more variation than t
environment (Table 2). However, the space repr
sented by PCNM vectors better described the va
ability
of
goldenrod
distribution
(Table 2). particular, the first CCA axis adequately modell
the distribution of E. graminifolia (Fig. 6, upp
panel, high positive scores), while the second CC
axis well represented the contrast between the plo
invaded by S. gigantea (high positive scores) and
altissima (moderate and negative scores). Results However, the space repre-
sented by PCNM vectors better described the vari-
ability
of
goldenrod
distribution
(Table 2). In
particular, the first CCA axis adequately modelled
the distribution of E. graminifolia (Fig. 6, upper
panel, high positive scores), while the second CCA
axis well represented the contrast between the plots
invaded by S. gigantea (high positive scores) and S. altissima (moderate and negative scores). The values
of PCNM vectors that significantly shaped goldenrod
distribution plotted against geographical coordinates Table 2 Results of canonical correspondence analysis (CCA)
(R2, F and p) of the entire dataset and topsoil subset
R2
F
p
Entire dataset
Environment
0.031
6.531
0.005
Distance
0.081
10.053
0.005
Space
0.169
6.531
0.005
Topsoil subset
Environment
0.161
4.152
0.005
Distance
0.121
3.402
0.005
Space
0.208
4.786
0.005
123 Table 2 Results of canonical correspondence analysis (CCA)
(R2, F and p) of the entire dataset and topsoil subset The ordination of the topsoil subset showed that
goldenrod distributions were significantly shaped by
C/N ratio and Mg and the percentage of silt in the soil
among the environmental components. The shortest
distances to initial infestation sites (distance) of E. graminifolia (minEut) and S. canadensis (minCan), as
well as the four PCNM vectors, also significantly
influenced goldenrod distribution, when the distance
and space group of factors were analysed (Table 2). The distribution of S. gigantea, S. altissima and E. 12 575 Plant Ecol (2016) 217:565–582 graminifolia correlated with the CCA axis calculated
The first ordination axis again reflected the contrast
Fig. 6 Results of CCA ordination for the entire dataset. On the
upper panel, the values of site scores of CCA with exclusively
spatial variables as constraints plotted against geographical
coordinates of plots are shown. The lower panel shows the
results for the combined model (space, environment and
distance)
of
alien
goldenrod
distribution:
the
variation
partitioning results on the left and the CCA biplot on the right. minCan the shortest distance to the initial infestation location
from S. canadensis s.l. plots; minEut the shortest distance to the
initial infestation location from E. graminifolia plots, E. gram E. graminifolia, S. alti S. altissima, S. cana S. canadensis, S. giga S. gigantea, V2–V30 PCNM vectors Fig. 6 Results of CCA ordination for the entire dataset. On the
upper panel, the values of site scores of CCA with exclusively
spatial variables as constraints plotted against geographical
coordinates of plots are shown. Results The lower panel shows the
results for the combined model (space, environment and
distance)
of
alien
goldenrod
distribution:
the
variation partitioning results on the left and the CCA biplot on the right. minCan the shortest distance to the initial infestation location
from S. canadensis s.l. plots; minEut the shortest distance to the
initial infestation location from E. graminifolia plots, E. gram E. graminifolia, S. alti S. altissima, S. cana S. canadensis, S. giga S. gigantea, V2–V30 PCNM vectors The first ordination axis again reflected the contrast
between the sites invaded by E. graminifolia and the
remaining species, while the second axis represented
the differentiation between S. altissima and S. gigan-
tea (Fig. 7, lower panel). The details of the topsoil
subset analysis are shown in the Online Resource E. graminifolia correlated with the CCA axis calculated
from the PCNM vectors (Table 3). We also found that
silt and Mg, as well as minCan and minEut, were
correlated with the site score of the first CCA axis
calculated using the space components (Table 3). Finally, the combined model of goldenrod distribu-
tions explained 28.3 % of the dataset variation (Fig. 7,
lower panel). The pure space component explained
more variation than solely environment fraction and
distance, while the fraction of shared variation was
relatively high (Fig. 7, lower panel). The combined
model explained the distribution variabilities of
46.6 % of E. graminifolia, 33.3 % of S. altissima,
19.9 % of S. gigantea and 10.3 % of S. canadensis. graminifolia correlated with the CCA axis calculated
from the PCNM vectors (Table 3). We also found that
silt and Mg, as well as minCan and minEut, were
correlated with the site score of the first CCA axis
calculated using the space components (Table 3). Finally, the combined model of goldenrod distribu-
tions explained 28.3 % of the dataset variation (Fig. 7,
lower panel). The pure space component explained
more variation than solely environment fraction and
distance, while the fraction of shared variation was
relatively high (Fig. 7, lower panel). The combined
model explained the distribution variabilities of
46.6 % of E. graminifolia, 33.3 % of S. altissima,
19.9 % of S. gigantea and 10.3 % of S. canadensis. Solidago altissima and S. gigantea Solidago altissima and S. gigantea The frequently observed patchy distributions of inva-
sive plant species can result from both dispersal 3 Plant Ecol (2016) 217:565–582 576 Table 3 Correlations
between species data,
environmental variables and
the shortest distances
significantly shaping
species distribution, from
one side, and canonical axes
derived from CCA analysis
with PCNM vectors as
explanatory variables, on
the other
The correlations were tested
separately for the entire
dataset as well as topsoil
subset
Components
Entire dataset
Topsoil subset
CCA1
CCA2
CCA1
CCA2
S. altissima
Z = -13.332
Z = -11.001
Z = -6.335
Z = -5.739
p \ 0.001
p \ 0.001
p \ 0.001
p \ 0.001
S. canadensis
n.s. Z = -5.353
Z = -1.263
Z = 0.4496
p \ 0.001
p = 0.206
p = 0.653
S. gigantea
Z = 12.825
Z = 12.733
Z = 6.876
Z = -7.104
p \ 0.001
p \ 0.001
p \ 0.001
p \ 0.001
E. graminifolia
Z = 5.413
Z = -5.251
Z = 4.725
Z = 2.498
p \ 0.001
p \ 0.001
p \ 0.001
p = 0.012
Precipitation
n.s. Z = -3.2814
–
–
p = 0.001
Canopy
Z = 2.182
n.s. –
–
p = 0.029
C/N
–
–
Z = 1.8002
Z = 0.654
p = 0.072
p = 0.513
Mg
–
–
Z = -2.688
Z = 0.968
p = 0.007
p = 0.333
Silt
–
–
Z = -3.824
Z = 1.614
p = 0.001
p = 0.106
minEut
Z = -6.337
Z = -2.508
Z = -4.633
Z = 1.672
p \ 0.001
p = 0.012
p \ 0.001
p = 0.094
minCan
Z = 4.636
Z = 1.767
Z = 3.954
Z = -1.717
p \ 0.001
p = 0.077
p \ 0.001
p = 0.086 essential for successful invasion is competition with
resident vegetation (Levine et al. 2003; Vila´ and
Weiner 2004; van Kleunen et al. 2010; Gioria and
Osborne 2014; Kuebbing and Nun˜ez 2015). Based on
the results, there is no reason to assume that native
vegetation in areas invaded by these two taxa differs. The results of ANOSIM and NMDS reveal that the
two species did not differ with respect to the
composition of co-occurring vascular plant species. On the other hand, the distributions of these species
were well modelled using geographical space. Solidago altissima and S. gigantea Specif-
ically, the location of initial infestation sites, prior to
the rapid spread phase on invasion (Fig. 1b), was well
correlated with the present-day distribution of spe-
cies. This suggests that the observed pattern resulted
from a limitation in the local distribution within the
invaded ranges. However, these species are known to
have efficient long-range dispersal (Weber 2000;
Weber and Jakobs 2005). We suggest that priority
effect limited goldenrod distributions (Shulman et al. 1983; Vannette and Fukami 2014); that is, the limitation and lack of suitable habitats (Warren et al. 2013). Local dispersal, which leads to infilling gaps
within the invaded range, drives the landscape cover-
age of the invader and, usually, its environmental
impact (Wangen and Webster 2006; Miller and
Matlack 2010; Warren et al. 2013; Bartha et al. 2014). Because of this, the observation of aggregated
patterning without other analysis does not provide an
explanation of the ecological processes and limita-
tions, which determine the observed distribution
(Warren et al. 2013). In contrast to previous analyses regarding the
differentiation
of
ecological
niches
between
S. altissima and S. gigantea, our study reveals little
differentiation of climatic and soil conditions, and
none in the invaded habitats. Despite the fact that in
the native range, S. altissima is considered as
preferring drier sites than S. gigantea (Weber and
Jakobs 2005; Weber 2000), we did not find such
environmental preferences in the studied region based
both on TWI and plant species composition. A factor 123 123 Plant Ecol (2016) 217:565–582 577 Fig. 7 Results of CCA ordination for topsoil subset. On the
upper panel, the values of site scores of CCA with exclusively
spatial variables as constraints plotted against geographical
coordinates of plots are shown. The lower panel shows the
results for the combined model (space, environment and
distance)
of
alien
goldenrod
distribution:
the
variation
partitioning results on the left and the CCA biplot on the right. minCan the shortest distance to the initial infestation location
from S. canadensis s.l. plots; minEut the shortest distance to the
initial infestation location from E. graminifolia plots, E. gram E. graminifolia, S. alti S. altissima, S. cana S. canadensis, S. giga S. gigantea, V1–V18 PCNM vectors Fig. 7 Results of CCA ordination for topsoil subset. On the
upper panel, the values of site scores of CCA with exclusively
spatial variables as constraints plotted against geographical
coordinates of plots are shown. Euthamia graminifolia Euthamia graminifolia and Nowak 2007), suggesting that the species may
overcome the dispersal barrier to invasion (Szymura
and Szymura 2013). This species outstand from the other goldenrod species
as it was under-represented on abandoned lands, key
habitats of invasive Solidago in Central Europe and
did not tend to avoid forests. This species was also
more frequent within linear habitats such as road
verges and unpaved roads. This suggests that the
species is more shade tolerant than the others and is
perhaps less efficient in colonisation and occupation of
open habitats. In contrast, in the native range, it is a
typical species of open prairie landscape (Parker et al. 1993; Lake et al. 2014). In garden experiments, this
species is highly competitive and can significantly
decrease the biomass of S. altissima and S. gigantea
through underground competition (Szymura and Szy-
mura 2016). Therefore, its under-representation in
open areas is more likely attributed to the limited long-
range distribution of this species and to low seed
germination in the new range. The proportion of
germinating E. graminifolia seeds in the native range
of Canada reaches 78 % (White et al. 2009), but in
Europe, very few mature seeds could germinate,
compared to the much higher percentages of S. altissima and S. gigantea (Voser-Huber 1983; Szy-
mura 2012). Moreover, many of the abandoned fields
in the studied region were already occupied by S. gigantea, which would additionally limit the expan-
sion of E. graminifolia. Low germination of E. graminifolia is compensated by efficient re-growth
by rhizomes, which comprises a significant part of the
clonal biomass (Szymura and Szymura 2015). Thus,
its over-representation on linear habitats can be
attributed to the vegetative spread along road verges
and unpaved roads. Ineffective long-range dispersal
also explained the limited range of its distribution,
which did not expand considerably since the initial
establishment in Europe (Guzikowa and Maycock
1986; Weber 2001). There were also likely fewer E. graminifolia introduction sites, compared to the other
species as it is considered less ornamental than S. altissima and S. gigantea and has been less frequently
used in horticulture (Weber and Schmid 1998; Weber
2001). In Silesia, its propagules were probably trans-
ported accidentally along with ornamental trees into
the local arboretum (Dajdok and Nowak 2007). E. Euthamia graminifolia graminifolia is sporadically reported from open aban-
doned fields and new localities of this species have
been found (Kompała-Baba and Baba 2006; Dajdok y
)
The results suggest differences in ecological niche
of E. graminifolia compared to the Solidago species. However, the fraction of variability shared by space,
environment and distance in the topsoil model was
high. Thus, the presence of E. graminifolia on more
shaded habitats, on sandy soils with a high C/N ratio
and low content of K and Mg, as well as higher
minimal temperature, cannot unequivocally be con-
sidered as its ecological optima. The observed pattern
could also result from both limited distribution and
priority effects. The potential European range of E. graminifolia, determined on the basis of its climatic
preferences, is comparable to other goldenrods (We-
ber 2001). In North America, this species occupies
areas with slightly lower temperatures than the areas
occupied by S. altissima and S. gigantea (Weber
2001). In Silesia, we observed an opposite temperature
reaction. However, these differences appear to be
caused mostly by its limited distribution, because E. graminifolia sites were exclusively within the lowland
part of the region, where the minimal temperatures are
higher than those in highland areas. Moreover, the
differences in soil chemical composition can be
attributed to typical properties of forest soils, on
which E. graminifolia was most commonly found,
while the remaining species often invaded more fertile
post-agricultural lands. Solidago altissima and S. gigantea The lower panel shows the
results for the combined model (space, environment and
distance)
of
alien
goldenrod
distribution:
the
variation partitioning results on the left and the CCA biplot on the right. minCan the shortest distance to the initial infestation location
from S. canadensis s.l. plots; minEut the shortest distance to the
initial infestation location from E. graminifolia plots, E. gram E. graminifolia, S. alti S. altissima, S. cana S. canadensis, S. giga S. gigantea, V1–V18 PCNM vectors 1999a). In the case of S. altissima, the proportion of
seeds germinated in patches of vegetation was
0.008 %; however, the emergence of seedlings in
artificially created openings was high (Meyer and
Schmid 1999a). The authors concluded that offspring
coming from seeds did not influence the dynamics of
established populations of S. altissima but, most
probably, are important in colonisation of new, open
habitats with disturbed soil. As a result, we observed
regions dominated by either S. altissima or S. gigantea. The association of S. gigantea with river
valleys seems to be the effect of early colonisation
patterns, rather than strict preferences for wet habitats
in Central Europe. goldenrod species that first occupied the particular
area blocked the establishment of further species from
seeds. In subsequent stages, the populations produce a
large number of diaspores that increase the probabil-
ity of invasion of the remaining suitable habitats
within
the
neighbourhood
(Meyer
and
Schmid
1999a). Results of garden experiments showed that
the two species are similar with respect to their
competitiveness (Szymura and Szymura 2016); thus,
the competitive exclusion of one species by another is
not likely during later stages of invasion. In addition,
the probability that new species could establish from
seeds in a dense, well-established stand is minimal
(Hartnett and Bazzaz 1985; Meyer and Schmid 12 123 123 Plant Ecol (2016) 217:565–582 578 Solidago canadensis O´ va´ri M, Komoly C, Sutyinszki Z, Szabo´ G, Csatho´ AI,
Juha´sz M, Penksza K, Molna´r Z (2014) Impact of mid-
successional dominant species on the diversity and pro-
gress of succession in regenerating temperate grasslands. Appl Veg Sci 17:201–213 O´ va´ri M, Komoly C, Sutyinszki Z, Szabo´ G, Csatho´ AI,
Juha´sz M, Penksza K, Molna´r Z (2014) Impact of mid-
successional dominant species on the diversity and pro-
gress of succession in regenerating temperate grasslands. Appl Veg Sci 17:201–213 Beaudry JR (1963) Studies on Solidago L. VI. Additional
chromosome numbers of taxa of the genus Solidago. Can J
Genet Cytol 5(2):150–174 Beaudry JR, Chabot DL (1957) Studies on Solidago L. I. S. altissima L. and S. canadensis L. Contrib Inst Bot Univ
Montr Que 70:65–72 Borcard D, Legendre P (2002) All-scale spatial analysis of
ecological data by means of principal coordinates of
neighbor matrices. Ecol Model 153:51–68 Borcard D, Legendre P, Drapeau P (1992) Partialling out the
spatial
component
of
ecological
variation. Ecology
73:1045–1055. doi:10.2307/1940179 Borcard D, Legendre P, Avois-Jacquet C, Tuomisto H (2004)
Dissecting the spatial structure of ecological data at mul-
tiple scales. Ecology 85:1826–1832 Bornkamm R (2007) Spontaneous development of urban woody
vegetation on differing soils. Flora 202:695–704 Cain ML (1990) Patterns of Solidago altissima ramet growth
and mortality: the role of below-ground ramet connections. Oecologia 82(2):201–209 Carson WP, Root RB (2000) Herbivory and plant species
coexistence: community regulation by an outbreaking
phytophagous insect. Ecol Monogr 70(1):73–99 Catford JA, Jansson R, Nilsson C (2009) Reducing redundancy
in invasion ecology by integrating hypotheses into a single
theoretical framework. Divers Distrib 15:22–40 Chamberlain SA, Bronstein JL, Rudgers JA (2014) How context
dependent are species interactions? Ecol Lett 17:881–890 Chapuis-Lardy L, Vanderhoeven S, Dassonville N, Koutika LS,
Meerts P (2006) Effect of the exotic invasive plant Sol-
idago gigantea on soil phosphorus status. Biol Fertil Soils
42(6):481–489 Chytry´ M, Jarosik V, Pysek P, Hajek O, Knollova´ I, Tichy´ L,
Danihelka J (2008) Separating habitat invasibility by alien
plants
from
the
actual
level
of
invasion. Ecology
89:1541–1553 Acknowledgments
The study was supported by Grants: NCN
(N N305 401438), the Wrocław University of Environmental
and Life Sciences, and the University of the Wrocław. The
authors thank the Editor and Anonymous Reviewers for their
comments, which helped us to improve the manuscript. Dajdok Z, Nowak A (2007) Solidago graminifolia (L.) Elliott in
Poland—spreading and habitat preferences. Solidago canadensis In: Tokarska-
Guzik B et al (eds) Plant invasions: human perception,
ecological impacts and management. Backhuys Publishers,
Leiden, pp 101–116 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 unre-
stricted 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 Com-
mons license, and indicate if changes were made. Del Fabbro C, Gu¨sewell S, Prati D (2013) Allelopathic effects of
three plant invaders on germination of native species: a
field study. Biol Invasions 16:1035–1042 Dray S, Legendre P, Peres-Neto PR (2006) Spatial modelling: a
comprehensive framework for principal coordinate analy-
sis
of
neighbour
matrices
(PCNM). Ecol
Model
196:483–493 Essl F, Dullinger S, Rabitsch W, Hulme PE, Hu¨lber K, Jarosˇı´k
V, Kleinbauer I, Krausmann F, Ku¨hn I, Nentwig W, Vila`
M, Genovesi P, Gherardi F, Desprez-Loustau ML, Roques
A, Pysˇek P (2011) Socioeconomic legacy yields an inva-
sion debt. Proc Natl Acad Sci USA 108(1):203–207 Solidago canadensis Solidago is known as one of the most complex genera
of higher plants, and its variability has further
increased due to hybridisation, introgression and
ecological factors (Beaudry and Chabot 1957; Beau-
dry 1963; Weber 1997). In the native range, S. canadensis consists of diploid (2n = 18) and tetra-
ploid plants (2n = 36), while S. altissima consists of
hexaploid (2n = 54), triploid (2n = 27) and tetraploid
(2n = 36) cytotypes (Semple et al. 1981, 1984, 2015). Both taxa, S. canadensis var. canadensis and S. altissima, were introduced from North America to
Europe (Wagenitz 1979; Weber 1997). However, in
Europe, the taxonomical status of S. canadensis and S. altissima is unclear, because both are represented
exclusively by diploids (Weber 2000). These species
can form hybrids in the native range. Hybridisation has 12 123 Plant Ecol (2016) 217:565–582 579 also been observed in Europe between S. canadensis
s.l. and native S. virgaurea (Pliszko 2013; Migdałek
et al. 2014). Thus, it could be also hypothesised that
the random pattern of S. canadensis distribution,
overlapping extensively with the ranges of S. altissima
and S. gigantea, could be related to the hybrid nature
of this species. However, the populations examined
here were checked for their chromosome number
(Szymura et al. 2015), and the results showed that all
S. canadensis and S. altissima were exclusively
diploids, whereas S. gigantea was tetraploid. These
findings allow us to reject the hypothesis about the
hybrid nature of local S. canadensis. The traits used to
distinguish S. altissima and S. canadensis primarily
refer to the presence or absence of hairs on stems and
leaves as well as leaf epidermis (Weber 1997; Semple
and Cook 2006; Szymura and Wolski 2011). There-
fore, they could not be considered as functional traits,
which influence the fitness and competitiveness of
individuals, and are likely not under natural selection. S. altissima and S. canadensis did not differ in
functional traits, biomass production and allocation,
as well as ploidy levels (Szymura and Szymura 2013,
2015; Szymura et al. 2015). These findings, in
combination with results of this study, suggest that
all the Solidago plants with more or less hairy stems in
Central Europe may be considered as one complex
species, S. canadensis s.l., without differences in their
ecology and most likely their environmental impact. References Abrahamson WG, Dobley KB, Houseknecht HR, Pecone CA
(2005) Ecological divergence among five co-occurring
species of old-field goldenrods. Plant Ecol 177:43–56
Bartha S, Szentes S, Horva´th A, Ha´zi J, Zimmermann Z, Molna´r
C, Dancza I, Margo´czi K, Pa´l RW, Purger D, Schmidt D, Abrahamson WG, Dobley KB, Houseknecht HR, Pecone CA
(2005) Ecological divergence among five co-occurring
species of old-field goldenrods. Plant Ecol 177:43–56 Fenesi A, Va´ga´si CI, Beldean M, Fo¨ldesi R, Kolcsa´r LP, Shapiro
JT, To¨ro¨k E, Kova´cs-Hostya´nszki A (2015a) Solidago
canadensis
impacts
on
native
plant
and
pollinator 12 3 3 580 Plant Ecol (2016) 217:565–582 CGIAR-CSI SRTM 90m database. http://srtm.csi.cgiar. org. Accessed Sept 2015 CGIAR-CSI SRTM 90m database. http://srtm.csi.cgiar. org. Accessed Sept 2015 communities in different-aged old fields. Basic Appl Ecol
16(4):335–346 Fenesi A, Gere´d J, Meiners SJ, To´thme´re´sz B, To¨ro¨k P,
Ruprecht E (2015b) Does disturbance enhance the com-
petitive effect of the invasive Solidago canadensis on the
performance of two native grasses? Biol Invasions
17(11):3303–3315 Johnson LE, Brawley SH, Adey WH (2012) Secondary spread
of invasive species: historic patterns and underlying
mechanisms of the continuing invasion of the European
rockweed Fucus serratus in eastern North America. Biol
Invasions 14(1):79–97
´ Ka˛cki Z, S´liwinski M (2012) The Polish Vegetation Database:
structure, resources and development. Acta Soc Bot Pol
81(2):75–79 Fraser CI, Banks SC, Waters JM (2015) Priority effects can lead
to underestimation of dispersal and invasion potential. Biol
Invasions 17(1):1–8 Kardol P, Souza L, Classen AT (2013) Resource availability
mediates the importance of priority effects in plant com-
munity
assembly
and
ecosystem
function. Oikos
122(1):84–94 Fukami T (2010) Community assembly dynamics in space. In:
Verhoef HA, Morin PJ (eds) Community ecology: pro-
cesses, models, and applications. Oxford University Press,
Oxford, pp 45–54 Gioria M, Osborne BA (2014) Resource competition in plant
invasions: emerging patterns and research needs. Front
Plant Sci 5:501 Kompała-Ba˛ba A, Ba˛ba W (2006) Solidago graminifolia (L.)
Elliott on anthropogenic sites of the Silesian Upland
Poland. Biodivers Res Conserv 3–4:329–332 Grman E, Suding KN (2010) Within-year soil legacies con-
tribute to strong priority effects of exotics on native Cali-
fornia grassland communities. Restor Ecol 18(5):664–670 Kuebbing SE, Nun˜ez MA (2015) Negative, neutral, and positive
interactions among nonnative plants: patterns, processes,
and
management
implications. Glob
Change
Biol
21(2):926–934 g
Gu¨sewell S, Zuberbu¨hler N, Clerc C (2005) Distribution and
functional traits of Solidago gigantea in a Swiss lakeshore
wetland. References Bot Helv 115(1):63–75 Kuebbing SE, Nun˜ez MA, Simberloff D (2013) Current mis-
match between research and conservation efforts: the need
to study co-occurring invasive plant species. Biol Conserv
160:121–129 Guzikowa M, Maycock PF (1986) The invasion and expansion
of three North American species of goldenrod (Solidago
canadensis L. sensu lato, S. gigantea Ait. and S. gramini-
folia (L.) Salisb.) in Poland. Acta Soc Bot Pol 55:367–384 Lake JC, Leishman MR (2004) Invasion success of exotic plants
in natural ecosystems: the role of disturbance, plant attri-
butes
and
freedom
from
herbivores. Biol
Conserv
117(2):215–226 Hartnett DC, Bazzaz FA (1985) The genet and ramet population
dynamics of Solidago canadensis in an abandoned field. J Ecol 73:407–413 Lake EC, Hough-Goldstein J, D’Amico V (2014) Integrating
management techniques to restore sites invaded by mile-a-
minute
weed,
Persicaria
perfoliata. Restor
Ecol
22(2):127–133 Hejda M, Pysˇek P, Jarosˇı´k V (2009) Impact of invasive plants on
the species richness, diversity and composition of invaded
communities. J Ecol 97(3):393–403 La´nı´kova´ D, Chytry´ M, Lososova´ Z (2009) Xerophilous ruderal
vegetation with biennial and perennial species. In: Chytry´
M (ed) Vegetation of the Czech Republic. 2 Ruderal weed
rock scree vegetation. Academia, Praha, pp 206–288 Hijmans RJ, Cameron SE, Parra JL, Jones PG, Jarvis A (2005)
Very high resolution interpolated climate surfaces for
global land areas. Int J Climatol 25(15):1965–1978 Hitchmough J, De La Fleur M, Findlay C (2004) Establishing
North American prairie vegetation in urban parks in
northern England. Part 1. Effect of sowing season, sowing
rate and soil type. Landsc Urban Plan 66:75–90 Levine JM, Vila M, Antonio CM, Dukes JS, Grigulis K, Lavorel
S (2003) Mechanisms underlying the impacts of exotic
plant invasions. Proc R Soc Lond B 270:775–781 Hollander M, Wolfe DA (1999) Nonparametric statistical
methods, 2nd edn. Wiley, New York Marchante H, Marchante E, Freitas H, Hoffmann JH (2015)
Temporal changes in the impacts on plant communities of
an invasive alien tree, Acacia longifolia. Plant Ecol
216(11):1481–1498 Hothorn T, Hornik K, van de Wiel MA, Zeileis A (2013)
Package ‘coin’. Conditional inference procedures in a
permutation test framework. http://cran.r-project.org/web/
packages/coin/coin.pdf. Accessed Sept 2015 Meyer A, Schmid B (1999a) Seed dynamics and seedling estab-
lishment in the invading perennial Solidago altissima under
different experimental treatments. J Ecol 87(1):28–41 Hulme PE, Pysˇek P, Pergl J, Jarosˇı´k V, Schaffner U, Vila` M
(2014) Greater focus needed on alien plant impacts in
protected areas. 585:103–112 (in Polish with English abstract) Szymura M, Szymura TH (2011) Rozmieszczenie nawłoci
(Solidago spp.) na obszarze Dolnego S´la˛ska oraz ich
wpływ na ro´ _znorodnos´c´ biologiczna˛ zasiedlanych fito-
cenoz. Acta Bot Sil 6:195–212 (in Polish with English
abstract) Putten WH, Bardgett RD, Bever JD, Bezemer TM, Casper BB,
Fukami T, Kardol P, Klironomos JN, Kulmatiski A, Sch-
weitzer JA, Suding KN (2013) Plant–soil feedbacks: the past,
the present and future challenges. J Ecol 101(2):265–276 Pysˇek P (1998) Alien and native species in Central European
urban floras: a quantitative comparison. J Biogeogr
25:155–163 Szymura M, Szymura TH (2013) Soil preferences and mor-
phological diversity of goldenrods (Solidago L.) from
south-western Poland. Acta Soc Bot Pol 82(2):107–115 Pysˇek P, Richardson DM (2008) Invasive plants. In: Jørgensen
SE, Fath BD (eds) Ecological engineering. Encyclopedia
of ecology, vol 3. Elsevier, Oxford, pp 2011–2020 Szymura M, Szymura TH (2015) Growth, phenology, and bio-
mass allocation of alien Solidago species in Central Eur-
ope. Plant Species Biol 30:245–256 Pysˇek P, Richardson DM (2010) Invasive species, environ-
mental change and management, and health. Annu Rev
Environ Resour 35:25–55 Szymura M, Szymura TH (2016) Interactions between alien
goldenrods (Solidago and Euthamia species) and compar-
ison with native species in Central Europe. Flora 218:51–61 Rhoads AF, Block TA (2000) The plants of Pennsylvania: an
illustrated manual. University of Pennsylvania Press,
Philadelphia Szymura M, Wolski K (2011) Leaf epidermis traits as tools to
identify Solidago L. taxa in Poland. Acta Biol Crac Bot
53(1):38–46 p
Ricklefs RE (2010) Dynamics of colonization and extinction on
islands: insights from Lesser Antillean birds. In: Losos JB,
Ricklefs RE (eds) The theory of island biogeography
revisited. Princeton
University
Press,
Princeton,
pp 388–414 Szymura M, Szymura TH, Kreitschitz A (2015) Morphological
and cytological diversity of goldenrods (Solidago L. and
Euthamia Nutt.) from south-western Poland. Biodivers Res
Conserv 38:41–49 Tokarska-Guzik B (2003) The expansion of some alien plant
species (neophytes) in Poland. In: Child L, Brock JH,
Brundu G, Prach K, Pysˇek P, Wade PM, Williamson M
(eds) Plant invasions: ecological treats and management
solutions. Backhuys Publishers, Leiden, pp 147–167 Rosenberg MS, Anderson CD (2011) PASSaGE: pattern anal-
ysis, spatial statistics and geographic exegesis. Version 2. Method Ecol Evol 2(3):229–232 Schlaepfer DR, Edwards PJ, Semple CJ, Billeter R (2008)
Cytogeography of Solidago gigantea (Asteraceae) and its
invasive ploidy level. J Biogeogr 35:2119–2127 Van Kleunen M, Weber E, Fischer M (2010) A meta-analysis of
trait differences between invasive and non-invasive plant
species. References Conserv Lett 7(5):459–466 Meyer A, Schmid B (1999b) Experimental demography of the
old-field perennial Solidago altissima: the dynamics of the
shoot population. J Ecol 87(1):17–27 p
( )
Hulme PE, Pauchard A, Pysˇek P, Vila` M, Alba C, Blackburn
TM, Bullock JM, Chytry´ M, Dawson W, Dunn AM, Essl F,
Genovesi P, Maskell LC, Meyerson LA, Nun˜ez MA, Pergl
J, Pescott OL, Pocock MJO, Richardson DM, Roy HE,
Smart SM, Sˇtajerova´ K, Stohlgren T, van Kleunen M,
Winter M (2015) Challenging the view that invasive non-
native plants are not a significant threat to the floristic
diversity of Great Britain. Proc Natl Acad Sci USA
112(23):E2988–E2989 Migdałek G, Kolczyk J, Pliszko A, Kos´cin´ska-Paja˛k M, Słomka
A (2014) Reduced pollen viability and achene develop-
ment in Solidago 9 niederederi Khek from Poland. Acta
Soc Bot Pol 83(3):251–255 Miller NP, Matlack GR (2010) Biodiversity research: popula-
tion expansion in an invasive grass, Microstegium vimi-
neum: a test of the channeled diffusion model. Divers
Distrib 16(5):816–826 Mirek Z, Pie˛kos´-Mirkowa H, Zaja˛c A, Zaja˛c M (2002) Flow-
ering plants and pteridophytes of Poland a checklist. In: Jarvis A, Reuter HI, Nelson A, Guevara E (2008) Hole-filled
SRTM for the globe version 4. Available from the 12 3 Plant Ecol (2016) 217:565–582 581 Mirek Z (ed) Biodiversity of Poland, vol 1. W. Szafer
Institute of Botany, Polish Academy of Sciences, Cracow Mirek Z (ed) Biodiversity of Poland, vol 1. W. Szafer
Institute of Botany, Polish Academy of Sciences, Cracow Semple JC, Brammall RA, Chmielewski J (1981) Chromosome
numbers of goldenrods, Euthamia and Solidago (Com-
positae–Asteraceae). Can J Bot 59:1167–1173 Moron´ D, Lenda M, Sko´rka P, Szentgyo¨rgyi H, Settele J,
Woyciechowski M (2009) Wild pollinator communities are
negatively affected by invasion of alien goldenrods in
grassland landscapes. Biol Conserv 142:1322–1332 Semple JC, Ringius GS, Leeder C, Morton G (1984) Chromo-
some numbers of goldenrods, Euthamia and Solidago
(Compositae–Asteraceae). II. Additional counts with
comments on cytogeography. Brittonia 36:280–292 Oksanen J, Kindt R, Legendre P, O’Hara B, Simpson GL,
Solymos P, Stevens MHH, Wagner H (2008) Vegan
community ecology package. R package version 1.15-0. http://cran.r-project.org/, http://vegan.rforger-project.org. Accessed Sept 2015 Semple JC, Rahman H, Bzovsky S, Sorour MK, Kornobis K,
Lopez Laphitz R, Tong L (2015) A multivariate morpho-
metric study of the Solidago altissima complex and S. canadensis (Asteraceae: Astereae). References Phytoneuron 2014(10):
1–31 Pal RW, Chen S, Nagy DU, Callaway RM (2015) Impacts of
Solidago gigantea on other species at home and away. Biol
Invasions 17(11):3317–3325 Sheppard AW, Shaw RH, Sforza R (2006) Top 20 environ-
mental weeds for classical biological control in Europe: a
review of opportunities, regulations and other barriers to
adoption. Weed Res 46(2):93–117 Parker IM, Mertens SK, Schemske DW (1993) Distribution of
seven native and two exotic plants in a tallgrass prairie in
southeastern Wisconsin: the importance of human distur-
bance. Am Midl Nat 130:43–55 Shulman MJ, Ogden JC, Ebersole JP, McFarland WN, Miller
SL, Wolf NG (1983) Priority effects in the recruitment of
juvenile coral reef fishes. Ecology 64:1508–1513 Pejchar L, Mooney HA (2009) Invasive species, ecosystem
services
and
human
well-being. Trends
Ecol
Evol
24:497–504 Sko´rka P, Lenda M, Tryjanowski P (2010) Invasive alien
goldenrods negatively affect grassland bird communities in
Eastern Europe. Biol Conserv 143:856–861 Peres-Neto P, Legendre P, Dray S, Borcard D (2006) Variation
partitioning of species data matrices: estimation and
comparison of fractions. Ecology 87:2614–2625 Szymura M (2012) Ocena zdolnos´ci do rozmna_zania generaty-
wnego i wegetatywnego nawłoci wyste˛puja˛cych w połud-
niowo-zachodniej Polsce. Zesz Nauk Uniw Przyr we Wroc
585:103–112 (in Polish with English abstract) Pliszko A (2013) A new locality of Solidago 9 niederederi
Khek (Asteraceae) in Poland. Biodivers Res Conserv
29:57–62 585:103–112 (in Polish with English abstract) Ecol Lett 13(2):235–245 Schmid B, Puttick GM, Burgess KH, Bazzaz FA (1988) Corre-
lations between genet architecture and some life history
features in three species of Solidago. Oecologia 75:459–464 Vannette RL, Fukami T (2014) Historical contingency in spe-
cies interactions: towards niche-based predictions. Ecol
Lett 17(1):115–124 Semple JC, Cook RE (2006) Solidago. In: Flora North America
Editorial Committee (ed) Flora of North America. Aster-
aceae, part 2. Astereae and Senecioneae, vol 20. Oxford
University Press, Oxford, pp 107–166 Vila´ M, Weiner J (2004) Are invasive species better competitors
than native plant species? Evidence from pair-wise
experiments. Oikos 105:229–238 12 3 3 3 582 Plant Ecol (2016) 217:565–582 Weber E, Jakobs G (2005) Biological flora of Central Europe:
Solidago gigantea Aiton. Flora 200:109–118 Vila` M, Espinar JL, Hejda M, Hulme PE, Jarosˇı´k V, Maron JL,
Pysˇek P (2011) Ecological impacts of invasive alien plants:
a meta-analysis of their effects on species, communities
and ecosystems. Ecol Lett 14:702–708 Weber E, Schmid B (1998) Latitudinal population differentia-
tion in two species of Solidago (Asteraceae) introduced
into Europe. Am J Bot 85:1110–1121 Voser-Huber MI (1983) Studien an eingebu¨rgerten Arten der
Gattung Solidago L. Diss Bot 68:1–97 White AL, Boutin C, Dalton RL, Henkelman B, Carpenter D
(2009) Germination requirements for 29 terrestrial and
wetland wild plant species appropriate for phytotoxicity
testing. Pest Manag Sci 65(1):19–26 Wagenitz G (1979) Solidago L. In: Hegi G (ed) Illustrierte Flora
von Mitteleuropa, vol VI (3.1). Carl Hanser, Mu¨nchen,
p 16–29 Wangen SR, Webster CR (2006) Potential for multiple lag phases
during biotic invasions: reconstructing an invasion of the
exotic tree Acer platanoides. J Appl Ecol 43(2):258–268 Whitham TG (1983) Host manipulation of parasites: within-
plant variation as a defense against rapidly evolving pests. In: Variable plants and herbivores in natural and managed
systems. Academic, New York, p 15–41 systems. Academic, New York, p 15–41 Warren RJ, Ursell T, Keiser AD, Bradford MA (2013) Habitat,
dispersal and propagule pressure control exotic plant
infilling within an invaded range. Ecosphere 4(2):1–12 Wise MJ, Abrahamson WG, Landis K (2006) Edaphic envi-
ronment, gall midges, and goldenrod clonal expansion in a
mid-successional old-field. Acta Oecol 30(3):365–373 Weber E (1997) Morphological variation of the introduced
perennial Solidago canadensis L. sensu lato (Asteraceae)
in Europe. 585:103–112 (in Polish with English abstract) Bot J Linn Soc 123:197–210 Young TP, Zefferman EP, Vaughn KJ, Fick S (2015) Initial
success of native grasses is contingent on multiple inter-
actions among exotic grass competition, temporal priority,
rainfall and site effects. AoB Plants 7:plu081 Weber E (2000) Biological flora of Central Europe: Solidago
altissima L. Flora 195:123–134 p
Zefferman EP (2015) Experimental tests of priority effects and
light availability on relative performance of Myriophyllum
spicatum and Elodea nuttallii propagules in artificial
stream channels. PLoS One 10(3):e0120248 Weber E (2001) Current and potential ranges of three exotic
goldenrods
(Solidago)
in
Europe. Conserv
Biol
15(1):122–128 Weber E (2003) Invasive plant species of the world. A reference
guide to environmental weeds. CABI Publishing, Oxon Weber E (2011) Strong regeneration ability from rhizome frag-
ments in two invasive clonal plants (Solidago canadensis
and S. gigantea). Biol Invasions 13(12):2947–2955 123 12
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Hierarchical Over-the-Air Federated Edge Learning
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24/10/2024 05:55
Hierarchical Over-the-Air Federated Edge Learning / Aygun, O.; Kazemi, M.; Gunduz, D.; Duman, T.
2022-:(2022), pp. 3376-3381. (Intervento presentato al convegno 2022 IEEE International Confere
Communications, ICC 2022 tenutosi a COEX, kor nel 2022) [10.1109/ICC45855.2022.9839230].
Terms of use:
The terms and conditions for the reuse of this version of the manuscript are specified in the publish
policy. For all terms of use and more information see the publisher's website.
Institute of Electrical and Electronics Engineers Inc.
This is the peer reviewd version of the followng article: 24/10/2024 05:55
Hierarchical Over-the-Air Federated Edge Learning / Aygun, O.; Kazemi, M.; Gunduz, D.; Duman, T. 2022-:(2022), pp. 3376-3381. (Intervento presentato al convegno 2022 IEEE International Confere
Communications, ICC 2022 tenutosi a COEX, kor nel 2022) [10.1109/ICC45855.2022.9839230]. Terms of use:
The terms and conditions for the reuse of this version of the manuscript are specified in the publish
policy. For all terms of use and more information see the publisher's website. Institute of Electrical and Electronics Engineers Inc. This is the peer reviewd version of the followng article: This is the peer reviewd version of the followng article: Hierarchical Over-the-Air Federated Edge Learning / Aygun, O.; Kazemi, M.; Gunduz, D.; Duman, T. M.. -
2022-:(2022), pp. 3376-3381. (Intervento presentato al convegno 2022 IEEE International Conference on
Communications, ICC 2022 tenutosi a COEX, kor nel 2022) [10.1109/ICC45855.2022.9839230]. Terms of use:
The terms and conditions for the reuse of this version of the manuscript are specified in the publishing
policy. For all terms of use and more information see the publisher's website. Institute of Electrical and Electronics Engineers Inc. e
s o use
The terms and conditions for the reuse of this version of the manuscript are specified in the publishing
policy. For all terms of use and more information see the publisher's website. 24/10/2024 05:55 I. INTRODUCTION Extensive amounts of collected data from various sources
such as mobile phones and Internet-of-things (IoT) sensors
have enabled the accelerating rise of machine learning (ML)
algorithms, aiming to assemble all the data in a cloud server
to obtain representative datasets for model training. This,
however, brings out growing concerns regarding the privacy,
cost, and latency of traditional ML algorithms. Firstly, data
owners have become more sensitive about sharing their data;
secondly, the increasing quality of data results in higher
communication costs; and finally, solutions that work in real-
time are faced with latency issues [1]. To overcome these
problems, a decentralized approach called federated learning
(FL) has been introduced, where the transmission of data is
not required since models are trained locally instead of using
a centralized server for training [2]. In order to make distant MUs more resilient to the chan-
nel effects, we propose hierarchical over-the-air federated
learning (HOTAFL), where MUs communicate with their
corresponding ISs through wireless links. In this setup, each
MU shares its local training result with its corresponding IS
through OTA (cluster) aggregation. After several local itera-
tions with the MUs in their clusters, the ISs send the results to
the PS to complete the global aggregation, which constitutes
one global iteration. We examine the performance of HOTAFL
and compare the results with those of the conventional FL and
error-free HFL both through analytical results and numerical
experiments. The results show that the proposed framework
outperforms conventional OTA FL and leads to a better model
accuracy and faster convergence. In FL, several data owners called mobile users (MUs)
are selected based on some criteria such as their computing
capability, data quality, available power, and location [3]. Each MU in the federation trains a local model using its
own data and computing power in every iteration. After each
MU completes its local stochastic gradient descent (SGD)
computation, only the weight updates are sent to a parameter
server (PS) that performs model aggregation and sends back
the updated global model to MUs for the next iteration. Despite its superiority over traditional ML, adverse channel
effects in wireless setups and increased communication costs The paper is organized as follows: in Sections II and III,
we introduce the HOTAFL framework as well as its com-
munication model. In Section IV, we provide a convergence
analysis of HOTAFL under convexity assumptions on the loss
functions. Ozan Ayg¨un’s research in this study is supported by Turkcell A.S. within the
framework of 5G and Beyond Joint Graduate Support Programme coordinated
by Information and Communication Technologies Authority. Hierarchical Over-the-Air Federated Edge Learning
Ozan Ayg¨un1, Mohammad Kazemi1, Deniz G¨und¨uz2 and Tolga M. Duman1
1Dept. of Electrical and Electronics Engineering, Bilkent University, Ankara, Turkey
2Dept. of Electrical and Electronic Engineering, Imperial College London, London, UK
{ozan, kazemi, duman}@ee.bilkent.edu.tr, d.gunduz@imperial.ac.uk have arisen some concerns about the feasibility of conventional
FL in practical scenarios. To address the communication cost
concerns, over-the-air (OTA) aggregation [4] has become a
popular method in wireless schemes thanks to its efficient
strategy that allocates all the users to the same bandwidth,
thereby handling the transmission and aggregation of the
gradient updates simultaneously (over the air). For this frame-
work, one approach to deal with the channel effects (particu-
larly when there is no transmit side channel state information)
is to increase the number of receive antennas at the PS [5]. Nevertheless, the disparity among the channel gains is still a
critical factor when some MUs are far away from the PS. Abstract—Federated learning (FL) over wireless communica-
tion channels, specifically, over-the-air (OTA) model aggregation
framework is considered. In OTA wireless setups, the adverse
channel effects can be alleviated by increasing the number of
receive antennas at the parameter server (PS), which performs
model aggregation. However, the performance of OTA FL is
limited by the presence of mobile users (MUs) located far away
from the PS. In this paper, to mitigate this limitation, we propose
hierarchical over-the-air federated learning (HOTAFL), which
utilizes intermediary servers (IS) to form clusters near MUs. We provide a convergence analysis for the proposed setup, and
demonstrate through theoretical and experimental results that
local aggregation in each cluster before global aggregation leads
to a better performance and faster convergence than OTA FL. v:2112.11167v1 [cs.LG] 21 Dec 2021 arXiv:2112.11167v1 [cs.LG] 21 Dec Index Terms—machine learning, over-the-air communication,
clustering, hierarchical federated learning. Recent developments on FL include device selection algo-
rithms [6], efficient communication schemes [4], [7]–[11], het-
erogeneity of data [12], and power and latency analysis [13],
[14]. Although Federated Averaging [2] is the most common
way to perform global aggregation in error-free setups, OTA
communication has been preferred for wireless FL [5], [12],
[15]. Furthermore, hierarchical federated learning (HFL) has
been gaining increasing attention, where the objective is to
utilize intermediate servers (IS) to form clusters to reduce
communication costs. There exist studies on HFL on latency
and power analysis [16], [17], resource allocation [18], [19],
and performance analysis for non-independent and identically
distributed (i.i.d.) data [20]. However, there is no work on
HFL with OTA taking into account practical wireless channel
models, which motivates this work. y
g
y
D. Gunduz acknowledges support from UK EPSRC through CHIST-ERA
project CONNECT (CHISTERA-18-SDCDN-001, EPSRC-EP/T023600/1). B. OTA Communication We now consider the scheme referred as OTA communica-
tions, for which the links between the users and the ISs are
wireless with OTA aggregation, however, the links between
ISs and the PS is assumed to be error-free. Since a common
wireless medium is used in local aggregations, noisy versions
of the model updates ∆θIS,c(t) are received at the ISs. In our
setup, the ISs are equipped with K antennas, and we assume
perfect channel state information (CSI) at the receivers. For
the k-th antenna, the received signal at the c-th IS can be
written as1 We consider a hierarchical and iterative approach to min-
imize (1) consisting of global, local, and user iterations. In
every cluster iteration, the MUs carry out τ user iterations on
their own, then send their model updates to their corresponding
ISs for local iteration. I local iterations are performed at the
IS in every cluster before all the local models are forwarded to
the PS for global aggregation. At the j-th user iteration of the
i-th local iteration, the weight update is performed employing
stochastic gradient descent (SGD) for the m-th user in the c-th
cluster as follows yi
IS,c,k(t) =
M
X
m=1
hi
m,c,k(t) ◦xi
m,c,k(t) + zi
IS,c,k(t),
(8) θi,j+1
m,c (t) = θi,j
m,c(t) −ηi,j
m,c(t)∇Fm,c(θi,j
m,c(t), ξi,j
m,c(t)), (2) (8) where ηi,j
m,c(t) is the learning rate, ∇Fm,c(θi,j
m,c(t), ξi,j
m,c(t))
denotes the stochastic gradient estimate for the weight vector
θi,j
m,c(t) and a randomly sampled batch of data samples ξi,j
m,c(t)
from the dataset of the m-th user in the c-th cluster at
the t-th global, i-th local and j-th user iteration. Initially,
θ1,1
m,c(t) = θi
IS,c(t), ∀i ∈[I] where [I] ≜{1, 2, . . . , I}, and
θ1
IS,c(t) = θP S(t), where θP S(t) is the global model at the PS
at the t-th global iteration and θi
IS,c(t) denotes the local model
of IS in the c-th cluster at the i-th local iteration. The purpose
of employing ISs is to accumulate the local model differences
within each cluster more frequently in smaller areas before
obtaining the global model θP S(t) for the next iteration. where ◦denotes the element-wise product, xi
m,c,k(t) ∈CN,
zi
IS,c,k(t) ∈CN with independent and identically distributed
(i.i.d.) entries zi,n
IS,c,k(t) ∼CN(0, σ2
z). 1Note that the setup here can be efficiently implemented in practice using
orthogonal frequency-division multiplexing (OFDM). II. SYSTEM MODEL After completing I local iterations in each cluster, ISs send
their model updates to the PS, which can be written as After completing I local iterations in each cluster, ISs send
their model updates to the PS, which can be written as The objective of HOTAFL is to minimize a loss function
F(θ) with respect to the model weight vector θ ∈R2N, where
2N is the model dimension. Our system consists of C clusters
each containing an IS and M MUs as depicted in Fig. 1. The
dataset of the m-th MU in the c-th cluster is denoted as Bm,c,
and we define B ≜PC
c=1
PM
m=1 |Bm,c|. We have ∆θP S,c(t) = θI+1
IS,c(t) −θP S(t). (6) (6) The global update rule is ∆θP S(t) =
1
C
PC
c=1 ∆θP S,c(t). Using recursion, we can conclude that The global update rule is ∆θP S(t) =
1
C
PC
c=1 ∆θP S,c(t). Using recursion, we can conclude that ∆θP S(t) =
1
MC
C
X
c=1
I
X
i=1
M
X
m=1
∆θi
m,c(t). (7) (7) F(θ) =
C
X
c=1
M
X
m=1
|Bm,c|
B
Fm,c(θ),
(1) (1) After the global aggregation, the model at the PS is updated
as θP S(t + 1) = θP S(t) + ∆θP S(t). where Fm,c(θ) ≜
1
|Bm,c|
P
u∈Bm,c f(θ, u), with f(θ, u) de-
noting the corresponding loss of u-th data sample. A. Ideal Communication We refer to the case where all the communication among
all the units is error-free as the ideal communication scenario. In this case, after performing SGD, each MU calculates its
model difference to be sent to its corresponding IS as ∆θi
m,c(t) = θi,τ+1
m,c (t) −θi
IS,c(t). (3) (3) Then, the local aggregation at the c-th cluster is performed as Then, the local aggregation at the c-th cluster is performed as ∆θi
IS,c(t) = 1
M
M
X
m=1
∆θi
m,c(t),
(4)
θi+1
IS,c(t) = θi
IS,c(t) + ∆θi
IS,c(t). (5) (4) conclude the paper in Section VI. (5) I. INTRODUCTION We present our numerical results in Section V, and MU
Local Aggregation
Global Aggregation
Cluster
IS
PS
Fig. 1: HOTAFL system model. Also, note that ∇Fm,c(θi,j
m,c(t), ξi,j
m,c(t)) is an unbiased estima-
tor of ∇Fm,c(θi,j
m,c(t)), i.e., Eξ
∇Fm,c(θi,j
m,c(t), ξi,j
m,c(t))
=
∇Fm,c(θi,j
m,c(t)), where the expectation is over the random-
ness due to the SGD. B. OTA Communication Knowing the CSI perfectly, the c-th IS combines the
received signals as yi
IS,c(t) = 1
K
PK
k=1
PM
m=1 hi
m,c,k(t)
∗
◦
yi
IS,c,k(t). For the n-th symbol, it can be written as θP S(t + 1) = θP S(t) + ∆ˆθP S(t),
(17)
where ∆ˆθP S(t) =
∆ˆθ1
P S(t) ∆ˆθ2
P S(t) · · · ∆ˆθ2N
P S(t)
T . (17) n
S,c(t)=Pt
M
X
m=1
1
K
K
X
k=1
|hi,n
m,c,k(t)|2
∆θi,n,cx
m,c
(t)
|
{z
}
yi,n,sig
IS,c
(t) (signal term)
+ Pt
K
M
X
m=1
M
X
m′=1
m′̸=m
K
X
k=1
(hi,n
m,c,k(t))∗hi,n
m′,c,k(t)∆θi,n,cx
m′,c (t)
|
{z
}
yi,n,itf
IS,c
(t) (interference term)
+ 1
K
M
X
m=1
K
X
k=1
(hi,n
m,c,k(t))∗zi,n
c,k(t)
|
{z
}
yi,n,no
IS,c
(t) (noise term)
. (11) yi,n
IS,c(t)=Pt
M
X
m=1
1
K
K
X
k=1
|hi,n
m,c,k(t)|2
∆θi,n,cx
m,c
(t)
|
{z
}
yi,n,sig
IS,c
(t) (signal term) B. OTA Communication The channel coeffi-
cients are modelled as hi
m,c,k(t) =
p
βm,c gi
m,c,k(t), where
gm,c,k(t) ∈CN with entries gi,n
m,c,k(t) ∼CN(0, σ2
h) (i.e.,
Rayleigh fading), βm,c is the large-scale fading coefficient
modeled as βm,c = (dm,c)−p, where p represents the path
loss exponent, and dm,c denotes the distance between the m-
th user in the c-th cluster and the IS in that cluster. 1) Local Aggregation: In OTA communication, in order
to increase the spectral efficiency, the model differences are
grouped to form a complex vector ∆θi,cx
m,c ∈CN with the
following real and imaginary parts yn
P S(t) =
C
X
c=1
xn
P S,c(t)
(15)
=
C
X
c=1
I
X
i=1
Re
yi,n,sig
IS,c
(t)
PtMσ2
h
|
{z
}
yn
P S,1(t)
+
C
X
c=1
I
X
i=1
Re
yi,n,itf
IS,c
(t)
PtMσ2
h
|
{z
}
yn
P S,2(t)
+
C
X
c=1
I
X
i=1
Re
yi,n,no
IS,c (t)
PtMσ2
h
|
{z
}
yn
P S,3(t)
. (16) (15) ∆θi,re
m,c(t) ≜
∆θi,1
m,c(t), ∆θi,2
m,c(t), . . . , ∆θi,N
m,c(t)
T ,
(9a)
∆θi,im
m,c (t)≜
∆θi,N+1
m,c
(t), ∆θi,N+2
m,c
(t), . . . , ∆θi,2N
m,c (t)
T. (9b) Under the assumption that there is no inter-cluster interference,
the received signal for the k-th antenna in the c-th cluster at
the i-th local iteration can be represented as Under the assumption that there is no inter-cluster interference,
the received signal for the k-th antenna in the c-th cluster at
the i-th local iteration can be represented as +
C
X
c=1
I
X
i=1
Re
yi,n,no
IS,c (t)
PtMσ2
h
|
{z
}
yn
P S,3(t)
. (16) (16) yi
IS,c,k(t) = Pt
M
X
m=1
hi
m,c,k(t) ◦∆θi,cx
m,c(t) + zi
IS,c,k(t), (10) {z
yn
P S,3(t) The received signal at the PS is then recovered as ∆ˆθn
P S(t) =
1
C Re{yn
P S(t)}, ∆ˆθn+N
P S (t) =
1
C Im{yn
P S(t)}. Finally, the
global aggregation is performed as The received signal at the PS is then recovered as ∆ˆθn
P S(t) =
1
C Re{yn
P S(t)}, ∆ˆθn+N
P S (t) =
1
C Im{yn
P S(t)}. Finally, the
global aggregation is performed as where Pt is the transmit power constant at the t-th global
iteration. IV. CONVERGENCE ANALYSIS In this section, we present a convergence analysis of the
proposed HOTAFL algorithm. Define the optimal solution as
θ∗≜arg minθ F(θ), the minimum values of the total and the
local loss functions as F ∗= F(θ∗) and F ∗
m,c, respectively, and
the bias in the dataset as Γ ≜F ∗−PC
c=1
PM
m=1
Bm,c
B F ∗
m,c ≥
0. In addition, assume that the learning rate of the overall
system does not change in local iterations, i.e., ηi,j
m,c(t) = η(t). Therefore, we can write the global update rule as yIS,c
( )
g
+ Pt
K
M
X
m=1
M
X
m′=1
m′̸=m
K
X
k=1
(hi,n
m,c,k(t))∗hi,n
m′,c,k(t)∆θi,n,cx
m′,c (t)
|
} + Pt
K
M
X
m=1
M
X
m′=1
m′̸=m
K
X
k=1
(hi,n
m,c,k(t))∗hi,n
m′,c,k(t)∆θi,n,cx
m′,c (t)
|
{z
}
yi,n,itf
IS,c
(t) (interference term) {z
yi,n,itf
IS,c
(t) (interference term) + 1
K
M
X
m=1
K
X
k=1
(hi,n
m,c,k(t))∗zi,n
c,k(t)
|
{z
}
yi,n,no
IS,c
(t) (noise term)
. (11 θi,j+1
m,c (t) = θi,j
m,c(t) −η(t)∇Fm,c(θi,j
m,c(t), ξi,j
m,c(t)),
(18) (11) (11) which can also be written as which can also be written as yi,n,no
IS,c
(t) (noise term) θi,j+1
m,c (t)−θi,1
m,c(t) = −η(t)
j
X
l=1
∇Fm,c(θi,l
m,c, ξi,l
m,c(t)). (19) Aggregated model differences can be recovered by Assumption 1. All the loss functions are L-smooth and µ-
strongly convex; i.e., ∀v, w ∈R2N, ∀m ∈[M], ∀c ∈[C], Assumption 1. All the loss functions are L-smooth and µ-
strongly convex; i.e., ∀v, w ∈R2N, ∀m ∈[M], ∀c ∈[C], ∆ˆθi,n
IS,c(t) =
1
PtMσ2
h ¯βc
Re{yi,n
IS,c(t)},
(12a)
∆ˆθi,n+N
IS,c
(t) =
1
PtMσ2
h ¯βc
Im{yi,n
IS,c(t)},
(12b) (12a) ,
(12b)
model differ-
Fm,c(v)−Fm,c(w)≤⟨v−w,∇Fm,c(w)⟩+ L
2 ∥v −w∥2
2, (20)
Fm,c(v)−Fm,c(w)≥⟨v−w,∇Fm,c(w)⟩+ µ
2 ∥v −w∥2
2. (21) (12b) where ¯βc = PM
m=1 βm,c. After estimating the model differ-
ence values, the cluster model update is written as Assumption 2. The expected value of the squared l2 norm of
the stochastic gradients are bounded; i.e., ∀j ∈[τ], i ∈[I], θi+1
IS,c(t) = θi
IS,c(t) + ∆ˆθi
IS,c(t),
(13) (13) (13) Eξ
h
∇Fm,c(θi,j
m,c(t), ξi,j
m,c(t))
2
2
i
≤G2,
(22)
which translates to ∀n∈[2N], Eξ
∇Fm,c(θi,j,n
m,c , ξi,j,n
m,c (t))
≤G. Theorem 1. V. SIMULATION RESULTS We consider a hierarchical system with one PS and C = 4
non-overlapping clusters, each containing one IS with K =
5MC receive antennas and M = 5 MUs. Users are randomly
placed in the clusters in such a way that their distance to the
PS is between 0.5 and 3, while having a distance between
0.5 and 1 with their corresponding IS. We also define α =
PM
m=1
PC
c=1 dm,c
PD
d=1 dd
as a measure of relative closeness of the MUs
to their corresponding IS compared to the PS, where dm,c is
the distance between m-th user in c-th cluster to the c-th IS and
dd is the distance between the d-th user and the PS. α is set
to 0.4 in the simulations. We use MNIST [21] and CIFAR-10
[22] datasets with Adam optimizer [23], and considered both
i.i.d. and non-i.i.d. data distributions. In the i.i.d. case, data
samples are randomly distributed among MUs, while in the
non-i.i.d. case, the training data is divided into 5MC groups
each consisting of data with only one label. Then, 5 groups
are assigned to each user randomly. For CIFAR10, we use the
neural network given in [5] with 2N = 307498 whereas for
MNIST, we employ a one-layer neural network with 784 input
and 10 output neurons with 2N = 7850. We consider a hierarchical system with one PS and C = 4
non-overlapping clusters, each containing one IS with K =
5MC receive antennas and M = 5 MUs. Users are randomly
placed in the clusters in such a way that their distance to the
PS is between 0.5 and 3, while having a distance between
0.5 and 1 with their corresponding IS. We also define α =
PM
m=1
PC
c=1 dm,c
PD
d=1 dd
as a measure of relative closeness of the MUs Proof: Let us define auxiliary variable v(t + 1) =
θP S(t) + ∆θP S(t). Then, we have ∥θP S(t+1)−θ∗∥2
2 =∥θP S(t+1)−v(t+1) + v(t+1)−θ∗∥2
2
= ∥θP S(t + 1) −v(t + 1)∥2
2 + ∥v(t + 1) −θ∗∥2
2
+ 2⟨θP S(t + 1) −v(t + 1), v(t + 1) −θ∗⟩. (25) P
d=1 dd
to their corresponding IS compared to the PS, where dm,c is
the distance between m-th user in c-th cluster to the c-th IS and
dd is the distance between the d-th user and the PS. IV. CONVERGENCE ANALYSIS Since the third term in Y (a) is independent of η(a),
even for lim
t→∞η(t) = 0, we have lim
t→∞E[F(θP S(t))] −F ∗̸= 0. This term is also inversely proportional to M, C, and K. +
σ2
zIN
P 2a M 2C2Kσ2
h
M
X
m=1
C
X
c=1
βm,c
¯β2c Lemma 1. We have E
h
θP S(t + 1) −v(t + 1)
2
2
i
≤η2(t)τ 2G2I
M 2C2
M
X
m1=1
C
X
c1=1
β2
m1,c1
K ¯
β2c1
+
M
X
m2=1
C
X
c2=1
A1I
+
M
X
m=1
M
X
m′=1
m′̸=m
C
X
c=1
η2(t)τ 2G2Iβm,cβm′,c
M 2C2K ¯β2c
+
σ2
zIN
P 2
t M 2C2Kσ2
h
M
X
m=1
C
X
c=1
βm,c
¯β2c
. (26)
Proof: See Appendix A. E
h
θP S(t + 1) −v(t + 1)
2
2
i
≤η2(t)τ 2G2I
M 2C2
M
X
m1=1
C
X
c1=1
β2
m1,c1
K ¯
β2c1
+
M
X
m2=1
C
X
c2=1
A1I
+
M
X
m=1
M
X
m′=1
m′̸=m
C
X
c=1
η2(t)τ 2G2Iβm,cβm′,c
M 2C2K ¯β2c Three scenarios are considered: baseline with error-free
transmissions, FL with OTA aggregation over a wireless
medium, and HOTAFL. We set the total number of global
iterations T to 200, the mini-batch size to |ξi
m,c(t)| = 500,
σ2
h = 1 and the path loss exponent p to 4. The noise variance
is σ2
z = 10 for the MNIST, σ2
z = 1 for the CIFAR-10 training. Also, the power multiplier is set to Pt = 1 + 10−2t for
HOTAFL, Pt = 1.5 + 10−2t for conventional FL, t ∈[T]. Three scenarios are considered: baseline with error-free
transmissions, FL with OTA aggregation over a wireless
medium, and HOTAFL. We set the total number of global
iterations T to 200, the mini-batch size to |ξi
m,c(t)| = 500,
σ2
h = 1 and the path loss exponent p to 4. The noise variance
is σ2
z = 10 for the MNIST, σ2
z = 1 for the CIFAR-10 training. Also, the power multiplier is set to Pt = 1 + 10−2t for
HOTAFL, Pt = 1.5 + 10−2t for conventional FL, t ∈[T]. (26) Proof: See Appendix A. IV. CONVERGENCE ANALYSIS In HOTAFL, for 0 ≤η(t) ≤min{1,
1
µτI }, the Eξ
h
∇Fm,c(θi,j
m,c(t), ξi,j
m,c(t))
2
2
i
≤G2,
(22) Eξ
h
∇Fm,c(θi,j
m,c(t), ξi,j
m,c(t))
2
2
i
≤G2,
(22)
translates to ∀n∈[2N], Eξ
∇Fm,c(θi,j,n
m,c , ξi,j,n
m,c (t))
≤G. (22) where ∆ˆθi
IS,c(t) =
∆ˆθi,1
IS,c(t) ∆ˆθi,2
IS,c(t) · · · ∆ˆθi,2N
IS,c (t)
T . where ∆ˆθi
IS,c(t) =
∆ˆθi,1
IS,c(t) ∆ˆθi,2
IS,c(t) · · · ∆ˆθi,2N
IS,c (t)
T . 2) Global Aggregation: This part is similar to the case of
ideal communication. The only difference is that the aggre-
gated signals are estimates of the actual model differences. Letting xP S,c(t) be the transmitted signal from the c-th IS, its
n-th symbol can be written as Theorem 1. In HOTAFL, for 0 ≤η(t) ≤min{1,
1
µτI }, the
global loss function can be upper bounded as E
∥θP S(t) −θ∗∥2
2
≤
t−1
Y
a=1
X(a)
∥θP S(0)−θ∗∥2
2+
t−1
X
b=1
Y (b)
t−1
Y
a=b+1
X(a), (23)
h
X( )
(1
( )I (
( )(
1)))
d xn
P S,c(t) = ∆θn
P S,c(t) + j∆θn+N
P S,c (t). (14) (14) Then, using (6), (11), (14) and recursion, the received signal
for 1≤n≤N (similarly for N+1≤n≤2N) can be written as where X(a) = (1 −µη(a)I (τ −η(a)(τ −1))) and where X(a) = (1 −µη(a)I (τ −η(a)(τ −1))) and Corollary 1. Assuming L-smoothness, after T global itera-
tions, the loss function can be upper bounded as Y (a)=η2(a)τ 2G2I
M 2C2
M
X
m1=1
C
X
c1=1
β2
m1,c1
K ¯
β2c1
+
M
X
m2=1
C
X
c2=1
A1I
+
M
X
m=1
M
X
m′=1
m′̸=m
C
X
c=1
η2(a)τ 2G2Iβm,cβm′,c
M 2C2K ¯β2c
+
σ2
zIN
P 2a M 2C2Kσ2
h
M
X
m=1
C
X
c=1
βm,c
¯β2c
+ (1 + µ(1 −η(a)) η2(a)IG2 τ(τ −1)(2τ −1)
6
+ η2(a)I(τ 2 + τ −1)G2 + 2η(a)I(τ −1)Γ,
(24)
with A1 = 1 −βm1,c1
¯βc1
−βm2,c2
¯βc2
+ βm1,c1βm2,c2
¯βc1 ¯βc2
. E [F(θP S(T)) −F ∗] ≤L
2 E
h
∥θP S(T) −θ∗∥2
2
i
≤L
2
T −1
Y
n=1
X(n)
∥θP S(0)−θ∗∥2
2+ L
2
T −1
X
p=1
Y (p)
T −1
Y
n=p+1
X(n). (28) Remark. Lemma 2. We have Lemma 2. We have E
h
v(t+1)−θ∗
2
2
i
≤(1−µη(t)I(τ −η(t)(τ−1)))E
h
θP S(t)−θ∗
2
2
i
+ (1 + µ(1 −η(t)) η2(t)IG2 τ(τ −1)(2τ −1)
6
+ η2(t)I(τ 2 + τ −1)G2 + 2η(t)I(τ −1)Γ. (27) Accuracy plots are presented in Figs. 2-4, where ¯P is
the average transmit power. The results show that bringing
the servers closer to the users enhances learning accuracy
significantly. One reason for the improved performance is
that the cluster structure enables the MUs share their model
differences with a local server closer than the PS, reducing
the adverse effects of the large-scale wireless channel effects. Accuracy plots are presented in Figs. 2-4, where ¯P is
the average transmit power. The results show that bringing
the servers closer to the users enhances learning accuracy
significantly. One reason for the improved performance is
that the cluster structure enables the MUs share their model
differences with a local server closer than the PS, reducing
the adverse effects of the large-scale wireless channel effects. Another reason is that MUs receive updated models even
without communicating with the PS due to local aggregations. We also observe that although more initial power is given
to FL, the user updates do not reflect on the global model
as much as HOTAFL does due to the effects of the wireless
channel. More local iterations enables faster convergence but
uses more transmit power due to the accumulating nature of
IS. Even though the noise variance is high when compared to (27) Proof: The proof is similar to that of Lemma 2 in [5]. Proof: The proof is similar to that of Lemma 2 in [5]. Lemma 3. E [⟨θP S(t + 1) −v(t + 1), v(t + 1) −θ∗⟩] = 0. Proof: We have E[⟨θP S(t+1)−v(t+1), v(t+1)−θ∗⟩]=
E
h
⟨∆ˆθP S(t)−∆θP S(t),θP S(t)+∆θP S(t)−θ∗⟩
i
. Then, know-
ing that channel realizations are independent of the user
and cluster updates at the same global iteration t, we have
E
h
⟨∆ˆθP S(t)−∆θP S(t),θP S(t)+∆θP S(t)−θ∗⟩
i
=0. Lemma 3. E [⟨θP S(t + 1) −v(t + 1), v(t + 1) −θ∗⟩] = 0. Proof: We have E[⟨θP S(t+1)−v(t+1), v(t+1)−θ∗⟩]=
E
h
⟨∆ˆθP S(t)−∆θP S(t),θP S(t)+∆θP S(t)−θ∗⟩
i
. Then, know-
ing that channel realizations are independent of the user
and cluster updates at the same global iteration t, we have
E
h
⟨∆ˆθP S(t)−∆θP S(t),θP S(t)+∆θP S(t)−θ∗⟩
i
=0. V. SIMULATION RESULTS α is set
to 0.4 in the simulations. We use MNIST [21] and CIFAR-10
[22] datasets with Adam optimizer [23], and considered both
i.i.d. and non-i.i.d. data distributions. In the i.i.d. case, data
samples are randomly distributed among MUs, while in the
non-i.i.d. case, the training data is divided into 5MC groups
each consisting of data with only one label. Then, 5 groups
are assigned to each user randomly. For CIFAR10, we use the
neural network given in [5] with 2N = 307498 whereas for
MNIST, we employ a one-layer neural network with 784 input
and 10 output neurons with 2N = 7850. Next, we provide upper bounds on the three terms of (25). Next, we provide upper bounds on the three terms of (25). h
i
Recursively iterating through the results of Lemmas 1, 2,
and 3 concludes the theorem. Lemma 2. We have MNIST data with τ = 1. 0
25
50
75
100
125
150
175
200
Number of Global Iterations,t
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Accuracy
HOTAFL Ideal, I=4
HOTAFL Ideal, I=2
FL Ideal
HOTAFL, I = 4, ̄P = 3.29
HOTAFL, I = 2, ̄P = 2.76
FL, ̄P = 4.80 g. 4: Test accuracy for i.i.d. CIFAR 10 data with τ
5. 0
25
50
75
100
125
150
175
200
Number of Global Iterations,t
0
1
2
3
4
5
Upper bound, [F(θPS(t))] −F*(×10−3)
FL
FL Ideal
HOTAFL, I = 2
HOTAFL Ideal, I = 2
HOTAFL, I = 4
HOTAFL Ideal, I = 4 0
75
100
125
1
Number of Global Iterations,t Fig. 3: Test accuracy for non-i.i.d. MNIST data with τ = 3. Fig. 5: Convergence rate for i.i.d. MNIST data with τ = 1. the transmit power, deploying 5MC = 100 receive antennas
almost mitigates the noise and the interference terms [5]. Increasing τ compensates the accuracy under more complex
data structure. In Fig. 5, we compare the convergence rates
of conventional FL and HOTAFL using the upper bound in
(28), with 2N = 7850, L = 10, µ = 1, G2 = 1, Γ = 1, η(t) =
5·10−2−2 · 10−5t, Pt = 1 + 10−2t, β = 3, ∥θP S(0) −θ∗∥2
2 = the transmit power, deploying 5MC = 100 receive antennas
almost mitigates the noise and the interference terms [5]. Increasing τ compensates the accuracy under more complex
data structure. In Fig. 5, we compare the convergence rates
of conventional FL and HOTAFL using the upper bound in
(28), with 2N = 7850, L = 10, µ = 1, G2 = 1, Γ = 1, η(t) =
5·10−2−2 · 10−5t, Pt = 1 + 10−2t, β = 3, ∥θP S(0) −θ∗∥2
2 =
103. It can be seen that the convergence rate of HOTAFL is
very close to that of the ideal case, and it becomes almost the
same when the number of local iterations is increased. VI. CONCLUSIONS where A1 = 1 −βm1,c1
¯βc1
−βm2,c2
¯βc2
+ βm1,c1βm2,c2
¯βc1 ¯βc2 In this work, we have proposed HOTAFL where ISs are
employed to create clusters to bring the server-side closer to
the areas where MUs are more densely located. Our framework
includes OTA cluster aggregations, where the MUs send their
model updates to the ISs through a wireless channel with
path loss and fading. We have examined the performance and
convergence rate of HOTAFL through theoretical limits as
well as model training where MNIST and CIFAR-10 datasets
are used with both i.i.d. and non-i.i.d. data distributions. The
results show that employing a cluster-based hierarchical model
outperforms the conventional FL. Proof: Using (7) and (12), we have E
∆ˆθn
P S,1(t) −∆θn
P S(t)
2
= E
h
1
M 2C2
M
X
m1=1
M
X
m2=1
C
X
c1=1
C
X
c2=1
I
X
i1=1
I
X
i2=1
∆θi1,n
m1,c1(t)
× ∆θi2,n
m2,c2(t)
1 −
1
Kσ2
h ¯βc1
K
X
k1=1
|hi1,n
m1,c1,k1(t)|2 E
∆ˆθn
P S,1(t) −∆θn
P S(t)
2
= E
h
1
M 2C2
M
X
m1=1
M
X
m2=1
C
X
c1=1
C
X
c2=1
I
X
i1=1
I
X
i2=1
∆θi1,n
m1,c1(t)
× ∆θi2,n
m2,c2(t)
1 −
1
Kσ2
h ¯βc1
K
X
k1=1
|hi1,n
m1,c1,k1(t)|2
−
1
Kσ2
h ¯βc2
K
X
k2=1
|hi2,n
m2,c2,k2(t)|2
+
1
K2σ4
h ¯β2c1
K
X
k1=1
K
X
k2=1
|hi1,n
m1,c1,k1(t)|2|hi2,n
m2,c2,k2(t)|2i
. (31) −
1
Kσ2
h ¯βc2
K
X
k2=1
|hi2,n
m2,c2,k2(t)|2
+
1
K2σ4
h ¯β2c1
K
X
k1=1
K
X
k2=1
|hi1,n
m1,c1,k1(t)|2|hi2,n
m2,c2,k2(t)|2i
. (31) Lemma 2. We have 2N
X
n=1
E
∆ˆθn
P S,1(t) −∆θn
P S(t)
2
=
1
M 2C2
M
X
m1=1
C
X
c1=1
I
X
i1=1
β2
m1,c1
K ¯
β2c1
E
∆θi1
m1,c1(t)
2
2
+
M
X
m2=1
C
X
c2=1
I
X
i2=1
2N
X
n=1
A1E
∆θi1,n
m1,c1(t)∆θi2,n
m2,c2(t)
,
(30)
where A1 = 1 −βm1,c1
¯βc1
−βm2,c2
¯βc2
+ βm1,c1βm2,c2
¯βc1 ¯βc2
Proof: Using (7) and (12), we have 2N 2
103. It can be seen that the convergence rate of HOTAFL is
very close to that of the ideal case, and it becomes almost the
same when the number of local iterations is increased. (30) Lemma 2. We have h
i
Recursively iterating through the results of Lemmas 1, 2,
and 3 concludes the theorem. 0
25
50
75
100
125
150
175
200
Number of Global Iterations,t
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Accuracy
HOTAFL Ideal, I=4
HOTAFL Ideal, I=2
FL Ideal
HOTAFL, I = 4, ̄P = 29.40
HOTAFL, I = 2, ̄P = 27.35
FL, ̄P = 44.22
Fig. 4: Test accuracy for i.i.d. CIFAR-10 data with τ = 5. 0
25
50
75
100
125
150
175
200
Number of Global Iterations,t
0
1
2
3
4
5
Upper bound, [F(θPS(t))] −F*(×10−3)
FL
FL Ideal
HOTAFL, I = 2
HOTAFL Ideal, I = 2
HOTAFL, I = 4
HOTAFL Ideal, I = 4
Fig. 5: Convergence rate for i.i.d. MNIST data with τ = 1. Lemma 4. We have 0
25
50
75
100
125
150
175
200
Number of Global Iterations,t
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Accuracy
HOTAFL Ideal, I=4
HOTAFL Ideal, I=2
FL Ideal
HOTAFL, I = 4, ̄P = 0.10
HOTAFL, I = 2, ̄P = 0.07
FL, ̄P = 0.24
Fig. 2: Test accuracy for i.i.d. MNIST data with τ = 1. 0
25
50
75
100
125
150
175
200
Number of Global Iterations,t
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Accuracy
HOTAFL Ideal, I=4
HOTAFL Ideal, I=2
FL Ideal
HOTAFL, I = 4, ̄P = 3.29
HOTAFL, I = 2, ̄P = 2.76
FL, ̄P = 4.80
Fig. 3: Test accuracy for non-i.i.d. MNIST data with τ = 3. 0
25
50
75
100
125
150
175
200
Number of Global Iterations,t
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Accuracy
HOTAFL Ideal, I=4
HOTAFL Ideal, I=2
FL Ideal
HOTAFL, I = 4, ̄P = 0.10
HOTAFL, I = 2, ̄P = 0.07
FL, ̄P = 0.24 0
25
50
75
100
125
150
175
200
Number of Global Iterations,t
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Accuracy
HOTAFL Ideal, I=4
HOTAFL Ideal, I=2
FL Ideal
HOTAFL, I = 4, ̄P = 29.40
HOTAFL, I = 2, ̄P = 27.35
FL, ̄P = 44.22 Fig. 4: Test accuracy for i.i.d. CIFAR-10 data with τ = 5. Fig. 2: Test accuracy for i.i.d. MNIST data with τ = 1. 1 0 Fig. 2: Test accuracy for i.i.d. In the following lemmas, we will bound each of these terms. Summing over all the symbols and using the independence of
channel coefficients result in (30). Lemma 6. [14] D. Liu and O. Simeone, “Privacy for free: Wireless federated learning
via uncoded transmission with adaptive power control,” IEEE J. Sel. Areas Commun., vol. 39, no. 1, pp. 170–185, 2021. 2N
X
n=1
E
∆ˆθn
P S,3(t)
2
=
σ2
zIN
P 2
t M 2C2Kσ2
h
M
X
m=1
C
X
c=1
βm,c
¯β2c
. (34) pp
[15] M. M. Amiri, D. Gunduz, S. R. Kulkarni, and H. Vincent Poor,
“Convergence of federated learning over a noisy downlink,” IEEE Trans. Wireless Commun., pp. 1–16, 2021. Proof: Using the independence of channel coefficients,
for 1 ≤n ≤N, we have [16] M. S. H. Abad, E. Ozfatura, D. Gunduz, and O. Ercetin, “Hierarchical
federated learning across heterogeneous cellular networks,” in ICASSP
2020-2020 IEEE International Conference on Acoustics, Speech and
Signal Processing (ICASSP). IEEE, 2020, pp. 8866–8870. E
∆ˆθn
P S,3(t)
2
= E
h
M
X
m=1
C
X
c=1
I
X
i=1
K
X
k=1
1
PtMCKσ2
h ¯βc
× Re
hi,n
m,c,k(t)
∗zi,n
c,k(t)
2i
= E
h
M
X
m=1
C
X
c=1
I
X
i=1
K
X
k=1
1
P 2
t M 2C2K2σ4
h ¯β2c
×
Re
hi,n
m,c,k(t)
∗zi,n
c,k(t)
2i
=
σ2
zI
2P 2
t M 2C2Kσ2
h
M
X
m=1
C
X
c=1
βm,c
¯β2c
. (35) E
∆ˆθn
P S,3(t)
2
= E
h
M
X
m=1
C
X
c=1
I
X
i=1
K
X
k=1
1
PtMCKσ2
h ¯βc
× Re
hi,n
m,c,k(t)
∗zi,n
c,k(t)
2i pp
[17] L. Liu, J. Zhang, S. Song, and K. B. Letaief, “Client-edge-cloud
hierarchical federated learning,” in ICC 2020-2020 IEEE International
Conference on Communications (ICC). IEEE, 2020, pp. 1–6. [18] S. Luo, X. Chen, Q. Wu, Z. Zhou, and S. Yu, “HFEL: Joint edge asso-
ciation and resource allocation for cost-efficient hierarchical federated
edge learning,” IEEE Trans. Wireless Commun., vol. 19, no. 10, pp. 6535–6548, 2020. × Re
hi,n
m,c,k(t)
zi,n
c,k(t)
i
= E
h
M
X
m=1
C
X
c=1
I
X
i=1
K
X
k=1
1
P 2
t M 2C2K2σ4
h ¯β2c
×
Re
hi,n
m,c,k(t)
∗zi,n
c,k(t)
2i
=
σ2
zI
2P 2
t M 2C2Kσ2
h
M
X
m=1
C
X
c=1
βm,c
¯β2c
. (35) [19] J. Wang, S. Wang, R.-R. Chen, and M. Ji, “Local averaging helps: Hi-
erarchical federated learning and convergence analysis,” arXiv preprint
arXiv:2010.12998, 2020. [20] C. Briggs, Z. APPENDIX A For the n-th symbol, we have ∆ˆθn
P S(t) = P3
l=1 ∆ˆθn
P S,l(t),
so using the independence of channel coefficients, we have E
||θP S(t+1)−v(t+1)||2
2
= E
∆ˆθP S(t)−∆θP S(t)
2
2
=
2N
X
n=1
(E
∆ˆθn
P S,1(t)−∆θn
P S(t)
2
+
3
X
l=2
E
∆ˆθn
P S,l(t)
2
. (29) Summing over all the symbols and using the independence of
channel coefficients result in (30). In the following lemmas, we will bound each of these terms. Lemma 5. We have [2] B. McMahan, E. Moore, D. Ramage, S. Hampson, and B. A. y. Arcas, “Communication-Efficient Learning of Deep Networks from
Decentralized Data,” Proceedings of the 20th International Conference
on Artificial Intelligence and Statistics (AISTATS), pp. 1273–1282, 2017. 2N
X
n=1
E
∆ˆθn
P S,2(t)
2
=
M
X
m=1
M
X
m′=1
m′̸=m
C
X
c=1
I
X
i=1
βm,cβm′,c
M 2C2K ¯β2c
E
∆θi
m′,c(t)
2
2
. (32) [3] D. Gunduz, D. B. Kurka, M. Jankowski, M. M. Amiri, E. Ozfatura,
and S. Sreekumar, “Communicate to earn at the edge,” IEEE Commun. Mag., vol. 58, no. 12, pp. 14–19, 2020. pp
[4] M. Mohammadi Amiri and D. Gunduz, “Machine learning at the wire-
less edge: Distributed stochastic gradient descent over-the-air,” IEEE
Trans. Signal Process., vol. 68, pp. 2155–2169, 2020. [5] M. M. Amiri, T. M. Duman, D. Gunduz, S. R. Kulkarni, and H. V. P. Poor, “Blind federated edge learning,” IEEE Trans. Wireless Commun.,
vol. 20, no. 8, pp. 5129–5143, 2021. Proof: For 1 ≤n ≤N, using the independence of
channel coefficients, we have pp
[6] M. M. Amiri, S. R. Kulkarni, and H. V. Poor, “Federated learning with
downlink device selection,” arXiv preprint arXiv:2107.03510, 2021. E
∆ˆθn
P S,2(t)
2
= E
h
M
X
m=1
M
X
m′=1
m′̸=m
C
X
c=1
I
X
i=1
1
MCKσ2
h ¯βc E
∆ˆθn
P S,2(t)
2
= E
h
M
X
m=1
M
X
m′=1
m′̸=m
C
X
c=1
I
X
i=1
1
MCKσ2
h ¯βc
×
K
X
k=1
Re
hi,n
m,c,k(t)
∗hi,n
m′,c,k(t)∆θi,n
m′,c(t)
2i
= E
h
M
X
m=1
M
X
m′=1
m′̸=m
C
X
c=1
I
X
i=1
βm,cβm′,c
2M 2C2K ¯β2c
×
∆θi,n
m′,c(t)
2 +
∆θi,n+N
m′,c
(t)
2 + ∆θi,n
m,c(t)∆θi,n
m′,c(t)
−∆θi,n+N
m,c
(t)∆θi,n+N
m′,c
(t)
i
(33) [7] G. Zhu, Y. Wang, and K. Lemma 6. Fan, and P. Andras, “Federated learning with hierarchical
clustering of local updates to improve training on non-iid data,” in 2020
International Joint Conference on Neural Networks (IJCNN). IEEE,
2020, pp. 1–9. (35) pp
[21] Y. LeCun, “The MNIST database of handwritten digits,” http://yann. lecun. com/exdb/mnist/, 1998. The same result holds for N + 1 ≤n ≤2N. Combining the
two results concludes the lemma. [22] A. Krizhevsky et al., “Learning multiple layers of features from tiny
images,” 2009. Combining the results in Lemmas 4, 5, and 6 and applying
Assumption 2 with (19) completes the proof of Lemma 1. [23] D. P. Kingma and J. Ba, “Adam: A method for stochastic optimization,”
arXiv preprint arXiv:1412.6980, 2014. APPENDIX A Huang, “Broadband analog aggregation for
low-latency federated edge learning,” IEEE Trans. Wireless Commun.,
vol. 19, no. 1, pp. 491–506, 2020. [8] G. Zhu, Y. Du, D. G¨und¨uz, and K. Huang, “One-bit over-the-air
aggregation for communication-efficient federated edge learning: Design
and convergence analysis,” IEEE Trans. Wireless Commun., vol. 20,
no. 3, pp. 2120–2135, 2021. pp
[9] M. Chen, N. Shlezinger, H. V. Poor, Y. C. Eldar, and S. Cui,
“Communication-efficient federated learning,” Proceedings of the Na-
tional Academy of Sciences, vol. 118, no. 17, 2021. [10] B. Tegin and T. M. Duman, “Blind federated learning at the wireless
edge with low-resolution adc and dac,” IEEE Trans. on Wireless Com-
mun., 2021. ×
∆θi,n
m′,c(t)
2 +
∆θi,n+N
m′,c
(t)
2 + ∆θi,n
m,c(t)∆θi,n
m′,c(t)
−∆θi,n+N
m,c
(t)∆θi,n+N
m′,c
(t)
i
(33) [11] ——, “Federated learning over time-varying channels,” in IEEE Global
Communications Conference (GLOBECOM), Madrid, Spain, Dec. 2021. [12] T. Sery, N. Shlezinger, K. Cohen, and Y. Eldar, “Over-the-air federated
learning from heterogeneous data,” IEEE Trans. Signal Process., vol. 69,
pp. 3796–3811, 2021. Obtaining the expressions for N + 1 ≤n ≤2N in a similar
manner and combining the two, results in (32). [13] C. T. Dinh, N. H. Tran, M. N. Nguyen, C. S. Hong, W. Bao, A. Y. Zomaya, and V. Gramoli, “Federated learning over wireless networks:
Convergence analysis and resource allocation,” IEEE/ACM Transactions
on Networking, vol. 29, no. 1, pp. 398–409, 2020. Lemma 6. REFERENCES [1] W. Y. B. Lim, N. C. Luong, D. T. Hoang, Y. Jiao, Y. C. Liang, Q. Yang,
D. Niyato, and C. Miao, “Federated learning in mobile edge networks:
A comprehensive survey,” IEEE Commun. Surveys Tuts., vol. 22, no. 3,
pp. 2031–2063, 2020. [1] W. Y. B. Lim, N. C. Luong, D. T. Hoang, Y. Jiao, Y. C. Liang, Q. Yang,
D. Niyato, and C. Miao, “Federated learning in mobile edge networks:
A comprehensive survey,” IEEE Commun. Surveys Tuts., vol. 22, no. 3,
pp. 2031–2063, 2020.
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Supplementary Figure 1 from Phosphoproteomics Identifies Driver Tyrosine Kinases in Sarcoma Cell Lines and Tumors
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Bayesian Semiparametric Density Deconvolution in the Presence of Conditionally Heteroscedastic Measurement Errors
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S.1 S.1 SUPPLEMENTARY MATERIALS Supplementary Materials to
Bayesian Semiparametric Density Deconvolution in the
Presence of Conditionally Heteroscedastic Measurement
Errors Abhra Sarkar and Bani K. Mallick
Department of Statistics, Texas A&M University, 3143 TAMU, College Station, TX 77843-3143
USA
abhra@stat.tamu.edu and bmallick@stat.tamu.edu
John Staudenmayer
Department of Mathematics and Statistics, University of Massachusetts, Amherst, MA
01003-9305 USA
jstauden@math.umass.edu
Debdeep Pati
Department of Statistics, Florida State University, Tallahassee, FL
32306-4330 USA
debdeep@stat.fsu.edu
Raymond J. Carroll
Department of Statistics, Texas A&M University, 3143 TAMU, College Station, TX 77843-3143
USA
carroll@stat.tamu.edu SUPPLEMENTARY MATERIALS S.2 Quadratic B−spline Bases
t3 = A
t4
t5
t6
t7
t8
t9 = B
0.0
0.5
1.0
Figure S.1: Plot of 9 quadratic (q = 2) B-splines on [A, B] defined using 11 knot points that divide
[A, B] into K = 6 equal subintervals. Quadratic B−spline Bases Figure S.1: Plot of 9 quadratic (q = 2) B-splines on [A, B] defined using 11 knot points that divide
[A, B] into K = 6 equal subintervals. −4
−2
0
2
4
0.0
0.1
0.2
0.3
0.4
0.5
Density
Scaled error −4
−2
0
2
4
0.0
0.1
0.2
0.3
0.4
0.5
Density
Scaled error
Figure S.2: Skew-normal densities with mean=0, variance=1 and varying skewness parameter λ. The solid line is the density of SN(· | 0, 1, 0), the special case of standard normal distribution. The dashed line is the density of SN(· | 0, 1, 7). The dotted line is the density of SN(· | 0, 1, ∞)
corresponding to the special case of a half-normal density. Figure S.2: Skew-normal densities with mean=0, variance=1 and varying skewness parameter λ. The solid line is the density of SN(· | 0, 1, 0), the special case of standard normal distribution. The dashed line is the density of SN(· | 0, 1, 7). The dotted line is the density of SN(· | 0, 1, ∞)
corresponding to the special case of a half-normal density. Table S.1: Combined p-values for 4! = 24 nonparametric tests of association between Wj1 and
Cj2j3j4 = {(Wj2 −Wj3)/(Wj2 −Wj4)} for various j1 ̸= j2 ̸= j3 ̸= j4 for 25 regularly consumed
dietary components for which daily intakes were recorded in the EATS study. See Section 3 for
additional details. S.1
Quadratic B-Splines Used to Model Variance Func-
tions in Section 2 of the Main Paper Consider knot-points t1 = t2 = t3 = A < t4 < · · · < B = tK+3 = tK+4 = tK+5, where t3:(K+3) are
equidistant with δ = (t4 −t3). For j = 3, 4, . . . , (K + 2), define b2,j(X)
=
{(X −tj)/δ}2/2
if tj ≤X < tj+1,
−{(X −tj+1)/δ}2 + (X −tj+1)/δ + 1/2
if tj+1 ≤X < tj+2,
{1 −(X −tj+2)/δ}2
if tj+2 ≤X < tj+3,
0
otherwise. b2,j(X)
=
{(X −tj)/δ}2/2
−{(X −tj+1)/δ}2 + (X −tj+1)/δ + 1/2
{1 −(X −tj+2)/δ}2
0 Also define b2,1(X)
=
{1 −(X −t1)/δ}2/2
if t3 ≤X < t4,
0
otherwise. b2,2(X)
=
−{(X −t3)/δ}2 + (X −t4)/δ + 1/2
if t3 ≤X < t4,
{1 −(X −t4)/δ}2/2
if t4 ≤X < t5,
0
otherwise. b2,K+1(X)
=
{(X −tK+1)/δ}2/2
if tK+1 ≤X < tK+2,
−{(X −tK+2)/δ}2 + (X −tK+2)/δ + 1/2
if tK+2 ≤X < tK+3,
0
otherwise. b2,K+2(X)
=
{(X −tK+2)/δ}2/2
if tK+2 ≤X < tK+3,
0
otherwise. if t3 ≤X < t4,
otherwise. if t3 ≤X < t4,
if t4 ≤X < t5,
otherwise. Supplementary Materials to
Bayesian Semiparametric Density Deconvolution in the
Presence of Conditionally Heteroscedastic Measurement
Errors SUPPLEMENTARY MATERIALS S.3 Dietary
Component
P-value combined from 4!=24 tests
Truncation Limit ς = 0.05
Truncation Limit ς = 0.50
Kendall’s
τ Test
Spearman’s
ρ Test
Kendall’s
τ Test
Spearman’s
ρ Test
1
Calcium
1
1
0.511
0.984
2
Carbohydrate
1
1
0.824
1
3
Carotene
1
1
0.816
0.993
4
Cholesterol
1
1
0.978
1
5
Copper
1
1
0.982
1
6
Monosaturated Fat
1
1
0.777
1
7
Polysatuared Fat
1
1
1
1
8
Saturated Fat
1
1
0.987
1
9
Fiber
1
1
0.627
0.995
10
Folate
1
1
1
1
11
Iron
1
1
0.996
1
12
Magnesium
1
1
1
1
13
Niacin
1
1
0.910
0.999
14
Phosphorus
0.986
1
0.769
0.986
15
Potassium
1
1
0.989
1
16
Protein
1
1
0.969
1
17
Riboflavin
1
1
1
1
18
Sodium
1
1
0.856
0.999
19
Thiamin
1
1
1
1
20
Vitamin A
1
1
0.999
1
21
Vitamin B6
1
1
0.985
1
22
Vitamin B12
1
1
0.999
1
23
Vitamin C
0.980
1
0.507
0.970
24
Vitamin E
1
1
1
1
25
Zinc
1
1
1
1
able S.1: Combined p-values for 4! = 24 nonparametric tests of association between Wj1 a
j2j3j4 = {(Wj2 −Wj3)/(Wj2 −Wj4)} for various j1 ̸= j2 ̸= j3 ̸= j4 for 25 regularly consum
ietary components for which daily intakes were recorded in the EATS study. See Section 3 f
dditional details. SUPPLEMENTARY MATERIALS S.4 S.4 S.1
Quadratic B-Splines Used to Model Variance Func-
tions in Section 2 of the Main Paper S.2
Additional Simulation Experiments In this section, we present the results of additional simulation experiments when the true density
of interest is a normalized mixture of B-splines: f 3
X(X) ∝P7
k=1 b2,k(X)ck with c = (c1, . . . , c7)T =
(0, 0, 2, 0.1, 1, 0, 0)T and equidistant knots on [−2, 6]. The normalizing constant was estimated
by numerical integration on a grid of 500 equidistant points in [−2, 6]. The true values of X
were generated from f 3
X using the inverse cumulative distribution function method. We recall
that the SRB approach of Staudenmayer, et al. (2008) models fX by normalized mixture of B-
splines and assumes normality of the scaled errors. The SRB approach and the three methods
we proposed in the main paper are compared over a factorial combination of three sample sizes
(n = 250, 500, 1000), nine different types of distributions for the scaled errors (Table 1 and Figure
1), and one variance function v(X) = (1 + X/4)2. For each subject, mi = 3 replicates were
simulated. The estimated MISEs are presented in Table S.2. Results for error distribution (i) are
summarized in Figure S.3. The results show that the deconvolution approaches proposed in Section 2 of the main paper
outperform the SRB model in all 27 (3 × 9) cases, even in scenarios when the measurement errors
were normally distributed and hence the truth actually conformed to the SRB model. This may SUPPLEMENTARY MATERIALS S.5 be attributed to the fact that Models I, II and III estimate fX by a flexible infinite mixture model,
where the number of mixture components that are ‘active’ in the data is inferred semiautomatically
from the data making it an adaptive data dependent approach. On the other hand, the SRB
model estimates the density of interest by a mixture of normalized B-Splines with a fixed number
of components. Model III, we recall, also relaxes parametric assumptions on the measurement
errors, accommodating skewness, multimodality and heavy tails and resulting in huge reductions
in MISE over other models when the measurement errors are heavy-tailed. Table S.2: Mean integrated squared error (MISE) performance of density deconvolution models
described in Section 2 of this article (Models I, II and III) compared with the model of Stauden-
mayer, et al. (2008) (Model SRB) for different scaled error distributions when the true density of
interest is a mixture of splines. The true variance function was v(X) = (1 + X/4)2. See Section
S.2 for additional details. The minimum value in each row is highlighted. True and estimated densities of interest True and estimated densities of interest −2
0
2
4
6
0.0
0.1
0.2
0.3
0.4
X
Density
True and estimated densities of interest
−4
−2
0
2
4
0.0
0.4
0.8
Scaled error
Density
True, estimated and assumed densities of errors
−2
−1
0
1
2
3
4
5
0
2
4
6
8
10
X
Variance function
True and estimated variance functions
Figure S.3: Results for heavy-tailed error distribution (i) with sample size n=1000 corresponding
to 25th percentile MISE. The true density fX is a normalized mixture of B-splines. See Section
S.2 for additional details. The top panel shows the estimated densities under different models. The bottom left panel shows estimated densities of scaled errors under Model-II (dashed line) and
Model-III (solid bold line) superimposed over a standard Normal density (solid line). The bottom
right panel shows estimated variance functions under different models. For the top panel and the
bottom right panel, the solid thin line is for Model-I; the dashed line is for Model-II; the solid bold
line is for Model-III; and the dot-dashed line is for the Model of Staudenmayer, et al. (2008). In
all three panels the bold gray lines represent the truth. −2
0
2
4
6
0.0
0.1
0.2
0.3
0.4
X
Density −4
−2
0
2
4
0.0
0.4
0.8
Scaled error
Density
True, estimated and assumed densities of errors
−2
−1
0
1
2
3
4
5
0
2
4
6
8
10
X
Variance function
True and estimated variance functions −2
−1
0
1
2
3
4
5
0
2
4
6
8
10
X
Variance function
True and estimated variance functions True, estimated and assumed densities of errors −4
−2
0
2
4
0.0
0.4
0.8
Scaled error
Density
True, estimated and assumed densities of errors True and estimated variance functions Density X Figure S.3: Results for heavy-tailed error distribution (i) with sample size n=1000 corresponding
to 25th percentile MISE. The true density fX is a normalized mixture of B-splines. See Section
S.2 for additional details. The top panel shows the estimated densities under different models. The bottom left panel shows estimated densities of scaled errors under Model-II (dashed line) and
Model-III (solid bold line) superimposed over a standard Normal density (solid line). The bottom
right panel shows estimated variance functions under different models. S.2
Additional Simulation Experiments True Error
Distribution
Sample Size
MISE ×1000
SRB
Model1
Model2
Model3
(a)
250
8.66
4.58
4.74
4.68
500
4.80
3.63
3.74
3.87
1000
4.03
2.57
2.75
2.68
(b)
250
9.13
5.77
4.38
4.48
500
5.12
3.76
3.53
3.56
1000
4.68
2.83
2.50
2.72
(c)
250
6.35
4.74
4.35
4.16
500
6.08
3.15
3.85
3.07
1000
3.93
2.54
2.96
1.93
(d)
250
6.31
5.17
5.95
3.61
500
3.70
3.91
6.36
2.70
1000
2.92
2.75
7.08
2.03
(e)
250
8.73
5.74
5.31
4.06
500
7.42
5.63
3.70
3.01
1000
7.99
3.37
2.35
1.90
(f)
250
8.86
5.32
5.39
5.19
500
4.64
3.87
3.83
3.12
1000
3.31
2.47
3.00
2.35
(g)
250
22.77
12.51
12.61
3.45
500
19.66
17.66
17.09
2.25
1000
40.55
22.66
16.36
1.50
(h)
250
11.15
6.61
6.38
3.96
500
8.34
9.38
7.18
3.22
1000
13.69
9.91
7.98
2.03
(i)
250
17.49
12.25
13.55
3.28
500
32.99
20.40
15.19
2.42
1000
40.67
19.47
12.18
1.17
Table S.2: Mean integrated squared error (MISE) performance of density deconv
described in Section 2 of this article (Models I, II and III) compared with the mo
mayer, et al. (2008) (Model SRB) for different scaled error distributions when the
interest is a mixture of splines. The true variance function was v(X) = (1 + X/4
S.2 for additional details. The minimum value in each row is highlighted. Table S.2: Mean integrated squared error (MISE) performance of density deconvolution models
described in Section 2 of this article (Models I, II and III) compared with the model of Stauden-
mayer, et al. (2008) (Model SRB) for different scaled error distributions when the true density of
interest is a mixture of splines. The true variance function was v(X) = (1 + X/4)2. See Section
S.2 for additional details. The minimum value in each row is highlighted. Table S.2: Mean integrated squared error (MISE) performance of density deconvolution models
described in Section 2 of this article (Models I, II and III) compared with the model of Stauden-
mayer, et al. (2008) (Model SRB) for different scaled error distributions when the true density of
interest is a mixture of splines. The true variance function was v(X) = (1 + X/4)2. See Section
S.2 for additional details. The minimum value in each row is highlighted. SUPPLEMENTARY MATERIALS S.6 True and estimated densities of interest For the top panel and the
bottom right panel, the solid thin line is for Model-I; the dashed line is for Model-II; the solid bold
line is for Model-III; and the dot-dashed line is for the Model of Staudenmayer, et al. (2008). In
all three panels the bold gray lines represent the truth.
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https://openalex.org/W2896239200
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https://hal.science/hal-01472903/document
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English
| null |
Design Patterns in Beeping Algorithms (extended abstract)
|
HAL (Le Centre pour la Communication Scientifique Directe)
| 2,016
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cc-by
| 15,014
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Design Patterns in Beeping Algorithms (extended
abstract)
Arnaud Casteigts, Yves Métivier, John Michael Robson, Akka Zemmari
To cite this version:
Arnaud Casteigts, Yves Métivier, John Michael Robson, Akka Zemmari. Design Patterns in Beeping
Algorithms (extended abstract). 20th International Conference on Principles of Distributed Systems
(OPODIS), Dec 2016, Madrid, Spain. hal-01472903 To cite this version:
Arnaud Casteigts, Yves Métivier, John Michael Robson, Akka Zemmari. Design Patterns in Beeping
Algorithms (extended abstract). 20th International Conference on Principles of Distributed Systems
(OPODIS), Dec 2016, Madrid, Spain. hal-01472903 To cite this version: Arnaud Casteigts, Yves Métivier, John Michael Robson, Akka Zemmari. Design Patterns in Beeping
Algorithms (extended abstract). 20th International Conference on Principles of Distributed Systems
(OPODIS), Dec 2016, Madrid, Spain. hal-01472903 Abstract We consider networks of processes which interact with beeps. In the basic model defined by
Cornejo and Kuhn [5], which we refer to as the BL variant, processes can choose in each round
either to beep or to listen. Those who beep are unable to detect simultaneous beeps. Those
who listen can only distinguish between silence and the presence of at least one beep. Stronger
variants exist where the nodes can also detect collision while they are beeping (BcdL) or listening
(BLcd), or both (BcdLcd). Beeping models are weak in essence and even simple tasks are difficult
or unfeasible with them. This paper starts with a discussion on generic building blocks (design patterns) which seem
to occur frequently in the design of beeping algorithms. They include multi-slot phases: the
fact of dividing the main loop into a number of specialised slots; exclusive beeps: having a
single node beep at a time in a neighbourhood (within one or two hops); adaptive probability:
increasing or decreasing the probability of beeping to produce more exclusive beeps; internal
(resp. peripheral) collision detection: for detecting collision while beeping (resp. listening); and
emulation of collision detection: for enabling this feature when it is not available as a primitive. We then provide algorithms for a number of basic problems, including colouring, 2-hop colour-
ing, degree computation, 2-hop MIS, and collision detection (in BL). Using the patterns, we
formulate these algorithms in a rather concise and elegant way. Their analyses (in the full ver-
sion) are more technical, e.g. one of them relies on a Martingale technique with non-independent
variables; another improves that of the MIS algorithm in [8] by getting rid of a gigantic constant
(the asymptotic order was already optimal). Finally, we study the relative power of several variants of beeping models. In particular,
we explain how every Las Vegas algorithm with collision detection can be converted, through
emulation, into a Monte Carlo algorithm without, at the cost of a logarithmic slowdown. We
prove that this slowdown is optimal up to a constant factor by giving a matching lower bound. Digital Object Identifier 10.4230/LIPIcs... Keywords. Beeping models, Design patterns,
Collision detection, Colouring, 2-hop colouring, Degree computation, Emulation. Design Patterns in Beeping Algorithms
(extended abstract)∗ A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari LaBRI, University of Bordeaux
{acasteig, metivier, robson, zemmari}@labri.fr LaBRI, University of Bordeaux
{acasteig, metivier, robson, zemmari}@labri.fr HAL Id: hal-01472903
https://hal.science/hal-01472903v1
Submitted on 21 Feb 2017 L’archive ouverte pluridisciplinaire HAL, est
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entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. ∗This research has been supported by ANR projects DESCARTES (ANR-16-CE40-0023) and ESTATE
(ANR-16-CE25-0009-03). A full version is available on arXiv (http://arxiv.org/abs/1607.02951)
licensed under Creative Commons License CC-BY
Leibniz International Proceedings in Informatics
Schloss Dagstuhl – Leibniz-Zentrum für Informatik, Dagstuhl Publishing, Germany 1.1
Contributions The contributions of this paper are manifold. As a warm-up, we start by identifying generic
building blocks (design patterns) which seem to occur often in the design of beeping algo-
rithms. Then we present a number of algorithms for various graph problems which improve
upon previous solutions. Finally, we generalise existing emulation techniques for using col-
lision detection if it is not available, and we prove them optimal w.h.p. up to a constant
factor. Due to space limitations, this version of the paper omits (in its core) most complexity
analyses and some proofs. However, both are available respectively in Appendix A and B of
the present paper, as well as in the arXiv version whose reference is given in the first page. 1.1.1
Design patterns. We identify a number of common building blocks in beeping algorithms, including multi-slot
phases: the fact of dividing the main loop into a (typically constant) number of slots having
specific roles (e.g., contention among neighbours, collision detection, termination detection);
exclusive beeps: the fact of having a single node beep at a time in a neighbourhood (within
one or two hops, depending on the needs); adaptive probability: increasing or decreasing
the probability of beeping in order to maximise the number of exclusive beeps; internal
(resp. peripheral) collision detection: the fact of detecting collision while beeping (resp. listening); and emulation of collision detection: the fact of detecting collisions even when it
is not available as a primitive. As we show in the paper, these patterns make it possible to
formulate the algorithms in a rather concise and elegant way. (extended abstract) (extended abstract) In beeping models, the only communication capabilities offered to the nodes are to beep
or to listen. Several variants exist. In [5], a node that beeps is unable to detect whether other
nodes have beeped simultaneously. When listening, it can distinguish between silence or the
presence of at least one beep, but it cannot distinguish between one and several beeps. In
Section 6 of [1], beeping nodes can detect whether other nodes are beeping simultaneously. In [10] and Section 4 of [1], yet another variant is considered where listening nodes can tell
the difference between silence, one beep, and several beeps. In this paper, we denote the ability to detect collision while beeping (internal collision) by
Bcd and that of detecting collision while listening (peripheral collision) by Lcd. The absence
of such ability is denoted by B and L, respectively. The existing models can be reformulated
using the cartesian product of these capabilities. Hence, the basic model introduced by
Cornejo and Kuhn in [5] is BL; the model considered by Afek et al. in [1] (Section 6) and
Jeavons et al. in [8] is BcdL; and the model considered in [10] and in Section 4 of [1] is BLcd. To the best of our knowledge, BcdLcd was only used in a previous work of the authors [3]. Although some variants are stronger than others, all beeping models remain extremely
weak in essence. Yet, they are relevant to account for real-world applications or phenomena. For instance, they reflect the features of a network at the lowest levels (physical and MAC
layers), where a node can probe or emit signals, with or without collision detection. At
a higher level of abstraction, beeping models also reflect some communication patterns in
biology [4, 1, 9]. 1
Introduction Distributed computing is concerned with various assumptions, like the structure of the
network (trees, rings, planar graphs, etc.) or knowledge available to the nodes (network
size, identifiers, port numbering, etc.). Another important aspect is the size of messages,
which may range from unbounded, to logarithmic size, to constant size. As a natural goal is to reduce assumptions as much as possible. Typically, when a
problem is solved in some strong model, the community strives to solve it in weaker models. In a recent series of works [5, 10, 1, 7, 8, 6], new models were explored that are even weaker
than constant size messages. They are called beeping models. XX:2 Design Patterns in Beeping Algorithms (extended abstract) 1.1.2
Algorithms and analyses for basic graph problems. We present, or analyse algorithms for a number of basic graph problems, including colouring,
2-hop colouring, degree computation, Maximal Independent Set (MIS) and 2-hop MIS. Quite A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari XX:3 A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari
Model
Time (# slots)
Message size
Knowledge
# colours
BcdL
O(log n
+
∆)
expected and w.h.p. ≃1 bit
(BcdL beeps)
None
O(log n + ∆)
BcdL
O K(log n + log2 K)
w.h.p. ≃1 bit
(BcdL beeps)
Upper
bound
K
on
the max degree of G
K
Table 1 Randomised Las Vegas colouring algorithms on graphs with n vertices. Table 1 Randomised Las Vegas colouring algorithms on graphs with n vertices. often, the design of algorithms is easier and more natural if collision detection is assumed
as a primitive, e.g., in BcdLcd or BcdL. Furthermore, emulation techniques such as those
described later in this paper enable safe and automatic translations of algorithms into weaker
models like BL. For this reason, our algorithms are expressed using whichever model is the
most convenient. First, we present a Las Vegas (i.e. guaranteed result, uncertain time) colouring algorithm
in the BcdL model, with time complexity of O(log n + ∆) slots w.h.p., where ∆is the
maximum degree in G. Its analysis relies on a martingale technique with non-independent
random variables, which makes use of a result by Azuma [2] (details in appendix). In fact,
the phenomenon is quite ubiquitous in beeping models: the algorithm terminates in the
first moment when every node has produced an exclusive beep at least once within its (1-
hop) neighbourhood. This stopping time is made more complex by the use of the adaptive
probability pattern mentioned above. Another algorithm for 2-hop colouring is given, this
time in the BcdLcd model, with slot complexity O(log n+∆2) w.h.p. Both algorithms require
no knowledge on G. However, both can result in arbitrarily many colours (in fact, one per
slot). If the nodes know an upper bound K ≥∆, a different strategy is proposed that uses at
most K + 1 colours. However, the slot complexity becomes O(K(log n + log2 K)) w.h.p. for
colouring (trade K for K2 in the 2-hop variant). Note that this complexity is not thought
to be tight. The results are summarised on Table 1. 1.1.3
Collision detection and emulation techniques. Classical considerations on symmetry breaking in anonymous beeping networks, see for
example [1] (Lemma 4.1), imply that there is no Las Vegas internal collision detection
algorithm in the beeping models BL and BLcd. Likewise, there is no Las Vegas peripheral
collision detection algorithm in the beeping models BL and BcdL. Since collision detection is
required to detect exclusive beeps with certainty, and this pattern is central in most beeping
algorithms, this implies that a large range of algorithms cannot exist in a Las Vegas version
in these models. We study the cost of detecting collision when it is not available, typically in BL, and
present generic techniques to emulate collision detection probabilistically in order to trans-
form Las Vegas algorithms with collision detection into Monte Carlo algorithms (uncertain
result, guaranteed time) in BL. These techniques generalise that of Algorithm 3 in [1],
where a similar strategy is encapsulated into the algorithm. We show how, given 0 < ϵ < 1,
any collision in the neighbourhood of a given node can be detected in O(log( 1
ϵ )) slots with
error at most ϵ, and similarly it can be detected in O(log n) slots w.h.p. Ensuring that this
is true for any node requires more time. By union bound, it holds that O(log( n
ϵ )) slots
are sufficient with error ϵ and that O(log n) slots are sufficient w.h.p. We prove that this
technique is essentially optimal (asymptotically and up to a constant factor) by giving a
matching lower bound. Precisely, we prove that some topologies require Ω(log n) slots to
break symmetries w.h.p. Finally, we provide two generic procedures that can be used in an
algorithm to emulate collision detection when it is not available (e.g. in BL). These proce-
dures are EmulateBcdinBL(), to detect collision while beeping, and EmulateLcdinBL(), to
detect collision while listening. We illustrate their use in the case of the computation of a
MIS given in BcdL, thus obtaining a Monte Carlo algorithm in BL. Design Patterns in Beeping Algorithms Design Patterns in Beeping Algorithms 1.2
Organisation of the paper In Section 2 we present the model and give further definitions. Section 3 introduces design
patterns in a tutorial manner. These patterns are then used in Section 4 to describe the
various algorithms. For the sake of readability, the corresponding analyses are put together
in Section A. Finally, Section 5 presents our contribution on collision detection and emulation
techniques. An extra bibliography is provided in Section ?? on related questions. 1.1.2
Algorithms and analyses for basic graph problems. Based on the observation that degree computation is strongly related to 2-hop colouring,
we present an adaptation of the algorithm for this problem, with same slot complexity,
that is, O(log n + ∆2) w.h.p. In fact, the random process induced by this algorithm is
the same as that of colouring, except that it occurs in the square of the graph (whence
the ∆2 term). Algorithmically, the main loop contains more specialised slots (e.g., one
for peripheral collision reporting), but still a constant number of them, which keeps the
asymptotics unchanged. We then turn our attention to the 2-hop MIS problem, which shares
common traits and patterns with 2-hop colouring and degree computation and, regarding
the high-level purpose of each phase, with the MIS algorithm from [8]. The running time
is however shorter than that of 2-hop coulouring and degree computation (and the analysis
quite different) due to the fact that exclusive beeps cause whole neighbourhoods to terminate
at once. In fact, we prove that the slot complexity of this algorithm is O(log n) w.h.p. with
a “reasonable” constant factor of 76. Noteworthily, the number of phases (i.e. iterations of
the main loop) for the 2-hop MIS is exactly the same as what the analogue for classical MIS
would produce in the square of the graph. As a consequence, our analysis also improves
substantially that of the MIS algorithm presented in [8], where a gigantic constant factor (i.e. one larger than e25) is used. An earlier analysis in [11] yielded a better, yet huge constant
of 2 × 1011. Although constant factors are less meaningful in general, the gap in this case is
one between practical and unpractical running times. Furthermore, the contribution it not
as much in the constant itself than in the analysis techniques that achieve it. XX:4 Distributed Randomised Algorithm. A randomised (or probabilistic) algorithm is an algorithm which makes choices based on
given probability distributions. A distributed randomised algorithm is a collection of local
randomised algorithms (in our case, all identical). A Las Vegas algorithm is a randomised algorithm whose running time is not determin-
istic, but still finite with probability 1, and that always produces a correct result. A Monte
Carlo algorithm is a randomised algorithm whose running time is deterministic, but whose
result may be incorrect with a certain probability. Put differently, Las Vegas algorithms
have uncertain execution time but certain result, and Monte Carlo algorithms have cer-
tain execution time but uncertain result. Classical considerations on symmetry breaking in
anonymous beeping networks (see for instance Lemma 4.1 in [1]), imply that: ▶Remark. There is no Las Vegas (and a fortiori no deterministic) algorithm in BL which
allows a node to distinguish between an execution where it is isolated and one where it has
exactly one neighbour. From this remark we deduce that there is no Las Vegas counting algorithm in BL, which
advocates the use of stronger models. In what follows, we consider whichever model is
the most convenient and provide Las Vegas algorithms in these models. We then present
canonical emulation techniques to turn any such algorithm into a Monte Carlo one in BL. A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari addition, processors can perform an unrestricted amount of local computation in-between
two slots (in effect, our algorithms require little computation). ▶Remark. In general, nodes are active or passive. When they are active they beep or listen;
in the description of algorithms we say explicitly when a node beeps meaning that a non
beeping active node listens. The time complexity, also called slot complexity, is the maximum number of slots needed
until every node has terminated. Our algorithms are typically structured into phases, each
of which corresponds to a small (constant or logarithmic) number of slots. In the algorithm,
we specify which one is the current slot by means of a switch instruction with as many
case statements as there are slots in the phase. Phases repeat until some condition holds
for termination. ▶Remark. An algorithm given in a beeping model induces an algorithm in the (synchronous)
message passing model. Thus, given a problem, any lower bound on the round complexity
in the message passing model also holds for slot complexity in the beeping model. 3
Design patterns for beeping algorithms As a warm-up, this section presents a number of design patterns which seem to occur
frequently in the design of beeping algorithms. The concept of pattern refers here to reusable
solutions to common problems. These patterns are then used to describe algorithms in the
other sections. 2
Network Model and Definitions We consider a wireless network and we follow definitions given in [1] and [5]. The network
is anonymous: unique identifiers are not available to distinguish the processes. Possible
communications are encoded by a graph G = (V, E) where the nodes V represent processes
and the edges E represent pairs of processes that can hear each other. We denote by ∆the
maximum degree of G. The neighbourhood of a vertex v, denoted N(v), is the set of vertices
adjacent to v (at distance 1 from v). We define N(v) by including v itself in N(v). We also
use the set of vertices at distance at most 2 from v called the 2-neighbourhood of v and
denoted N2(v) (or N2(v) if it includes v). Finally, we write log n for the binary logarithm
of n. Time is divided into discrete synchronised time intervals (rounds) also called slots (follow-
ing the usual terminology in wireless networks). All processes wake up and start computation
in the same slot. In each slot, all processors act in parallel and either beep or listen. In XX:5 (extended abstract) goal is all the more difficult to achieve that the nodes cannot use identifiers nor even port
numbers in their basic exchanges. If we assume that a node that is beeping can detect
whether another node beeps simultaneously (Bcd), then this feature can be used to take
exclusive decision if indeed it beeps alone. We call this an exclusive beep. Algorithm 1
illustrates an empty shell of algorithm that relies on repeated attempts to produce exclusive
beeps. Most, if not all algorithms rely implicitly on this pattern as a basis. Algorithm 1: Exclusive beeps (using Bcd). repeat
beep with some probability;
if I beeped alone then
do something exclusive;
... until finished; Algorithm 1: Exclusive beeps (using Bcd). Algorithm 1: Exclusive beeps (using Bcd). Algorithm 1: Exclusive beeps (using Bcd). repeat
beep with some probability;
if I beeped alone then
do something exclusive;
... 2-hop exclusive beeps. For some problems like 2-hop colouring, 2-hop MIS, or computation of the degree (all dis-
cussed in this paper), the level of mutual exclusion offered by exclusive beeps is not sufficient
and the algorithm requires that a node be the only one to beep at distance 2. Assuming
collision can also be detected upon listening (Lcd), one can design a 2-slots pattern whereby
non-beeping neighbours report if they have heard more than one beep. Hence, if a node
produced an exclusive beep in the first slot, and none of its neighbours reported a collision
in the second, then it knows that it has produced a 2-hop exclusive beep (see Algorithm 2). Algorithm 2: Two-hops exclusive beeps (using BcdLcd). repeat
switch slot do
slot 1 // contending
beep with some probability;
slot 2 // detection of peripheral collision
if several neighbours beeped in slot 1 then
beep
after slot 2
if I beeped alone in slot 1 and no neighbour beeped in slot 2 then
do something 2-hop exclusive
... until finished; Algorithm 2: Two-hops exclusive beeps (using BcdLcd). repeat
switch slot do
slot 1 // contending
beep with some probability;
slot 2 // detection of peripheral collision
if several neighbours beeped in slot 1 then
beep
after slot 2
if I beeped alone in slot 1 and no neighbour beeped in slot 2 then
do something 2-hop exclusive
... until finished; Algorithm 2: Two-hops exclusive beeps (using BcdLcd). until finished; Exclusive beeps. Beeping algorithms operate in synchronous periods called slots, which are equivalent to the
concept of rounds in message passing models. Most problems in distributed computing
require some node v to take exclusive decisions at times (i.e., with respect to vertices of
N(v) or N2(v)), which requires some type of symmetry breaking. In beeping networks, this XX:6 Adaptive probability. As far as feasibility and expressivity are concerned, the next design pattern is not crucial. However, it plays a central role in terms of performance. Adaptive probability consists in
adapting the probability to beep in the next phase depending on the outcome of previous
phases. Typically, if a collision occurs, the probability is reduced, and if no one beeps, it is
increased. Since the nodes do not know how many neighbours are contending with them,
this technique proves useful in optimizing the odds of producing exclusive beeps. The values Algorithm 3: Adaptive beeping probability (using BcdLcd). Float p ←1/2 // say
repeat
beep with probability p;
if I beeped alone then
do something exclusive;
else
if no one beeped then
increase p;
else
decrease p;
until finished; Algorithm 3: Adaptive beeping probability (using BcdLcd). given to the probabilities in Algorithm 3 are left unspecified. There are several options. In this paper, we use a doubling/halving pattern, that is, p is increased to 2p (up to 1/2),
and it is decreased to p/2 (without limit). A similar doubling/halving pattern was used
in [11]. One could also increment or decrement the denominator of p as done in [3]. The
consequences of choosing one over the other are not discussed here. A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari which have not yet performed some action beep. If the slot remain silent, then a form of
local termination is detected: nodes are in a terminal state. which have not yet performed some action beep. If the slot remain silent, then a form of
local termination is detected: nodes are in a terminal state. Multi-slot phases. The example in Algorithm 2 illustrates another common aspect of beeping algorithms,
namely multi-slot phases. The expressivity of a single beep is rather poor, but several
combined slots can achieve elaborate behavior. In Algorithm 2, one slot is devoted to con-
tending and another to peripheral collision detection. The whole compound is then called
a phase. Another common task is termination detection. In a termination slot, all nodes XX:7 4.1
Colouring The colouring problem consists of assigning a colour to every node in the network, such that
no two neighbours have the same colour. We first consider the case that no extra information
is available to the nodes. Then we consider that (an upper bound on) the maximum degree
is known. 4
Algorithms for basic graph problems We now present algorithms for a number of problems, including colouring (with or without
knowledge on the degree), 2-hop colouring, computation of the degree and 2-hop MIS. These
algorithms are based on various combinations of the patterns presented in Section 3. All
algorithms are Las Vegas, and they rely on medium to strong primitives (BcdL to BcdLcd
models) depending on the needs. The adaptation of these algorithms in the weakest model
(BL) is discussed in Section 5. We also recall Jeavons et al.’s Las Vegas algorithm for the
MIS [8] problem and discuss its relations with our 2-hop MIS algorithm. Whenever using the adaptive probability pattern in algorithms, for generality, we stick
to the terms increase and decrease (as opposed to our analyses, in which these actions are
instantiated to doubling and halving the probability). Collision detection. Most algorithms in this paper use collision detection as a built-in primitive, referred to as
Bcd for detection on beeping and Lcd for detection on listening. However, this feature is
not always available as a primitive. An important question is the transformation of a (high-
level) algorithm using Bcd or Lcd (or both) into one that works in the weakest BL model. This question is the topic of Section 5, in which we study generic mechanisms to achieve this
goal. Essentially, each slot that requires collision detection can be replaced with a logarithmic
number of slots (in the size of various quantities depending on the desired guarantees) where
the ties are broken w.h.p. We provide dedicated procedures that generalise the technique
used internally to one of the algorithms in [1]. Besides complexity, the price to pay is that
the algorithm becomes Monte Carlo instead of Las Vegas, that is, the result is correct only
probabilistically (though possibly w.h.p.). We present a matching lower bound showing that
these procedures are essentially optimal. XX:8 Design Patterns in Beeping Algorithms A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari with time, thus it is at most the same (at most, because some phases may not produce
exclusive beeps). Colouring without knowledge. Informally, the algorithm proceeds as follows (see Algorithm 4 for details). Initially, every
node is uncoloured (nil). In every phase, each node increments a counter. Uncoloured nodes
contend with each other to produce an exclusive beep, and when one succeeds, it takes the
current value of the counter as its colour and retires. An adaptive probability is used to
regulate the probability of beeping among uncoloured nodes. Local termination (a node and
its neighbours are coloured) detection is not explicitly handled here, though we could add a
termination slot where uncoloured nodes are the only ones to beep. Algorithm 4: A Las Vegas colouring algorithm in BcdL (without knowledge). Float p ←1/2;
Integer colour ←nil;
Integer counter ←0;
repeat
beep with probability p;
if I beeped alone then
colour ←counter
else
if no one beeped then
increase p;
else
decrease p;
counter ←counter + 1;
until colour ̸= nil; The running time of this algorithm is of O(log n + ∆) phases w.h.p as well as on average
(none of both imply the other trivially). Note that this is also the number of slots, since each
phase consists of a constant number of slots. As for the number of colours, it is incremented XX:9 A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari 4.2
2-hop colouring A 2-hop colouring of a graph G is a colouring such that any two nodes at distance ≤2 have
different colours. In other words, it is a colouring of the square of G, the graph where an
edge exists between nodes which are neighbours in G or share a common neighbour in G. Colouring with a bound K on the maximum degree ∆. If a bound K ≥∆is known, then one can obtain a better colouring using at most K + 1
colours. The algorithm follows the same lines as Algorithm 4, i.e. a colour counter is
incremented in each phase, and its current value is chosen by those nodes who produced
an exclusive beep. The main difference (see Algorithm 5 for details) is that only those
colours within {0, . . . , K} are considered and thus the counter is incremented modulo K +1. Conflicts of colours are avoided by keeping a phase idle if the corresponding value was already
taken in the past (locally). To do so, when a node takes a colour, it re-beeps in a new slot
called confirmation slot to inform its neighbours that they must remove the current colour
from their list of authorized colours. Accordingly, the uncoloured will contend in a phase
only if the current colour is still available (otherwise, they wait). An adaptive probability
is used similarly to Algorithm 4, except that idle phases are not considered as silent (the
probability is not updated in these phases). Algorithm 5: A Las Vegas colouring algorithm in BcdL (knowing K ≥∆). Colours = {0, · · · , K};
Float p ←1/2;
Integer colour ←nil;
Integer counter ←0;
repeat
if counter ∈Colours then
switch slot do
slot 1 // contending
beep with probability p
slot 2 // confirmation
if I beeped alone in slot 1 then
colour ←counter;
beep;
else
if no one beeped then
increase p;
else
decrease p;
if someone beeped in slot 2 then
Colours ←Colours \ {counter}
counter ←(counter + 1) mod (K + 1);
until colour ̸= nil; Algorithm 5: A Las Vegas colouring algorithm in BcdL (knowing K ≥∆). Regarding performance, the only difference between this algorithm and Algorithm 4 is
that a growing number of phases are idle in each neighbourhood, inflicting a slow down
to the algorithm. Managing the dependencies here proved more difficult and we “only”
managed to prove that the number of phases is O
K(log n + log2 K)
w.h.p. However, the
algorithm is believed to be faster. (extended abstract) 4.3
Degree computation Let us recall that 2-hop exclusive beeps allow a node v to perform an exclusive action within
a radius of distance 2. This feature was used in Section 4.2 to assign unique colours. At it
turns out, the pattern is very versatile and it can be used to count the degree of a node as
well. The strategy consists in replacing the colour-related action in slot 2 (second if-then
block) by an action aiming at having v counted in the degree of its neighbours (then v stops
contending and keeps on reporting collisions, as before). Precisely, a new confirmation slot is
inserted wherein v re-beeps if indeed it produced a 2-hop exclusive beep. Upon hearing the
confirmation beep, all of v’s neighbours increment a local counter that eventually amounts
to their degree. Termination proceeds in the same way as for the 2-hop-colouring algorithm
(i.e. uncounted nodes beep in a termination slot). Up to a constant factor which accounts for the additional confirmation slot in each phase,
the running time of this algorithm is again O(log n + ∆2) w.h.p. A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari Once we realize that the execution produced here is the same as what Algorithm 4
would produce in the square of G, analysis of this algorithm is straightforward. The only
difference is that the maximal number of contenders of a node becomes ∆2 instead of ∆. Thus Algorithm 6 takes O(log n + ∆2) phases (and slots) w.h.p., and the number of colours
cannot exceed the same value. 4.4
Jeavons et al.’s Las Vegas Algorithm for the MIS in BcdL We recall here Jeavons et al.’s Las Vegas Algorithm for the MIS [8]. This algorithm uses an
adaptive probability to maximize the frequency of exclusive beeps (with a doubling/halving
pattern for p, starting at 1/2). If a node v produces an exclusive beep, it enters the MIS
(by the end of the first slot), then it uses a confirmation slot to inform its neighbours, all
of which terminate together with v. Since the whole neighbourhood shuts down at once,
the algorithm progresses faster than, for instance, the basic colouring algorithm discussed
above. This algorithm was already proven by Jeavons et al. to terminate within O(log n)
slots with a huge constant factor (larger than e25). With a bound K on the maximum degree ∆. The same idea can be applied as in the 1-hop variant, i.e., taking colours between 0 and
K2 + 1 (instead of K + 1) and incrementing the counter accordingly (mod K2 + 1). As a
result, at most K2 + 1 colours are used, with time complexity O(K2(log n + log2 K)) w.h.p. 2-hop colouring without knowledge. A similar strategy is used as in Algorithm 4 (colouring), except that exclusive beeps are
replaced with 2-hop exclusive beeps. Whenever a node produces such a beep, it takes the
current value of the counter as colour. Since no other node has beeped within distance 2,
the colouring is legal. Contrary to the 1-hop colouring, the collaboration of a node remains
crucial even after it becomes coloured. Indeed, this node must keep on reporting peripheral
collisions to its neighbours. As a result, instead of retiring from computation, coloured nodes
keep on listening until all of their neighbours are coloured, which is detected using an extra
termination slot. Details are given in Algorithm 6. Four slots are used in total, the first
two being devoted to the management of 2-hop exclusive beeps (see Section 3 for details). The third slot manages a (2-hop) adaptive probability based on beeps heard at distance one
(slot 1) or at distance two (slot 3 itself). Finally, slot 4 is the termination slot. Algorithm 6: A Las Vegas 2-hop-colouring algorithm in BcdLcd (without knowledge). Float p ←1/2;
Integer colour ←nil;
Integer counter ←0;
repeat
switch slot do
slot 1 // contending slot
if colour = nil then
beep with probability p;
slot 2 // peripheral collision detection (and consequences)
if several neighbours beeped in slot 1 then
beep
if I beeped alone in slot 1 and heard no beep in slot 2 then
colour ←counter
slot 3 // adaptive probability
if someone beeped in slot 1 then
beep
if colour = nil then
if no beep heard in slot 1 nor 3 then
increase p
else
decrease p
slot 4 // termination slot
if colour = nil then
beep
counter ←counter + 1
until no beep heard in slot 4; Algorithm 6: A Las Vegas 2-hop-colouring algorithm in BcdLcd (without knowledge). XX:11 A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari ▶Lemma 1. Let v be a node. If a collision occurs in the neighbourhood of v, then v detects
it in O
log( 1
ϵ )
sub-phases (slots) with probability at least 1 −ϵ, and in O (log n) sub-phases
(slots) with probability 1 −o
1
n2
. 5
Collision detection and emulation techniques In Section 4, we have considered collision detection as a built-in primitive. Depending on
the algorithms, we assumed that collision detection was possible while beeping (Bcd) or
while listening (Lcd). This assumption is convenient because it allows one to design Las
Vegas algorithms for all the considered problems. Unfortunately, we know since [1] that
no Las Vegas algorithms can be designed for most problems without collision detection,
that is, in the BL model. One has to turn to Monte Carlo instead, which means that the
result is correct only with some probability (possibly w.h.p.). In this section, we investigate
the cost of building a probabilistic collision detection primitive in the BL model, inspired
by a technique from [1]. Then we adapt it into two generic emulation procedures, one for
detecting collision while beeping, the other while listening. These procedures can then be
used to translate any Las Vegas algorithml in BcdL, BLcd, or BcdLcd, into a Monte Carlo
algorithm in BL. The cost is a logarithmic slowdown of the execution, which we prove is
essentially optimal (for sufficiently large n). (extended abstract) within a (small) constant factor. Interestingly, our analysis of this algorithm improves much
over that of [8], taking the huge constant down to 76 (i.e., making the algorithm practical). within a (small) constant factor. Interestingly, our analysis of this algorithm improves much
over that of [8], taking the huge constant down to 76 (i.e., making the algorithm practical). 5.1
Collision detection The impossibility for a node in BL to distinguish between begin alone or having neighbours
has strong implications. For instance, in the colouring problem, it means that two neighbours
could possibly end up with the same colour. In the MIS problem, two neighbours could enter
the MIS. In fact, there is no guarantee on the correctness of basic patterns like exclusive beeps
or 2-hop exclusive beeps, which are at the basis of most (if not all) Las Vegas algorithms. We present a (Monte Carlo) algorithm for detecting collisions in BL. This procedure
generalises the technique used in Algorithm 3 of [1], which consists of replacing each slot that
requires collision detection in the original model, with several BL slots in which symmetries
are probabilistically broken. Of course, the more slots, the more reliable the detection. 4.5
Computing a 2-hop MIS In this problem, we must select a set of nodes (the MIS) such that no pair of selected
nodes are within distance 2 and no node can be added further to the set. This algorithm
is a combination of those of other 2-hop algorithms seen above, and Jeavons et al’s MIS
algorithm. That is, the same structure of algorithm is used as for 2-hop colouring or degree
computation, except that whenever a node produces a 2-hop exclusive beep, it enters the
MIS and informs its neighbours (using the confirmation slot) that they will not be in the
MIS. This algorithm takes the same number of phases than what the (1-hop) MIS algorithm
would produce in the square of the graph, that is, O(log n) w.h.p.. The number of slots
is higher due to using additional slots for managing 2-hop exclusive beeps, but it remains The algorithm. The same argument, combined with the second claim of Lemma
1 proves the second claim. ◀ The algorithm. Assume a collision occurs between some nodes u1 and u2 in the neighbourhood of
v (one of them being possibly v itself). It is detected if u1 and u2 choose a different slot
in at least one of the k sub-phases. The probability that this does not happen is
1
2
k. This probability is less than ϵ (resp. o
1
n2
) for any k ≥log( 1
ϵ ) (resp. 2 log(n)). Observe
that if collisions occur between more than two nodes in the neighbourhood of v, this cannot
decrease the odds of a successful detection (to the contrary, the odds can only increase). ◀ Proof. Assume a collision occurs between some nodes u1 and u2 in the neighbourhood of
v (one of them being possibly v itself). It is detected if u1 and u2 choose a different slot
in at least one of the k sub-phases. The probability that this does not happen is
1
2
k. This probability is less than ϵ (resp. o
1
n2
) for any k ≥log( 1
ϵ ) (resp. 2 log(n)). Observe
that if collisions occur between more than two nodes in the neighbourhood of v, this cannot
decrease the odds of a successful detection (to the contrary, the odds can only increase). ◀ ▶Corollary 2. Let G be a graph. If collisions occur in the neighbourhood of an arbitrary
number of nodes, then all of them detect collision after at most O
log( n
ϵ )
sub-phases (slots)
with probability at least 1 −ϵ, and after at most O (log n) sub-phases (slots) w.h.p. Proof. Assume collisions occur in G and let T denote the number of sub-phases before all
concerned nodes detect collision. Clearly T = max{Tv | v ∈V }, where Tv is the time it
takes to any node v to decide collision. By the same argument as in the proof of Lemma 1,
together with union bound, it holds that Pr
T > log
n
ϵ
≤
n × Pr
Tv > log
n
ϵ
(1)
=
n ×
1
2log( n
ϵ ) = ϵ
(2) (1) (2) which proves the first claim. The same argument, combined with the second claim of Lemma
1 proves the second claim. ◀ which proves the first claim. The algorithm. Each slot that requires collision detection (Bcd or Lcd) is replaced with a number of sub-
phases, each consisting of two BL slots. For instance, if a node wishes to beep with collision
detection in the original algorithm, it will choose one of the two slots (u.a.r.) in each of the
sub-phases and will beep in that slot (listen in the other). If it hears a beep while listening in
the other slot, then an internal collision is detected. Similarly, if a node wishes to listen with
collision detection in the original algorithm, it will listen in both slots of each sub-phase. A peripheral collision is detected if a beep is heard in both slots of a same sub-phase. The
procedure is detailed by Algorithm 7, where k is the number of sub-phases used. False positives never happen, but real collisions might still go unnoticed, with probability
inversely related to k. We are interested in determining how large k should be to guarantee
that a given node detects a collision in its neighbourhood with a given probability. The
stronger question asks how many sub-phases are required to guarantee that none of the
nodes fails to detect a collision. XX:13 A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari Algorithm 7: Collision detection algorithm in BL (with parameter k)
Boolean collision ←false;
Integer i ←0;
while i < k do
if v wishes to beep then
Flip a coin;
if heads then
beep in slot 1;
listen in slot 2;
else
listen in slot 1;
beep in slot 2;
if another beep was heard then
collision ←true
else
listen in both slots;
if beeps are heard in both slots then
collision ←true;
i ←i + 1;
return collision; Algorithm 7: Collision detection algorithm in BL
Boolean collision ←false;
Integer i ←0;
while i < k do
if v wishes to beep then
Flip a coin;
if heads then
beep in slot 1;
listen in slot 2;
else
listen in slot 1;
beep in slot 2;
if another beep was heard then
collision ←true
else
listen in both slots;
if beeps are heard in both slots then
collision ←true;
i ←i + 1;
return collision; Algorithm 7: Collision detection algorithm in BL (with parameter k) Proof. 5.2
Emulation procedures Based on this tie-breaking mechanism, we define two probabilistic emulation procedures
whose purpose is to replace beep or listen instructions with collision detection in BL. XX:14
Design Patterns in Beeping Algorithms (extended abstract) (extended abstract) (extended abstract) Both are Monte Carlo in the sense that detection is only guaranteed with some proba-
bility. The first procedure, EmulateBcdinBL(), is given by Algorithm 8 and the second,
EmulateLcdinBL(), by Algorithm 9. Both procedures are parametrized by an integer k > 1,
which accounts for the number of sub-phases that are used in each invocation of the proce-
dure (k controls the error bound). They return true if a collision has been detected, false
otherwise. Both are Monte Carlo in the sense that detection is only guaranteed with some proba-
bility. The first procedure, EmulateBcdinBL(), is given by Algorithm 8 and the second,
EmulateLcdinBL(), by Algorithm 9. Both procedures are parametrized by an integer k > 1,
which accounts for the number of sub-phases that are used in each invocation of the proce-
dure (k controls the error bound). They return true if a collision has been detected, false
otherwise. Before the execution each vertex generates a sequence s of k random bits (u.a.r.) which
will be the ones used in each sub-phase. The reason why this is made once at the beginning
rather than in each invocation is a technicality that relates to preventing an additional
union bound in the analysis (more k would be needed to guarantee that each invocation is
successful if the numbers are drawn every time). Algorithm 8: A Procedure to emulate a Bcd in the BL model. Procedure EmulateLcdinBL(in : Integer k, Array s; out : Boolean collision)
Boolean collision ←false;
Integeri ←0;
repeat
if s[i] then beep in slot 1; listen in slot 2;
else listen in slot 1; beep in slot 2;
if another beep was heard then collision ←true;
i ←i + 1
until i = k;
End Procedure Algorithm 8: A Procedure to emulate a Bcd in the BL model. Procedure EmulateLcdinBL(in : Integer k, Array s; out : Boolean collision)
Boolean collision ←false; if another beep was heard then collision ←true; End Procedure Algorithm 9: A Procedure to emulate a Lcd in the BL model. Using the procedures. In the listings of our algorithms (see Section 4), listen instructions are implicit. By default,
a node listens if it does not beep. Emulation procedures should be used explicitly for both
beep and listen primitives, in order for the nodes to remain synchronized (since each of them
takes logarithmically many rounds to be carried out). Therefore, whenever a node calls
EmulateBcdinBL or EmulateLcdinBL, the other nodes should call one of these or wait the
corresponding amount of time. Likewise, the procedures should not be interrupted even after
a collision with a given neighbor is detected, to preserve synchrony with other neighbours
or farther nodes. (extended abstract) Procedure EmulateLcdinBL(in : Integer k; out : Boolean beep, Boolean collision)
Boolean beep ←false;
Boolean collision ←false;
Integer i ←0;
repeat
switch slot do
slots 1 and 2
listen
end of phase:
if a beep was heard in any slot then
beep ←true
if a beep was heard in both slots then
collision ←true
i ←i + 1
until i = k;
End Procedure until i = k; End Procedure Hence, the value of k depends on the bound we require on the probability of error, a
straightforward adaptation of the above analysis gives us the values of Lemma 3. ▶Lemma 3. For any ε > 0, and any n > 0: 1. if k = ⌈log
1
ε
⌉, the procedures are correct for a given node with probability 1 −ε XX:15 A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari 4ms
2ms
1s 2s
2ms + 2s
4s
2ms + 4s
... 2ms + 2is
2is
... ... 2ms + 1s
... Figure 1 The wheel gadget used in the proof of optimality for emulation. 4ms
2ms
1s 2s
2ms + 2s
4s
2ms + 4s
... 2ms + 2is
2is
... ... 2ms + 1s
... 2s Figure 1 The wheel gadget used in the proof of optimality for emulation. 2. if k = ⌈log
n
ε
⌉, the procedures are correct for any node with probability 1 −ε
3. if k = ⌈2 log(n)⌉, the procedures are correct for any node w.h.p. Observe that in general, the size of the network n is not known to the nodes, which is an
obstacle to achieving the second and third types of guarantees. However, it is reasonable in
practice to assume that the nodes know an upper bound on n, e.g., when a network of wireless
sensors is deployed. The upper bound may even be loose without much consequence: so
long as it is polynomial in n, the slowdown factor remains of the same order. 5.3
Optimality of the emulation In this section we prove that the emulation procedures presented in Section 5.2 are essentially
optimal (i.e. asymptotically and up to a constant factor), namely, we prove a Ω(log n) lower
bound on the number of slots required to detect collision in some graphs called wheels. A
(m, s)-wheel, illustrated in Figure 1, is a graph W = (V, E) such that V = u1, . . . u4ms,
the edges E are all the (ui−1, ui) (arithmetic modulo 4ms) plus m spokes, that is edges
(uis, u(i+2m)s) (1 ≤i ≤2m), where the wheel can be odd (all spokes with i odd) or even
(all spokes with i even). The even and odd (m, s)-wheels are isomorphic. We consider only
situations in which all vertices uis are in the same state, a state in which they wish to
beep and all other vertices are in the same internal state, a state in which they do not wish
to beep. Thus vertices at the ends of spokes and no others must conclude that there is a
collision. The slot complexity of any algorithm which detects collision in such a graph with
high probability is to be Ω(log n). Due to space limitations, the full proofs are relegated to
Appendix B. We provide, however, a minimal sentence of insight for each. Considering a computation of a collision detecting algorithm on a wheel, we define, for
any t > 0, bi
t as the signal (beep or not) from ui to all its neighbours at time t, and, for any (extended abstract) t ≥0, Bi
t the sequence bi
1 · · · bi
t . Then, we define the event Et for a spoke uis, u(i+2m)s as
follows: Et =
n
(Bis
t = B(i+2m)s
t
) ∧(Bis+1
t
= B(i+2m)s+1
t
) ∧(Bis−1
t
= B(i+2m)s−1
t
)
o
. ▶Lemma 4. For any t (0 ≤t < s), it holds that Pr (Et) ≥2−3t. The proof proceeds by induction on t, with base case t = 0. (Full proof in Appendix B.) The proof proceeds by induction on t, with base case t = 0. (Full proof in Appendix B.)
If Et holds for the spoke (uis, u(i+2m)s), we say that the spoke fails to break symmetry
within time t. This happens with probability at least 2−3t and, if it happens, the existence
of the spoke has had no influence on the computation up to time t. In particular, whenever
uis beeped, u(i+2m)s also beeped and so neither has ever heard the other beep. ▶Theorem 5. For any Monte Carlo algorithm A which detects collision in W, if A halts in
less than log2 n/4 rounds with probability greater than 3/4 then for some situations in some
wheels, A gives incorrect results for some vertices with probability greater than 1/4. The proof proceeds using the wheel gadget of Figure 1 and Lemma 4 to characterize the
rate at which the symmetry induced by the spokes can be broken. (Full proof in Appendix B.) ▶Corollary 6. The complexity of a Monte Carlo algorithm which detects collision with high
probability in the BL model is Ω(log n). ▶Corollary 6. The complexity of a Monte Carlo algorithm which detects collision with high
probability in the BL model is Ω(log n). A
Complexity analysis This appendix section provides the analysis and proofs of the running time complexity of
the algorithms presented in Section 4. A.1
Colouring algorithm without knowledge Informally the execution progresses as follows. There is a first period of adjustment in which
the probabilities will converge towards “good values”. Then the probability that an exclusive
beep is produced in a given phase in a given neighbourhood remains bounded in some ways. Loosely speaking, the final bound is essentially obtained by a repetition of the corresponding
periods ∆times. More precisely, we prove the following theorem: ▶Theorem 7. There are constants α, β and γ such that for any graph G = (V, E) of n
vertices and maximum degree ∆, the number of phases of Algorithm 4 to colour all the nodes
in G is: 1. less than α(∆+ log n) with probability 1 −o
n−1
, 2. less than β(∆+ log n) on average, 3. less than γ(∆+log n) with probability 1−o (n−c), for any c > 1. (This result is stronger
than 1, but the proof is more difficult, which is why we keep both.) 3. less than γ(∆+log n) with probability 1−o (n−c), for any c > 1. (This result is stronger
than 1, but the proof is more difficult, which is why we keep both.) 3. less than γ(∆+log n) with probability 1−o (n−c), for any c > 1. (This result is stronger
than 1, but the proof is more difficult, which is why we keep both.) References 1
Y. Afek, N. Alon, Z. Bar-Joseph, A. Cornejo, B. Haeupler, and F. Kuhn. Beeping a maximal
independent set. Distributed Computing, 26(4):195–208, 2013. 2
K. Azuma. Weighted sums of certain dependent random variables. Tohoku Mathematical
Journal, Second Series, 19(3):357–367, 1967. 3
A. Casteigts, Y. Métivier, J. Michael Robson, and A. Zemmari. Counting in one-hop
beeping networks. CoRR, abs/1605.09516, 2016. 4
J. Collier, N. Monk, P. Maini, and J. Lewis. Pattern formation by lateral inhibition with
feedback: a mathematical model of delta-notch intercellular signalling. Journal of Theo-
retical Biology, 183(4):429–446, 1996. 5
A. Cornejo and F. Kuhn. Deploying wireless networks with beeps. In DISC, pages 148–162,
2010. 6
S. Gilbert and C. Newport. The computational power of beeps. In Proc. of 29th Interna-
tional Symposium on Distributed Computing (DISC), 2015. 7
B. Huang and Th. Moscibroda. Conflict resolution and membership problem in beeping
channels. In DISC, pages 314–328, 2013. 8
P. Jeavons, A. Scott, and L. Xu. Feedback from nature: simple randomised distributed
algorithms for maximal independent set selection and greedy colouring. Distributed Com-
puting, DOI 10.1007/s00446-016-0269-8, 2016. 9
S. Navlakha and Z. Bar-Joseph. Distributed information processing in biological and com-
putational systems. Commun. ACM, 58(1):94–102, 2015. 10
J. Schneider and R. Wattenhofer. What is the use of collision detection (in wireless net-
works)? In DISC, pages 133–147, 2010. 11
A. Scott, P. Jeavons, and L. Xu. Feedback from nature: an optimal distributed algorithm
for maximal independent set selection. In PODC, pages 147–156, 2013. XX:17 A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari A.1.2
Available Neighbours. In a given round any ui has a well defined probability ai of being available that is able to
take the current colour since no neighbour claims it. These probabilities are far from being
independent. We will argue that, except for cases where pdec (the probability of a decrease
in d) is at least 2/5, the average decrease in M ′ is always minimised when all ai = 0. Consider a situation where pdec < 2/5 and some ai > 0. We can decrease ai to 0 with
no change to the other aj by adding an infinite number of vertices adjacent to ui but to
no other vertex in N(v). This will change the average increase/decrease in q and d; qi will
be halved instead of doubled with probability ai, decreasing q by 3qi/2 and so decreasing
f(q) by at most 6aiqi on average. The probability of a decrease in d is decreased by ai
times the probability that ui claims and no other uj takes the colour. This last probability
is the product of qi and the conditional probability that no other uj takes the colour given
that ui claims. But, since the probabilities of ui claiming and of some uj (j ̸= i) taking
the colour are negatively correlated or independent, this conditional probability is at most
the unconditional probability that no uj (j ̸= i) takes the colour and so greater than the
probability that no uj takes the colour, namely 1 −pdec ≥3/5, giving an average decrease
in d (respectively M ′) reduced by more than 3aiqi/5 (resp. 6aiqi). Thus the decrease in the measure is decreased more by the d component than it is
increased by the change in q. Repeating this process at most d times we arrive at a situation with a smaller mean
decrease in M ′ than the initial one and either pdec ≥2/5 or all ai = 0 so, to lower-bound
the decrease in M ′, we need only consider such situations. (extended abstract) if the degree of v decreases by more than 1, the change in M ′ only includes −10 in total
for the change in d rather than −10 for each neighbour removed; if v takes a colour, M ′ is decreased by just 10 whatever the values of p at v and its
neighbours; if v takes a colour, M ′ is decreased by just 10 whatever the values of p at v and its
neighbours; in a round where v is already coloured, M ′ is decreased by 1. This ensures that: if the algorithm has not terminated at v, M ′ ≥M, meaning that M ′ dominates M; if M ′ ≤0, the algorithm has terminated at v; M ′ decreases at each round by a value in [−3 . . . 11] (since, log p can only change by ±1,
f(q) can only change by up to ±2, and if d decreases because a neighbour u takes the
colour, u beeped in the round and so p has halved). M ′ decreases at each round by a value in [−3 . . . 11] (since, log p can only change by ±1,
f(q) can only change by up to ±2, and if d decreases because a neighbour u takes the
colour, u beeped in the round and so p has halved). A.1.1
Local Average Time Complexity. First, we give an overview. We define pv as the probability that vertex v claims the colour in
a given round and qv as the sum of p over all neighbours of v which we will call ui (1 ≤i ≤d)
where d is v’s degree in the residual graph (taken as 0 if v has been eliminated from the
graph). We define a measure M of the distance from a given situation to the goal where p =
1/2, qv ≤1/2, d = 0 as follows: M = −log(p) + f(q) + 10d where f is the function defined
as follows: f(x) = 4x if x ≤1, for x > 1, f is the piecewise linear approximation to 2 log2 4x wheref is interpolated
linearly between f(2i) = 2i + 4 and f(2i+1) = 2i + 6. We note the following properties of f which will be used in what follows: We note the following properties of f which will be used in what follows: f(x) is continuous for x > 0, except at powers of 2, f is differentiable with derivative ≤4, except at powers of 2, f is differentiable with derivative ≤4, f(x) −f(x/2) = 2 for x ≥1, f(x) −f(x/2) = 2x for x ≤1. We show that in any round, the mean decrease in M is at least 1. Then after a number
of rounds equal to the initial M (≤1 + 2 log(2d) + 10d), M is reduced on average to 0
unless the algorithm has already terminated at v and after O(log n) further rounds, the
algorithm has terminated at v with probability o(n−2) and so it has terminated everywhere
with probability o(n−1). Intuitively, we expect both p and q to decrease initially until q < 1/2, after which p will
re-ascend until it is at least close to 1/2 and then d will start to descend. We actually analyse the variation in a random variable M ′ which dominates the r.v. M. The r.v. M ′ is initially equal to M but its changes may be slightly different from those of
M in the following ways: q ≥1: q decreases to q/2, reducing f(q)
M ′ is decreased by at least 2 −1 = 1. M ′ is decreased by at least 2 −1 = 1. q ≥1: q decreases to q/2, reducing f(q) by 2 and log p can decrease by at most 1 so that
M ′ is decreased by at least 2 −1 = 1. A.1.4
Time Complexity w.h.p.. We define the sequence of r.v.’s (Mk)0≤k≤t as follows M ′
0 = M0 and for any k ≥1, Mk is the
value of M ′ after time k. We also define the sequence (Gk)0≤k≤t as the sequence of residual
graphs, i.e., G0 = G and Gk+1 is the graph obtained from Gk after round k +1 (each vertex
which succeeds in beeping alone is removed from the graph). Then for any k ≥1: E (Mk | G1, G2, · · · , Gk−1) ≤Mk−1 −1. (3) E (Mk | G1, G2, · · · , Gk−1) ≤Mk−1 −1. E (Mk | G1, G2, · · · , Gk−1) ≤Mk−1 −1. (3) Hence, (Mk)k≥0 is a super-martingale with respect to (Gk)k≥0. We define the r.v. Dk = Mk −Mk−1 for any k ≥1 and we denote µ = E (Dk). We also
introduce the r.v.:
4
3
+ 3 D′′
k = −
4
µ −3Dk + 3µ + 3
µ −3 . D′′
k = −
4
µ −3Dk + 3µ + 3
µ −3 . Then, it is easy to see that E (D′′
k) = −1 and Pr (−11 ≤D′′
k ≤3) = 1. N
d fi
th
(M ′′)
f ll
M ′′
M
d f
k ≥ Then, it is easy to see that E (D′′
k) = −1 and Pr (−11 ≤D′′
k ≤3) = 1. Then, it is easy to see that E (D′′
k) = −1 and Pr (−11 ≤D′′
k ≤3) = 1. Now, define the r.v. (M ′′
k )k≥0 as follows: M ′′
0 = M0 and for any k ≥1, M ′′
k = M ′′
k−1 +
D′′ + 1 Then (M ′′)k≥0 is a martingale with respect to (Gk)k≥0 Now, define the r.v. (M ′′
k )k≥0 as follows: M ′′
0 = M0 and for any k ≥1, M ′′
k = M ′′
k−1 +
D′′
k + 1. Then (M ′′
k )k≥0 is a martingale with respect to (Gk)k≥0. Now, define the r.v. (Mk )k≥0 as follows: M0 = M0 and for any k ≥1, Mk = Mk−1 +
D′′
k + 1. Then (M ′′
k )k≥0 is a martingale with respect to (Gk)k≥0. M ′′
k )k≥0 is a martingale with respect to (Gk)k≥0. We apply Theorem 18 of [2] to our martingale M ′′
t with expectation M0. A.1.4
Time Complexity w.h.p.. Since the
increments (D′′
k +1)k≥0 are in [−10..4] and have mean 0, their variance is upper bounded by
the case of a distribution with values −10 and 4 with probabilities 2/7 and 5/7 respectively,
giving variance of 40 and maximum discrepancy from the mean of 10. Applying the theorem
with t = 2M0 + 174 ln n and λ = t −M0, we see that the probability that Mt ≥0 is less
than Pr (M ′′
t ≥t) which is at most: e(−λ2/2(40t+10λ/3)), and we claim that this is o(n−2). This is because λ2/2(40t + 10λ/3) >> 2 ln n, i.e. λ2 >>
4 ln n(40t + 10λ/3). and we claim that this is o(n−2). This is because λ2/2(40t + 10λ/3) >> 2 ln n, i.e. λ2 >>
4 ln n(40t + 10λ/3). (Proof of this claim: λ2/13 >> 4 ln n(10λ/3) because λ >> 520 ln n/3;
12λ2/13 > 12(t2 −2M0t)/13 = 12t(t −2M0)/13 = 12t(174 ln n)/13 >> 160t ln n. Adding
these two gives the claim.) (Proof of this claim: λ2/13 >> 4 ln n(10λ/3) because λ >> 520 ln n/3;
12λ2/13 > 12(t2 −2M0t)/13 = 12t(t −2M0)/13 = 12t(174 ln n)/13 >> 160t ln n. Adding
these two gives the claim.) Then taking α = 174, this proves the first claim of Theorem 7. A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari q < 1: q decreases to q/2, decreasing f(q) by 2q while p doubles with probability at least
1 −q (and halves with probability at most q). This gives a mean decrease in M ′ of at
least 2q + (1 −2q) = 1. A.1.5
Average Time Complexity. We first prove the following lemma: A.1.3
The mean decrease in M ′. We consider cases depending on the value q. First note that if pdec ≥2/5, the mean decrease
in M ′ is at least 10(2/5) −2 −1 = 1. So in the other cases we suppose that all ai = 0 so
that q is halved. In the case where q < 1, we need to consider what happens when no ui claims the colour. If p < 1/2 this is that p increases, decreasing M ′ by 1; if p = 1/2 it is that, with probability
1/2, v takes the colour so that d decreases by 1, decreasing M ′ by 10 so that on average M ′
decreases by 5. Accordingly we suppose that the former happens. q ≥1: q decreases to q/2, reducing f(q) by 2 and log p can decrease by at most 1 so that
M ′ is decreased by at least 2 −1 = 1. XX:19 A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari ▶Lemma 8. Let v be any vertex in G, and t > 0. We have: ▶Lemma 8. Let v be any vertex in G, and t > 0. We have: Pr (v is not coloured at time t) ≤e−
3
260 (t−2M0). Proof. Let Tv denote the time before v gets coloured. Then, by discussions above, taking
λ = t −M0 in Theorem 18 of [2]: Pr (Tv −M0 > λ) ≤e
−
λ2
2(40t+ 10
3 λ) . XX:20 A.1.6
Time Complexity With Very High Probability. A.1.6
Time Complexity With Very High Probability. To prove the last claim, let c > 1 and take t = 260
3 (c + 1) ln n + 2M0 in Lemma 8, this gives: To prove the last claim, let c > 1 and take t = 260
3 (c + 1) ln n + 2M0 in Lemma 8, this gives: To prove the last claim, let c > 1 and take t = 260
3 (c + 1) ln n + 2M0 in Pr
Tv > 260
3 (c + 1) ln n + 2M0
≤
1
nc+1 = o
1
nc
. Thus, taking γ = 87 proves the last claim of Theorem 7. XX:20
Design Patterns in Beeping Algorithms On the other hand, λ ≥t −2M0 and hence, a simple computation yields: λ2 > 3
13(t −2M0)(4t + λ
3 ), λ2 > 3
13(t −2M0)(4t + λ
3 ), which proves the lemma. Back to Theorem 7. Let T denote the time before all the vertices in the graph are
coloured. Then: E (T)
=
X
t≥1
Pr (T ≥t) . Now, let t0 = 2M0 + 260
3 ln n then: E (T)
=
t0
X
t=1
Pr (T ≥t) +
X
t>t0
Pr (T > t)
≤
2M0 + 260
3 ln n +
X
t>t0
Pr (T > t) . On the other hand, for any t > 0: On the other hand, for any t > 0: Pr (T > t)
≤
X
v∈V
Pr (Tv > t) ≤ne−
3
260 (t−2M0). Yielding: E (T)
≤
2M0 + 260
3 ln n + n
X
t>t0
e−
3
260 (t−2M0)
=
2M0 + 260
3 ln n + n
X
t>0
e−
3
260 (t+t0−2M0)
=
2M0 + 260
3 ln n +
X
t>0
e−3t
260
=
2M0 + 260
3 ln n +
1
1 −e−
3
260 . Taking β = 87, this proves the second claim of Theorem 7. E (T)
≤
2M0 + 260
3 ln n + n
X
t>t0
e−
3
260 (t−2M0)
=
2M0 + 260
3 ln n + n
X
t>0
e−
3
260 (t+t0−2M0)
=
2M0 + 260
3 ln n +
X
t>0
e−3t
260
=
2M0 + 260
3 ln n +
1
1 −e−
3
260 . Taking β = 87, this proves the second claim of Theorem 7. Taking β = 87, this proves the second claim of Theorem 7. A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari We have the following theorem: ▶Theorem 9. Let G be a graph of size n, let K be an upper bound on the maximum degree
of G. Algorithm 5 computes a K + 1 colouring of G in at most O
K(log n + log2 K)
slots
w.h.p. Proof. We consider the Colouring algorithm in which every node has the same upper bound
K on the maximum degree. We consider both the basic algorithm in which v uses the current
value of |Colours| to decide its beeping probability and also the modified algorithm in which
it uses the value at the start of the current cycle. We recall that by a cycle we mean K
phases considering the |Colours| colours. We consider Pk the probability that vertex v survives uncoloured over k cycles. In what follows: In what follows: i ranges over 1..k, c ranges over the Ci colours possible for v at the start of cycle i, u ranges over the neighbours of v still uncoloured at the start of cycle i, pu(i, c) is the probability that u beeps at colour c in cycle i. First we consider the probability p that v survives uncoloured in a single phase using a
colour c ∈colours(v). Then: p
=
Pr (v does not beep at colour c in cycle i) p
=
Pr (v does not beep at colour c in cycle i) +
Pr (v does beep and some neighbour u also beeps) , +
Pr (v does beep and some neighbour u also beeps) , but Pr (v does beep) ≥1/2Ci and the beeping probabilities of v and its neighbours are
independent giving: p
≤
(1 −1/2Ci) + Pr (some neighbour beeps) /2Ci
=
(1 −1/2Ci) (1 + Pr (some neighbour beeps) /(2Ci −1))
≤
(1 −1/2Ci)
1 +
X
u
pu(i, c)/(2Ci −1)
! . ≤
(1 −1/2Ci)
1 +
X
u
pu(i, c)/(2Ci −1)
! . After the first phase, pu(i, c) and Ci are random variables dependent on what has happened
so far, and we consider the tree of all possible executions up to k cycles, where each tree
node has its own value of p. It is easily shown by induction that Pk is upper bounded
by the maximum over all paths in this tree of the product of the values of p along the
path. A.2
Colouring algorithm knowing K ≥∆. ▶Remark. We can consider the modified colouring algorithm, deduced from Algorithm 5,
defined in the following way. By a cycle we mean K rounds considering the K colours. Now,
every vertex uses the value of |Colours| at the start of each cycle to decide the beeping
probability it uses throughout this cycle. XX:21 A.3
2-hop colouring To calculate a 2-hop colouring of a graph G, we need to calculate a colouring of the “square”
of G, that is the graph with the same vertices as G and an edge between any pair v and
w of vertices which either are neighbours in G or have a common neighbour in G. In this
context, Theorem 7 becomes: ▶Theorem 10. There are constants α, β and γ such that for any graph G = (V, E) of n
vertices and maximum degree ∆, the number of phases of Algorithm 6 to calculate a 2-hop
colouring in G is: 1. less than α(∆2 + log n) with probability 1 −o
n−1
, 2. less than β(∆2 + log n) on average, 3. less than γ(∆2 + log n) with probability 1 −o (n−c), for any c > 1. 3. less than γ(∆2 + log n) with probability 1 −o (n−c), for any c > 1. ▶Remark. The same transformation can be done starting from Algorithm 5 when we know
an upper bound of the maximum degree. ▶Remark. The same transformation can be done starting from Algorithm 5 when we know
an upper bound of the maximum degree. (extended abstract) Firstly base: in cycle i, v has Ci colours available and so has less than Ci neighbours;
each neighbour u has P
c base ≤1/2, giving, for this cycle, P
u
P
c base/(2Ci −1) ≤1/4 so
that P
i
P
u
P
c base/(2Ci −1) ≤k/4. i
u
c
Secondly δ: For the modified algorithm δ = 0. In the basic algorithm, a node uj
initially has K colours available and when (if) this number decreases from l to l −1, pu(i, c)
increases from 1/2l to 1/2(l −1) and this increase of 1/2l(l −1) affects δ only for the, at
most, l −1 colours still to be considered in this cycle so that P
c δ for a cycle is at most
P
l 1/2l, the sum being taken over those l for which the number of colours is reduced from
l. This gives an upper bound on P
i
P
c δ/(2Ci −1) of log K/2(2j + 1) since Ci > j and so
P
u
P
i
P
c δ/(2Ci −1) < P
j log K/2(2j + 1) < log2 K/4. j
Hence, by standard arguments, after k = O(log n + log2 K) cycles for the basic algo-
rithm or O(log n) cycles for the modified algorithm, v has probability o(1/n2) of remaining
uncoloured and the graph has probability o(1/n) of having any uncoloured node. A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari We fix a path which gives this maximum and bound the product for this path. We
have the probability of surviving cycle i ≤(exp(−1/2) ∗Q
c(1 + P
u pu(i, c)/(2Ci −1))) ≤
exp(−1/2+P
c
P
u pu(i, c)/(2Ci−1)) and so Pk ≤exp(−k/2+P
i
P
c
P
u pu(i, c)/(2Ci−1)). We will give an upper bound on P
i
P
c
P
u pu(i, c)/(2Ci −1). We number v’s neighbours in the initial graph from 1 to deg(v) in decreasing order of
their lifetime, that is the number of phases in which they remain uncoloured. Thus as long as uj is not coloured the degree of v in the residual graph is at least j and so
|colours(v)| > j. We write pu(i, c) as base+δ where base = 1/2Ci and δ is what has been added as a result
of colours(u) being decreased before colour c and we will bound P
i
P
u
P
c base/(2Ci −1)
and P
u
P
i
P
c δ/(2Ci −1) separately. A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari We finally write l(q) for log(5 max{q, 1/5}), that is l(q) = max{log(5q), 0}. Then, we have the following theorem: We finally write l(q) for log(5 max{q, 1/5}), that is l(q) = max{log(5q), 0}. Then, we have the following theorem: ▶Theorem 11. For any t ≥0 and for any vertex v, its probability of remaining active after
the next t phases is at most αt0−t for the constant α = 21/36 ≈1.01944. Proof. Note that α3 log q = q3 log α = q1/12. The proof will be by induction on t. We have
t0 ≥2, so that if t = 0, αt0−t > 1 and the claim is trivially true. Let t > 0. After one phase which does not add v or a neighbour to the MIS we have
by induction that the probability of remaining active for the following t −1 phases is at
most αt′
0−t+1 where t′
0 is the new value of t0, namely 3l(q′) −2 log p′. So we conclude that
the probability of survival is upper bounded by the mean of the random variable which is
αt′
0−t+1 if v survives the first phase and 0 otherwise. We refer to this mean as the bound
and note that it is dependent on what happens outside the neighbourhood of v. ◀ We will come back to the proof of the Theorem, but we first prove the following lemma: ▶Lemma 12. The bound is maximised when what happens outside the neighbourhood of v
is that every neighbour u of v is inhibited from joining the MIS by some external neighbour
beeping and no neighbour of v becomes inactive through another vertex (outside N(v)) joining
the MIS. Proof. Clearly a vertex ouside N(v) joining the MIS can only affect the bound by reducing
q which reduces the bound. Consider any external behaviour E in which some u is not inhibited; we will show that
the bound is increased or unchanged if the behaviour is changed to E′ in which u is inhibited
and there is no change for any other neighbours of v. A.4
Analysis of Jeavons et al.’s Las Vegas Algorithm for the MIS in
BcdL In [8], Jeavons et al. give and analyse a Las Vegas beeping algorithm to compute a MIS in the
model BcdL. They prove that for any graph G with n vertices, their algorithm terminates
in at most K0 log n phases, with probability at least 1 −o(n−1) and K0 ≥e25. The starting point of our work is the observation made by Scott et al. at the end of
Section 4 in [11]: “Our simulations show that in practice the constants are rather lower”. We verify this observation by proving that the number of phases taken by the Jeavons et al. algorithm on any graph with n vertices is at most 76 log n w.h.p. We first introduce some notation that we will use in this section. If a neighbour of v beeps (in a slot), we say that v is “inhibited” (in that slot). For any
vertex v, we define the following sum: qv =
X
u∈N(v)
pu. We also note q∗
v = max{qv, 1/5} and finally t0 = 3 log(5q∗
v) −2 log pv. We omit the
subscript v where there is no risk of ambiguity. XX:23 A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari (In a given graph there may be no
such E′ but we consider the maximum possible over any graph containing the neighbourhood
N(v).) We consider fixed beeping decisions of all vertices in N(v) except u and show that
with these decisions E′ gives a value of the bound greater than or equal to that of E. We
consider three cases: Some neighbour of v which is neither u nor a neighbour of u enters the independent set:
Note that this is determined by the fixed beeping decisions and the external behaviour
other than as it affects u. Hence this happens for E iffit also happens for E′ and in each
case the bound is 0. Some neighbour of u in N(v) beeps:
pu will be halved whether or not u is inhibited by E′ and so both p′ and q′ and the
probability of survival are the same for E and E′. The bound is identical in the two
cases. Otherwise:
Let the value of p′ be p0 if u does not beep and p1 if u does beep. p1 ≤p0. Let the value of q′ be q0 if u does not beep and is not inhibited, q1 if it beeps and is
inhibited and q2 if it does not beep and is inhibited. Note that if u beeps and is not
inhibited, u enters the independent set and v does not survive. We have q1 ≥q0/4 since,
at most, u’s beeping can result in a vertex w halving qw when otherwise it would have
doubled it. Similarly q2 ≥q0/4 and q2 ≥q0 −3pu/2 since the inhibition results in pu
being halved rather than potentially doubled. The bounds are thus puα3l(q1)−2 log(p1)−t+1 + (1 −pu)α3l(q2)−2 log(p0)−t+1 in the inhibited
case and (1 −pu)α3l(q0)−2 log(p0)−t+1 in the uninhibited case. We claim that the ratio of
the inhibited bound to the uninhibited is at least 1. This ratio ≥puα3l(q1)+(1−pu)α3l(q2)
(1−pu)α3l(q0) Let the value of q′ be q0 if u does not beep and is not inhibited, q1 if it beeps and is
inhibited and q2 if it does not beep and is inhibited. Note that if u beeps and is not
inhibited, u enters the independent set and v does not survive. We have q1 ≥q0/4 since,
at most, u’s beeping can result in a vertex w halving qw when otherwise it would have
doubled it. (extended abstract) (since p1 ≤p0)
Remember that pu is a power of 1/2. We consider four subcases: (since p1 ≤p0)
Remember that pu is a power of 1/2. We consider four subcases: (
)
Remember that pu is a power of 1/2. We consider four subcases: q0 ≤1/5: l(q1) = l(q2) = l(q0) = 0 and the ratio ≥(pu + 1 −pu)/(1 −pu) > 1. 1/5 < q0 and pu ≥1/8: We use the bounds q1 ≥q0/4 and q2 ≥q0/4 giving that the
ratio is at least (pu + 1 −pu)α−6/(1 −pu) = α−6/(1 −pu) ≥α−6(8/7) ≥1. 1/5 < q0 ≤4/5 and pu ≤1/16: We use the bounds q1 ≥q0/4 and q2 ≥q0 −3pu/2
and the fact that for 0 < x ≤15/32, (1 −x)1/12 > 1 −4/3(x/12) so that the ratio is
at least puα−6/(1 −pu) + (1 −3pu/2q0)3 log α ≥puα−6 + (1 −15pu/2)1/12 ≥puα−6 +
(1 −(15pu/2)/12 × (4/3)) ≥1 + pu(α−6 −5/6) > 1. q0 > 4/5 and pu ≤1/16: Using the same bounds as in the previous subcase the ratio
is greater than
pu
1−pu α−6 +α3(l(q0−3pu/2)−l(q0)) >
pu
1−pu α−6 +α3(l(4/5−3pu/2)−l(4/5)) and
this is the bound already used for the case with q0 = 4/5 and the same value of pu
and so is greater than or equal to 1. q0 > 4/5 and pu ≤1/16: Using the same bounds as in the previous subcase the ratio
is greater than
pu
1−pu α−6 +α3(l(q0−3pu/2)−l(q0)) >
pu
1−pu α−6 +α3(l(4/5−3pu/2)−l(4/5)) and
this is the bound already used for the case with q0 = 4/5 and the same value of pu
and so is greater than or equal to 1. This ends the proof that E′ gives a value for the bound at least as great as that for E. The
lemma is then proved by a simple induction on the number of uninhibited vertices. We return to the inductive proof. Using the lemma we will always take q′ = q/2 giving
probability of survival ≤α3l(q/2)−2 log p′−t+1. We consider five cases. q ≥2/5: We have l(q/2) = l(q)−1 and p′ ≥p/2 giving P(survival) ≤α3(l(q)−1)−2(log p−1)−t
= α3l(q)−2(log p)−t as claimed. q ≥2/5: We have l(q/2) = l(q)−1 and p′ ≥p/2 giving P(survival) ≤α3(l(q)−1)−2(log p−1)−t+
= α3l(q)−2(log p)−t as claimed. A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari Similarly q2 ≥q0/4 and q2 ≥q0 −3pu/2 since the inhibition results in pu
being halved rather than potentially doubled. XX:24
Design Patterns in Beeping Algorithms XX:24 (extended abstract) 1/5 ≤q < 2/5 and p < 1/2: The probability that a neighbour of v beeps is less than
q so that pv is doubled with probability at least 1 −q and halved in the remaining
cases. In all cases l(q/2) = 0. Hence P(survival) ≤α−2 log(p)−t+1((1 −q)α−2 + qα2)
and our claim is that it is at most α3 log(5q)−2 log(p)−t. That is the claim is valid since
(1 −q)α−1 + qα3 ≤α3 log(5q) in the range 1/5 ≤q < 2/5. (It is valid at q = 1/5 since
4α−1 +α3 < 5 and at q = 2/5 since 3α−1 +2α3 < 5α3; between these two limits, the left
hand side is linear and the right hand side ((5q)3 log α) has a negative second derivative
so the inequality holds there also.) 1/5 ≤q < 2/5 and p = 1/2: With probability greater than 1−q no neighbour of v beeps
and then v has probability 1/2 of entering the independent set; otherwise pv remains 1/2. On the other hand, if a neighbour does beep, pv becomes 1/4. In all cases l(q/2) = 0. Thus the probability of survival ≤α2−t+1((1 −q)/2 + qα2) and the claim is that it is at
most α3log2(5q)+2−t. That is the claim is valid if (1 −q)α/2 + qα3 ≤α3 log(5q) a weaker
condition than in the previous case. q < 1/5 and p < 1/2: The probability that a neighbour of v beeps is less than 1/5 so
that pv is doubled with probability at least 4/5 and halved in the remaining cases. In all
cases l(q) decreases or is unchanged. Hence P(survival) ≤α3l(q)−2 log(p)−t+1((4/5)α−2+
(1/5)α2) and this is less than α3l(q)−2 log p−t as claimed, again since 4α−1 + α3 < 5. q < 1/5 and p = 1/2: With probability greater than 4/5 no neighbour of v beeps and
then v has probability 1/2 of entering the independent set; otherwise pv remains 1/2. On the other hand, if a neighbour does beep, q decreases and pv becomes 1/4. Hence
P(survival) ≤(2α3l(q/2)−2 log(1/2)−t+1+α3l(q/2)−2 log(1/4)−t+1)/5 ≤α3l(q)−2 log(1/2)−t+1(2+
α2)/5 which is at most α3l(q)−2 log(1/2)−t as claimed since 2 + α2 < 5α−1. α2)/5 which is at most α3l(q)−2 log(1/2)−t as claimed since 2 + α2 < 5α−1. B
Missing proofs This appendix section provides the missing proofs of Section 5. Proof of Lemma 4. By induction on t. Clearly the claim is true for t = 0. We suppose
that Et−1 is true and we consider probabilities conditional on the values of Bt−1 for is −1,
is, is + 1, (i + 2m)s −1, (i + 2m)s and (i + 2m)s + 1. (
)
(
) (
)
(
)
(
)
We will show that the probability that bis
t = b(i+2m)s
t
and bis−1
t
= b(i+2m)s−1
t
and bis+1
t
=
b(i+2m)s+1
t
is at least 2−3. The three events:
(i+2
) The three events:
bis
t = s(i+2m)s
b
bis−1
t
= b(i+2m)s−1
t
bis+1
t
= b(i+2m)s+1
t
are independent. bis
t = s(i+2
b
( bt
sb
bis−1
t
= b(i+2m)s−1
t
bis+1
t
= b(i+2m)s+1
t t
b
bis−1
t
= b(i+2m)s−1
t t
t
bis+1
t
= b(i+2m)s+1
t are independent. For the first, uim and u(i+2m)s started in the same state and have sent and heard identical
signals. Thus they have the same probability of beeping at the next round and so have
probability at least 1/2 of either both beeping or neither. For the second, the two chains (u(i−1)s · · · uis−1) and (u(i+2m−1)s · · · u(i+2m)s−1) started
in the same states, have received the same signals from uis and u(i+2m)s, and have sent the
same signals. Thus, again the two vertices uis−1 and u(i+2m)s−1 have the same conditional
probability of beeping and so probability at least 1/2 of making the same choice. The argument for the third event is identical. This proves that the three events happen with probability at least 2−3 yielding that the
probability of event Et is lower bounded by 2−3t. ◀ Proof of Theorem 5. For simplicity we consider wheels (m, s) where s is a power of 2 and
m = 24s−2/s so that s = log2 n/4. We consider a computation on this wheel without
specifying whether it is the odd or even wheel. By Lemma 4, the probability that a given
spoke i breaks symmetry within time s −1 is at most 1 −23−3s < exp(−23−3s) and this
is independent for all spokes so that the probability that every spoke breaks symmetry in
the even case in time s −1, is at most exp(−23−3sm) = exp(−2s+1/s) < 1/4. A.5
The Case of the 2-hop MIS Las Vegas Algorithm in BcdLcd The 2-hop MIS algorithm simulates the MIS algorithm in the square of G; knowing that
the complexity depends only on the number of vertices of the graph we deduce from the
previous section: ▶Theorem 14. The number of phases taken by the 2-hop MIS algorithm on any graph with
n nodes is less than 76 log n w.h.p. A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari ▶Corollary 13. The number of phases taken by Jeavons et al.’s algorithm on any graph with
n nodes is, in fact, less than 76 log n w.h.p. Proof. Since initially pv = 1/2 and qv < n/2 where the graph has n vertices, we conclude
that t0 < 3 log(5n/2) −2 log(1/2) < 3 log n + 6 so that after t = 76 log2 n phases, every
vertex v has probability n−2 of still being active and therefore the algorithm has terminated
with probability 1 −o(n−1). ◀ ▶Remark. The number of phases before the probability is 1 −o(n−1) compares well with
the value of K0 ≥e25 proved in [8]. This completes the proof of the theorem. We end this section by the following corollary: We end this section by the following corollary: XX:25 A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari XX:26
Design Patterns in Beeping Algorithms XX:26 B
Missing proofs Hence the (extended abstract) probability that the algorithm halts and some spoke fails to break symmetry is greater than
1/2. If, in the even case, spoke i fails to break symmetry, vertex ui hears the same signals
from its neighbours in the odd and even cases and, so, if it terminates the algorithm in this
time, it has the same probability of deciding collision in the two cases. Hence it gives the
wrong response in one case with probability at least 1/2. Hence there is a vertex which gives
the wrong response in the odd or even case with probability greater than 1/4. probability that the algorithm halts and some spoke fails to break symmetry is greater than
1/2. If, in the even case, spoke i fails to break symmetry, vertex ui hears the same signals
from its neighbours in the odd and even cases and, so, if it terminates the algorithm in this
time, it has the same probability of deciding collision in the two cases. Hence it gives the
wrong response in one case with probability at least 1/2. Hence there is a vertex which gives
the wrong response in the odd or even case with probability greater than 1/4. Thus if an algorithm halts in time o(log n) with probability ≥3/4, for sufficiently large
n it halts in time less than s and so its probability of giving an incorrect result is at least
1/4 for some initial conditions. It follows that the same is true for any algorithm halting in
expected time o(log n). ◀
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https://openalex.org/W2559601967
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https://europepmc.org/articles/pmc5134072?pdf=render
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English
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Influence of posterior corneal astigmatism on postoperative refractive astigmatism in pseudophakic eyes after cataract surgery
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BMC ophthalmology
| 2,016
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cc-by
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© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. 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: konkonhotaru@yahoo.co.jp
1Department of Ophthalmology, University of Tsukuba Hospital, 2-1-1
Amakubo, Tsukuba, Ibaraki 305-8576, Japan
2Hitachi General Hospital, 2-1-1 Jonancho, Hitachi, Ibaraki 317-0077, Japan
Full list of author information is available at the end of the article Abstract Background: To examine the influence of posterior corneal astigmatism on postoperative refractive astigmatism in
pseudophakic eyes after cataract surgery. Methods: The study enrolled 64 pseudophakic eyes of 50 patients (71.8 ± 9.9 years old, mean ± standard deviation)
who had undergone phacoemulsification with non-toric IOL implantation. Refractive astigmatism was measured
using an auto ref-keratometer with a 0.01- diopter (D) scale. Two types of corneal astigmatism were calculated
using anterior segment optical coherence tomography; keratometric and total corneal astigmatism. Keratometric
astigmatism was obtained based on anterior corneal curvature alone and total corneal astigmatism was calculated
using both anterior and posterior corneal curvatures. The difference between refractive and corneal astigmatism
was computed as the vector difference using 1) refractive and keratometric astigmatism and 2) refractive and total
corneal astigmatism. Results: The mean refractive, keratometric, and total corneal astigmatism was 0.92 ± 0.48 D, 0.87 ± 0.44 D,
and 0.94 ± 0.46 D, respectively. The difference between refractive and keratometric astigmatism (0.70 ± 0.40 D, mean
vector of 0.30 D axis 164°) was significantly larger than the difference between refractive and total corneal astigmatism
(0.63 ± 0.38 D, mean vector of 0.12 D axis 137°) (P = .019). Conclusions: The difference between refractive and total corneal astigmatism, calculated using both anterior
and posterior corneal curvatures, was significantly smaller than the difference between refractive and keratometric
astigmatism using anterior corneal astigmatism alone, implying that the latter overestimates the true postoperative
refractive astigmatism and can cause cylindrical inaccuracy after cataract surgery. Keywords: Posterior astigmatism, Cataract surgery, Toric intraocular lens Keywords: Posterior astigmatism, Cataract surgery, Toric intraocular lens Influence of posterior corneal astigmatism
on postoperative refractive astigmatism in
pseudophakic eyes after cataract surgery aki Sano1,2,3*, Takahiro Hiraoka4, Yuta Ueno4, Hideo Itagaki2, Tomohiro Ogami3 and Tetsuro Osh Sano et al. BMC Ophthalmology (2016) 16:212
DOI 10.1186/s12886-016-0391-1 Sano et al. BMC Ophthalmology (2016) 16:212
DOI 10.1186/s12886-016-0391-1 Background accuracy of preoperative measurements of corneal astig-
matism is often discussed [2, 3]. It has been reported that uncorrected astigmatism of
greater than one diopter (D) in magnitude significantly
deteriorates uncorrected visual acuity in pseudophakic
eyes [1]. The introduction of toric intraocular lens (IOL)
technology has made it possible to offer better and more
stable uncorrected visual acuity to patients with astigma-
tism. In clinical practice, however, some patients still
present with postoperative refractive astigmatism of un-
known origin even with toric IOL implantation, and the Both anterior and posterior corneal curvatures con-
tribute to total corneal astigmatism [4], but less atten-
tion has been directed to posterior corneal curvature
[4–10]. This is because traditionally anterior and poster-
ior corneal surfaces in normal eyes were thought to be
almost parallel in shape. In addition, refractive power of
the posterior surface is much smaller than the anterior
surface due to the small difference in refractive index
between the corneal stroma and the aqueous humor. Thus, in general, ophthalmologists tended to believe that
detailed examination of posterior corneal shape and
curve is not necessary [7]. Therefore, keratometric * Correspondence: konkonhotaru@yahoo.co.jp
1Department of Ophthalmology, University of Tsukuba Hospital, 2-1-1
Amakubo, Tsukuba, Ibaraki 305-8576, Japan
2Hitachi General Hospital, 2-1-1 Jonancho, Hitachi, Ibaraki 317-0077, Japan
Full list of author information is available at the end of the article Sano et al. BMC Ophthalmology (2016) 16:212 Page 2 of 9 Page 2 of 9 corrected visual acuity of less than 0.8 (decimal) (20/25
snellen), history of toric IOL implantation, or cases with
surgical complication. Informed consent was obtained
from all patients. The research was approved by the
Institutional Review Board of the University of Tsukuba
Hospital and conducted according to the tenets of the
Declaration of Helsinki. astigmatism which is calculated based on anterior cor-
neal measurements only has been used clinically to rep-
resent
total
corneal
astigmatism,
assuming
a
fixed
posterior/anterior curvature ratio to estimate the contri-
bution of posterior corneal power [4]. Newer technologies, such as slit-scanning videokera-
toscope, Scheimpflug device, and anterior segment
optical coherence tomography (AS-OCT), are now
available for measuring anterior as well as posterior
corneal shapes [4]. Results obtained with these de-
vices
demonstrated
that
keratometric
astigmatism
calculated based on the measurements of anterior
corneal surface alone significantly differ from that
based on both anterior and posterior corneal mea-
surements [4, 5]. Background The posterior corneal surface tends
toward
against-the-rule
astigmatism
pattern
in
comparison
with
the
anterior
corneal
surface
[7]. Therefore,
in
eyes
with
with-the-rule
astigmatism,
keratometric astigmatism overestimates total corneal
astigmatism,
whereas
in
eyes
with
against-the-rule
astigmatism, keratometric astigmatism underestimates
total corneal astigmatism [4, 7, 10]. These discrepan-
cies seem to be explained by the fact that corneal
thickness profile is not uniform between horizontal
and vertical directions, i.e., the cornea is thicker in
the vertical than the horizontal directions [11]. Thus,
it is not always true that the anterior and posterior
corneal curvatures have a constant and linear rela-
tionship [9, 11], and posterior corneal astigmatism
should be at least partially responsible for postopera-
tive refractive astigmatism of unknown origin [2, 3]. The preoperative evaluation included measurements
of objective refractive power obtained using an auto ref-
keratometer (RC-5000, Tomey Corporation, Nagoya,
Japan) with a 0.01-D scale, axial length measured by
contact
applanation
ultrasound
(AL-1100,
Tomey
Corporation, Nagoya, Japan), and keratometric and total
corneal powers measured by an AS-OCT (SS-1000,
CASIA, Tomey Corporation, Nagoya, Japan). We took
automatically-calculated values of total corneal power
from the AS-OCT based on the actual measures of
anterior and posterior corneal power. A standard phacoemulsification technique was per-
formed through a 3-mm superior sclerocorneal one-
plane incision under topical anesthesia. Aspheric and
non-toric IOLs were implanted in all patients. One month after surgery, we measured postoperative
refractive power using the RC-5000 with a 0.01-D scale
and keratometric power and total corneal powers using
the AS-OCT, and then assessed the correlation among
postoperative refractive, keratometric, and total corneal
astigmatism. In addition, the vector differences between
postoperative refractive astigmatism and two types of
corneal astigmatism (keratometric and total corneal
astigmatism) were calculated and compared with each
other. In this study, all measurements were based on the
data from the annular ring with 3 mm in diameter
around the corneal apex. The above findings highlight the need to clarify the
effect of posterior corneal astigmatism on postoperative
refractive astigmatism after cataract surgery. Several pre-
vious reports have already demonstrated the relationship
between posterior corneal astigmatism and total corneal
astigmatism [4, 5, 12]. However, the relationship be-
tween posterior corneal astigmatism and postoperative
refractive astigmatism of unknown origin in patients
undergoing cataract surgery has not been examined in
detail. AS-OCT AS-OCT
The AS-OCT is a non-contact, non-invasive three-
dimensional imaging system based on the principle of
“Swept Source” OCT. This system uses light of 1,310-
nm wavelength and achieves resolutions of 10 μm (axial)
and 30 μm (transverse) to obtain 30,000 axial-scans per
second. The scan range diameter is 10.0 mm, and 16
radial cross-sectional images were obtained within 0.34 s
per measurement, with each image containing 512
measurement points [13–15]. All measurements were
taken by experienced examiners (MS and YU). Two im-
ages were obtained for each eye, and the better image
was selected for data analysis. Methods This study included consecutive eligible patients who
had undergone phacoemulsification with non-toric IOL
implantation at the University of Tsukuba Hospital from
November 2012 to March 2013. Patients who had cor-
neal or retinal disease and a history of ocular surgery
other than cataract
surgery or
ocular
injury were
excluded. Patients were also excluded if they met any of
the
following
criteria:
postoperative
decimal
best- Background In this study, we investigated the influence of pos-
terior corneal astigmatism on postoperative refractive
astigmatism in pseudophakic eyes with non-toric IOL
implantation. refractive index of the cornea ¼ 1:376 In this study, the AS-OCT was used to measure and
calculate keratometric and total corneal astigmatism. Keratometric astigmatism was calculated as the differ-
ence in keratometric power between the steepest and
flattest meridians, whereas total corneal astigmatism was
calculated based on total corneal power without regard
to keratometric power. Corneal astigmatism calculation Keratometric power was calculated using the kerato-
metric index (1.3375) and the radius of anterior corneal
curvature, while total corneal power was calculated Sano et al. BMC Ophthalmology (2016) 16:212 Page 3 of 9 Page 3 of 9 based on the refractive power of the anterior and poster-
ior corneal surface as well as corneal thickness. based on the refractive power of the anterior and poster-
ior corneal surface as well as corneal thickness. Vector difference between refractive and corneal
astigmatism In this study, we computed vector difference between
postoperative refractive astigmatism and each of two
types of corneal astigmatism (keratometric and total cor-
neal astigmatism) using equations below [16], and these
differences were compared with each other to simulate
the influence of actual posterior corneal astigmatism on
refractive astigmatism in pseudophakic eyes after non-
toric IOL implantation, in which internal astigmatism
induced by IOL itself is theoretically regarded as 0 D. Statistical analyses y
Two types of corneal astigmatism (keratometric and
total corneal astigmatism) were compared using the
paired t-test. The mean magnitude of difference between
refractive and keratometric astigmatism, and difference
between refractive and total corneal astigmatism was
also compared using the paired t-test. The mean differ-
ences in magnitude between refractive and each of two
types of corneal astigmatism according to the types of
preoperative keratometric astigmatism, such as ATR,
WTR or oblique astigmatism were compared using the
Wilcoxon signed-rank test. Using Pearson’s correlation
and Bland-Altman plots, the correlation between post-
operative refractive and two types of corneal astigmatism
were examined. In addition, after all patients were
divided into two groups based on the magnitude of
difference between postoperative refractive and total
corneal astigmatism, various parameters such as age,
spherical equivalent refraction, refractive astigmatism,
keratometric astigmatism, total corneal astigmatism, axial
length, and IOL power were compared between the two
groups with greater than 0.5 D and less than 0.5 D
difference using Student’s t-test. P values < 0.05 were
considered statistically significant. Statistical analyses
were carried out using StatView version 5.0 (SAS
Inc., Cary, NC). keratometric power ¼
1:3375 ‐ 1:0
ð
Þ=r
keratometric index ¼ 1:3375 keratometric power ¼
1:3375 ‐ 1:0
ð
Þ=r keratometric index ¼ 1:3375 r ¼ radius of anterior corneal curvature total corneal power ¼ Pa þ Pb d Pa Pb=1:376
Pa ¼ refractive power of the anterior cornea
Pb ¼ refractive power of the posterior cornea
d ¼ corneal thickness Pa ¼ refractive power of the anterior cornea Pb ¼ refractive power of the posterior cornea
d ¼ corneal thickness refractive index of the cornea ¼ 1:376 Results This study enrolled 64 eyes (33 right; 51.6%) of 50
patients (25 women; 50.0%) with a mean age of 71.8 ±
9.9 (SD: standard deviation) years (range 33 to 92 years). The age distribution of patients was shown in Fig. 1. The lenses implanted in the study group were SN60 WF
(50 eyes) (Alcon, Fort Worth, TX), iSert Micro251
(10 eyes) (HOYA, Tokyo, Japan) and ZCB00V (4 eyes)
(Abbott Medical Optics, Santa Ana, CA). xr ¼ refractive astigmatism Cos 2 axis
ð
Þ
yr ¼ refractive astigmatism Sin 2 axis
ð
Þ
xc ¼ corneal astigmatism Cos 2 axis
ð
Þ
yc ¼ corneal astigmatism Sin 2 axis
ð
Þ xr ¼ refractive astigmatism Cos 2 axis
ð
Þ
yr ¼ refractive astigmatism Sin 2 axis
ð
Þ
xc ¼ corneal astigmatism Cos 2 axis
ð
Þ
yc ¼ corneal astigmatism Sin 2 axis
ð
Þ Fig. 1 The age distribution of patients was shown In the formulas, the angle of the axis of astigmatism is
doubled to give the correct x and y values. Cylinder ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
xr ‐ xc
ð
Þ2 þ
yr ‐ yc
ð
Þ2
p
Angle ¼ 1=2 Arc tan y=x
ð
Þ
If x and y > 0
then Axis ¼ Angle
If x < 0
then Axis ¼ Angle þ 90
○
If x > 0 and y < 0 then Axis ¼ Angle þ 180
○ Cylinder ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
xr ‐ xc
ð
Þ2 þ
yr ‐ yc
ð
Þ2
p
Angle ¼ 1=2 Arc tan y=x
ð
Þ
If x and y > 0
then Axis ¼ Angle
If x < 0
then Axis ¼ Angle þ 90
○
If x > 0 and y < 0 then Axis ¼ Angle þ 180
○ Refractive astigmatism was corrected to the corneal
plane using the following equation: Fc = (1000 x Fs)/
{1000 - (Fs x d)} (Fc = refractive power (D) at the corneal
plane, Fs = refractive power (D) at the spectacle plane,
and d = vertex distance (12 mm)) [17]. Sano et al. BMC Ophthalmology (2016) 16:212 Sano et al. BMC Ophthalmology (2016) 16:212 Sano et al. Results BMC Ophthalmology (2016) 16:212 Page 4 of 9 Table 2 Postoperative patients’ data
Parameter
Mean ± SD
Range
Postoperative days (days)
36.0 ± 12.0
20–77
Spherical equivalent refraction (D)
−1.03 ± 1.42
−5.88–1.48
Refractive astigmatism (D)
0.92 ± 0.48
−2.28–−0.12
Keratometric astigmatism (D)
0.87 ± 0.44
0.18–2.46
Total corneal astigmatism (D)
0.94 ± 0.46
0.09–2.68
Difference between refractive and
keratometric astigmatism (D)
(Mean vector)
0.70 ± 0.40
(0.30 Axis 164°)
0.09–1.72
Difference between refractive and
total corneal astigmatism (D)
(Mean vector)
0.63 ± 0.38
(0.12 Axis 135°)
0.04–1.81
SD standard deviation, D diopter Table 1 shows preoperative patient data. The mean
magnitude of keratometric astigmatism was 0.76 ± 0.46
D (range 0.03 to 2.76 D), and the mean axial length was
23.67 ± 1.69 mm (range 20.98 to 27.55 mm). Astigma-
tism types were categorized as against-the-rule (ATR)
(steepest meridian 0 to 29° or 150 to 180°), with-the-rule
(WTR) (steepest meridian 60 to 119°), or oblique (stee-
pest meridian 30 to 59° or 120 to 149°), and the number
of eyes for each group was 23, 19, and 22, respectively. Table 2 Postoperative patients’ data Table 2 shows postoperative patient data. The mean
magnitude of objective refractive, keratometric and total
corneal astigmatism was 0.92 ± 0.48 D, 0.87 ± 0.44 D,
and 0.94 ± 0.46 D, respectively. Total corneal astigma-
tism was significantly larger than keratometric astigma-
tism
(P = .0015,
paired
t-test)
(Fig. 2). The
mean
magnitude of difference was 0.70 ± 0.40 D between
refractive and keratometric astigmatism, and 0.63 ± 0.38 D
between refractive and total corneal astigmatism, with a sig-
nificant difference between them (P = .019, paired t-test). correlation between refractive and total corneal astig-
matism (r = 0.598, P < .0001, Fig. 4) seemed stronger
than that between refractive and keratometric astig-
matism (r = 0.515, P < .0001, Fig. 3). Figures 5 to 6 are
the Bland-Altman plots showing the relation between
postoperative
refractive
and
two
types
of
corneal
astigmatism, with the mean values of individual mea-
surements plotted on the horizontal axis and the dif-
ferences of individual measurements plotted on the
vertical axis. It was found that the correlation be-
tween refractive and total corneal astigmatism (Fig. 6)
is stronger than that between refractive and kerato-
metric astigmatism (Fig. 5). Figures 7 to 9 show
doubled-angle plots for each astigmatism. Discussion Using AS-OCT, we examined the anterior and posterior
corneal curvatures in pseudophakic eyes after phacoe-
mulsification. There are several tools available for the
measurement of anterior and posterior corneal curva-
tures. Among them, OCT-based topography boasts the
shortest measurement time as well as high resolution. Tang et al. reported that the repeatability of corneal
power measurements obtained using a Fourier-domain
OCT system (RTVue, Optovue, Inc. Fremont, CA) was
comparable
to
that
of
measurements
obtained
by
Placido-ring topography [18]. Szalai et al. reported that
AS-OCT had better reliability for measurements of pos-
terior corneal power, anterior and posterior corneal
astigmatism, and apical pachymetry in comparison to
Scheimpflug imaging [13]. Because the repeatability and
reliability of OCT-based topography have been estab-
lished [13, 18], we used the AS-OCT to examine both
anterior and posterior corneal curvatures. Figure 11 shows difference in vector between postop-
erative refractive and total corneal astigmatism. The
mean vector was 0.12 D axis 135°. When compared
between Figs. 10 and 11, the mean difference in vec-
tor between refractive and total corneal astigmatism
was closer to 0 D. Table 3 shows the patient data for
two groups separated by the magnitude of difference
between
postoperative
refractive
and
total
corneal
astigmatism: greater than 0.5 D and less than 0.5 D,
respectively. Statistical analysis showed no differences
between the two groups in terms of patient age, post-
operative spherical equivalent, postoperative refractive,
keratometric,
and
total
corneal
astigmatism,
axial
length, or IOL power. Figure 11 shows difference in vector between postop-
erative refractive and total corneal astigmatism. The
mean vector was 0.12 D axis 135°. When compared
between Figs. 10 and 11, the mean difference in vec-
tor between refractive and total corneal astigmatism
was closer to 0 D. Table 3 shows the patient data for
two groups separated by the magnitude of difference
between
postoperative
refractive
and
total
corneal
astigmatism: greater than 0.5 D and less than 0.5 D,
respectively. Statistical analysis showed no differences
between the two groups in terms of patient age, post-
operative spherical equivalent, postoperative refractive,
keratometric,
and
total
corneal
astigmatism,
axial
length, or IOL power. Fig. 4 A significant correlation was observed between the magnitude
of postoperative refractive and total corneal astigmatism (Pearson’s
correlation coefficient; r = 0.598, P < .0001) In clinical practice, we sometimes encounter unex-
pected postoperative refractive astigmatism after cataract
surgery even with toric IOLs. Results 5 Differences between the magnitude of postoperative refractive
and keratometric astigmatism plotted against their average
(Bland-Altman plots) Results The mean
vector of postoperative refractive, keratometric, and
total corneal astigmatism was 0.42 D axis 175° (Fig. 7),
0.16 D axis 15° (Fig. 8), and 0.39 D axis 5° (Fig. 9),
respectively. Figure
10 shows
difference in
vector
between
postoperative
refractive
and
keratometric
astigmatism. The mean vector was 0.30 D axis 164°. We also calculated the mean difference in magnitude
and vector between refractive and each of two types of
corneal astigmatism according to the types of preopera-
tive keratometric astigmatism, such as ATR, WTR or
oblique astigmatism. The mean difference in magnitude
and vector between refractive and keratometric astigma-
tism were 0.58 ± 0.32 D (0.14 D Axis126°), 0.62 ± 0.31 D
(0.44 D Axis2°), 0.89 ± 0.48 D (0.52 D Axis165°), respect-
ively. The vector difference between refractive and total
corneal
astigmatism
were
0.62 ± 0.34
D
(0.29
D
Axis103°), 0.52 ± 0.28 D (0.22 D Axis5°), 0.75 ± 0.48 D
(0.32
D
Axis153°),
respectively. As
for
the
mean
difference in magnitude between postoperative refract-
ive and total corneal astigmatism, there were signifi-
cant
differences
in
eyes
with
WTR
or
oblique
astigmatism (P = .006, P = .007), but not in eyes with
ATR astigmatism (P = .330). Fig. 2 The mean magnitude of postoperative keratometric and total
corneal astigmatism was significantly different (P = .015, paired t-test) Fig. 2 The mean magnitude of postoperative keratometric and total
corneal astigmatism was significantly different (P = .015, paired t-test) Figures 3 and 4 show correlations between postopera-
tive refractive and two types of corneal astigmatism
(keratometric
and
total
corneal
astigmatism). The Table 1 Preoperative patients’ data
Parameter
Mean ± SD
Range
Age (year)
71.8 ± 9.9
33–92
Sex (male: female)
25: 25
Right: Left
33: 31
Keratometric astigmatism (D)
0.76 ± 0.46
0.03–2.76
Type of keratometric astigmatism (eyes)
ATR (0–29°, 150–180°)
23
WTR (60–119°)
19
oblique (30–59°, 120–149°)
22
Axial length (mm)
23.67 ± 1.69
20.98–27.55
SD standard deviation, D diopter, ATR against-the-rule, WTR with-the-rule Sano et al. BMC Ophthalmology (2016) 16:212 Page 5 of 9 Fig. 3 A significant correlation was observed between the
magnitude of postoperative refractive and keratometric astigmatism
(Pearson’s correlation coefficient; r = 0.515, P < .0001) Fig. 5 Differences between the magnitude of postoperative refractive
and keratometric astigmatism plotted against their average
(Bland-Altman plots) Fig. 5 Differences between the magnitude of postoperative refractive
and keratometric astigmatism plotted against their average
(Bland-Altman plots) Fig. Discussion When we use toric IOL in In clinical practice, we sometimes encounter unex-
pected postoperative refractive astigmatism after cataract
surgery even with toric IOLs. When we use toric IOL in
Fig. 4 A significant correlation was observed between the magnitude
of postoperative refractive and total corneal astigmatism (Pearson’s
correlation coefficient; r = 0.598, P < .0001)
Fig. 6 Differences between the magnitude of postoperative
refractive and total corneal astigmatism plotted against their
average (Bland-Altman plots) Fig. 6 Differences between the magnitude of postoperative
refractive and total corneal astigmatism plotted against their
average (Bland-Altman plots) Fig. 6 Differences between the magnitude of postoperative
refractive and total corneal astigmatism plotted against their
average (Bland-Altman plots) Fig. 6 Differences between the magnitude of postoperative
refractive and total corneal astigmatism plotted against their
average (Bland-Altman plots) Fig. 4 A significant correlation was observed between the magnitude
of postoperative refractive and total corneal astigmatism (Pearson’s
correlation coefficient; r = 0.598, P < .0001) Sano et al. BMC Ophthalmology (2016) 16:212 Page 6 of 9 Fig. 7 Double-angle plots of postoperative refractive astigmatism. The mean vector of the astigmatism (represented by the grey
rhombus, larger than the other plots) was 0.41 D axis 175° (each
ring = 0.5 D, outer ring = 3.0 D) Fig. 9 Double-angle plots of postoperative total corneal astigmatism. The mean vector of the astigmatism (represented by the grey rhombus
larger than the other plots) was 0.38 D axis 4° (each ring = 0.5 D, outer
ring = 3.0 D) Fig. 7 Double-angle plots of postoperative refractive astigmatism. The mean vector of the astigmatism (represented by the grey
rhombus, larger than the other plots) was 0.41 D axis 175° (each
ring = 0.5 D, outer ring = 3.0 D) Fig. 9 Double-angle plots of postoperative total corneal astigmatism. The mean vector of the astigmatism (represented by the grey rhombus
larger than the other plots) was 0.38 D axis 4° (each ring = 0.5 D, outer
ring = 3.0 D) cataract surgery, a particular IOL model is selected by
assuming that astigmatism is derived entirely from the
cornea and crystalline lens. That is to say, if other factors
producing astigmatism exist, they must cause unex-
pected postoperative refractive astigmatism. Analyzing
postoperative refractive astigmatism of unknown origin
may help more accurate astigmatism correction. Discussion BMC Ophthalmology (2016) 16:212 Page 7 of 9 Page 7 of 9 Fig. 11 Double-angle plots of difference in vector between
postoperative refractive and total corneal astigmatism. The mean
vector of the difference (represented by the grey rhombus larger
than the other plots) was 0.12 D axis 135° (each ring = 0.5 D, outer
ring = 3.0 D) investigated internal astigmatism in eyes implanted with
non-toric IOL, and reported that the mean vector of in-
ternal astigmatism, corresponding to difference between
refractive and keratometric astigmatism in our study,
was 0.24 D and 0.38 D, respectively, which coincided
approximately with our results (0.30 D). The mean difference in vector and magnitude between
postoperative refractive and total corneal astigmatism
was closer to 0 D as compared to that between postoper-
ative refractive and keratometric astigmatism. This sug-
gests that preoperative actual measurement of posterior
corneal astigmatism may lead to more accurate postop-
erative astigmatism correction. When we further exam-
ined depending on the types of astigmatism such as
ATR, WTR, oblique astigmatism, the mean differences
in magnitude and vector between postoperative refract-
ive and total corneal astigmatism was closer to 0 D in
eyes with WTR or oblique astigmatism, but not in eyes
with ATR astigmatism. We are unaware of the exact rea-
son why eyes with ATR astigmatism had no similar ten-
dency. This issue should be examined in a larger
population because the number of eyes with ATR astig-
matism was somewhat small in the current study. Although several previous reports have already men-
tioned the discrepancy in posterior corneal astigmatism
between actual and estimated values [4, 5], this is the
first report to elucidate the more detailed influences of
posterior corneal astigmatism on total refractive error in
pseudophakic eyes. Based on the current findings, it can
be concluded that incorporating the data of posterior
corneal curvature into preoperative IOL power calcula-
tion results in better refractive outcomes after cataract
surgeries with toric IOLs. Fig. 11 Double-angle plots of difference in vector between
postoperative refractive and total corneal astigmatism. The mean
vector of the difference (represented by the grey rhombus larger
than the other plots) was 0.12 D axis 135° (each ring = 0.5 D, outer
ring = 3.0 D) than that between postoperative refractive and kerato-
metric astigmatism (Figs. 3, 4, 5 and 6). Discussion Theoretically,
considering that astigmatism is derived entirely from the
cornea and crystalline lens, refractive astigmatism must
be equal to corneal astigmatism, in cases of non-toric
IOL insertion eyes. Therefore, the above findings imply
that the relationship between refractive and total corneal
astigmatism is more theoretical than that between re-
fractive and keratometric astigmatism. We also calcu-
lated the difference between postoperative refractive and
two types of corneal astigmatism (keratometric and total
corneal
astigmatism)
(Figs. 10
and
11). Difference
between refractive and keratometric astigmatism was
also referred to as “internal astigmatism” in some reports
[2, 6, 19]. Teus et al. [6] and Tejedor et al. [2] g
The examination of individual cases showed that 56.0
and 17.2% of eyes showed difference between postopera-
tive refractive and total corneal astigmatism of greater
than 0.5 D and 1.0 D, respectively. This means that ap-
proximately half of eyes that are planned to undergo
toric IOL implantation may exhibit astigmatism correc-
tion errors greater than 0.5 D postoperatively even if
actual measurement data of posterior corneal astigma-
tism is incorporated into preoperative IOL power calcu-
lation. We tried to find the associated factors which
cause the greater difference, but no predictive factors
could be identified (Table 3). The difference between
postoperative refractive astigmatism and total corneal
astigmatism may involve any unknown astigmatism
other than corneal and lenticular astigmatism (e.g., ret-
inal, vitreous) [18, 19]. Further studies should be con-
ducted to clarify the causes other than posterior corneal
astigmatism
which
induce
postoperative
refractive
errors. Table 3 Patients’ data of two groups
Parameter (postoperative data)
greater than
0.5 D
less than 0.5 D
P value
Total number (eyes)
36 (56%)
28 (44%)
-
Age (year)
73.6 ± 10.1
69.4 ± 9.4
.093
Spherical equivalent
refraction (D)
−0.80 ± 1.16
−1.33 ± 1.68
.143
Refractive astigmatism (D)
0.92 ± 0.55
0.91 ± 0.39
.868
Keratometric astigmatism (D)
0.93 ± 0.48
0.81 ± 0.38
.296
Total corneal astigmatism (D)
0.97 ± 0.50
0.90 ± 0.40
.553
Axial length (mm)
23.44 ± 1.45
23.97 ± 1.94
.209
IOL power (D)
21.38 ± 3.50
20.09 ± 3.67
.159
D diopter Table 3 Patients’ data of two groups
Parameter (postoperative data)
greater than
0.5 D
less than 0.5 D
P value Table 3 Patients’ data of two groups Table 3 Patients’ data of two groups There are some limitations in this study. Discussion In this
study, we researched influence of posterior corneal astigmatism on postoperative refractive astigmatism by
comparing keratometric and total corneal astigmatism in
pseudophakic eyes after non-toric IOL implantation. We
found that the mean magnitude of keratometric and
total corneal astigmatism were significantly different
(Fig. 2). Moreover, the correlation between postoperative
refractive and total corneal astigmatism was stronger Fig. 10 Double-angle plots of difference in vector between
postoperative refractive and keratometric astigmatism. The mean
vector of the difference (represented by the grey rhombus larger
than the other plots) was 0.30 D axis 164° (each ring = 0.5 D, outer
ring = 3.0 D) y,
p
Fig. 8 Double-angle plots of postoperative keratometric
astigmatism. The mean vector of the astigmatism (represented
by the grey rhombus larger than the other plots) was 0.16 D
axis 13° (each ring = 0.5 D, outer ring = 3.0 D)
Fig. 10 Double-angle plots of difference in vector between
postoperative refractive and keratometric astigmatism. The mean
vector of the difference (represented by the grey rhombus larger
than the other plots) was 0.30 D axis 164° (each ring = 0.5 D, outer
ring = 3.0 D) Fig. 8 Double-angle plots of postoperative keratometric
astigmatism. The mean vector of the astigmatism (represented
by the grey rhombus larger than the other plots) was 0.16 D
axis 13° (each ring = 0.5 D, outer ring = 3.0 D) Fig. 10 Double-angle plots of difference in vector between
postoperative refractive and keratometric astigmatism. The mean
vector of the difference (represented by the grey rhombus larger
than the other plots) was 0.30 D axis 164° (each ring = 0.5 D, outer
ring = 3.0 D) Fig. 10 Double-angle plots of difference in vector between
postoperative refractive and keratometric astigmatism. The mean
vector of the difference (represented by the grey rhombus larger
than the other plots) was 0.30 D axis 164° (each ring = 0.5 D, outer
ring = 3.0 D) Fig. 8 Double-angle plots of postoperative keratometric
astigmatism. The mean vector of the astigmatism (represented
by the grey rhombus larger than the other plots) was 0.16 D
axis 13° (each ring = 0.5 D, outer ring = 3.0 D) Fig. 8 Double-angle plots of postoperative keratometric
astigmatism. The mean vector of the astigmatism (represented
by the grey rhombus larger than the other plots) was 0.16 D
axis 13° (each ring = 0.5 D, outer ring = 3.0 D) Sano et al. Author details
1 1Department of Ophthalmology, University of Tsukuba Hospital, 2-1-1
Amakubo, Tsukuba, Ibaraki 305-8576, Japan. 2Hitachi General Hospital, 2-1-1
Jonancho, Hitachi, Ibaraki 317-0077, Japan. 3Seinan Medical Center hospital,
4 1Department of Ophthalmology, University of Tsukuba Hospital, 2-1-1
Amakubo, Tsukuba, Ibaraki 305-8576, Japan. 2Hitachi General Hospital, 2-1-1
Jonancho, Hitachi, Ibaraki 317-0077, Japan. 3Seinan Medical Center hospital,
2190 Sakaimachi, Sashimagun, Ibaraki 306-0433, Japan. 4Department of
Ophthalmology, Faculty of Medicine, University of Tsukuba, 2-1-1 Amakubo,
Tsukuba, Ibaraki 305-8576, Japan. Discussion First, we only
simulated the influence of posterior corneal astigmatism Sano et al. BMC Ophthalmology (2016) 16:212 Sano et al. BMC Ophthalmology (2016) 16:212 Page 8 of 9 on postoperative refractive astigmatism in pseudophakic
eyes after non-toric IOL implantation. Prospective stud-
ies should be conducted to compare the surgical out-
comes between eyes in which preoperative IOL power
calculation is done using total corneal power (including
actual measurements of the posterior corneal curvature)
and keratometric power (neglecting actual measurement
of the posterior corneal curvature). Second, the degree
of preoperative corneal astigmatism was relatively small
among our study population. Similar research will be
necessary also in eyes with larger corneal astigmatism,
because candidates for toric IOL implantation generally
have considerable corneal astigmatism. Third, we didn’t
consider the influence of tilt and dislocation of im-
planted IOLs on postoperative refraction. There is a
possibility that these factors affect postoperative refract-
ive astigmatism. Further studies should also be con-
ducted to elucidate this point. Ethics approval and consent to participate This is a clinical study, and the research was approved by the Institutional
Review Board of the University of Tsukuba Hospital and conducted
according to the tenets of the Declaration of Helsinki. The trial registration
(No. H 24–92) was requested on November 30, 2012. Written informed consent to participate was obtained for each subject prior
to the study. Abbreviations 6. Teus MA, Arruabarrena C, Hernández-Verdejo JL, Sales-Sanz A, Sales-
Sanz M. Correlation between keratometric and refractive astigmatism
in pseudophakic eyes. J Cataract Refract Surg. 2010;36(10):1671–5. doi:10.1016/j.jcrs.2010.05.010 [PubMed]. AS-OCT: Anterior segment optical coherence tomography; ATR: Against-
the-rule; D: Diopter; IOL: Intraocular lens; SD: Standard deviation; WTR: With-
the-rule 7. Ueno Y, Hiraoka T, Beheregaray S, Miyazaki M, Ito M, Oshika T. Age-related
changes in anterior, posterior, and total corneal astigmatism. J Refract Surg. 2014;30(3):192–7. doi:10.3928/1081597X-20140218-01 [PubMed]. 7. Ueno Y, Hiraoka T, Beheregaray S, Miyazaki M, Ito M, Oshika T. Age-related
changes in anterior, posterior, and total corneal astigmatism. J Refract Surg. 2014;30(3):192–7. doi:10.3928/1081597X-20140218-01 [PubMed]. Author’s contributions MS was responsible for, collection of data, analysis and interpretation of
results and wrote the first draft of the manuscript. YU participated in its
design and data collection. TH and HI and TO (Tomohiro Ogami) revised the
intellectual content of the draft. TO (Tetsuro Oshika) supervised the study. All
authors reviewed and approved the final manuscript. Conclusions This study showed that the relationship between refract-
ive and total corneal astigmatism is more intimate than
that between refractive and keratometric astigmatism in
pseudophakic eyes, and the vector difference is closer to
0 D in the former than in the latter. If we use actual
measurements of the posterior corneal curvature for
evaluations prior to cataract surgery, the amount of
unexpected postoperative refractive astigmatism might
be reduced, resulting in improved uncorrected visual
acuity. However, even after eliminating the discrepancy
between actual and estimated values of posterior corneal
astigmatism, some amount of postoperative refractive
astigmatism of unknown origin persisted. Our findings
warrant further investigation to find causes of unex-
pected astigmatism correction errors beyond posterior
corneal astigmatism in order to improve visual function
after toric IOL implantation. Received: 19 April 2016 Accepted: 24 November 2016 Received: 19 April 2016 Accepted: 24 November 2016 References 1. Hayashi K, Manabe S, Yoshida M, Hayashi H. Effect of astigmatism on visual
acuity in eyes with a diffractive multifocal intraocular lens. J Cataract Refract
Surg. 2010;36(8):1323–9. doi:10.1016/j.jcrs.2010.02.016 [PubMed]. 2. Tejedor J, Guirao A. Agreement between refractive and corneal astigmatism
in pseudophakic eyes. Cornea. 2013;32(6):783–90. doi:10.1097/ICO. 0b013e31826dd44b [PubMed]. 3. Frings A, Katz T, Steinberg J, Druchkiv V, Richard G, Linke SJ. Ocular residual
astigmatism: effects of demographic and ocular parameters in myopic laser
in situ keratomileusis. J Cataract Refract Surg. 2014;40(2):232–8. doi:10.1016/j. jcrs.2013.11.015 [PubMed]. 4. Koch DD, Ali SF, Weikert MP, Shirayama M, Jenkins R, Wang L. Contribution of posterior corneal astigmatism to total corneal
astigmatism. J Cataract Refract Surg. 2012;38(12):2080–7. doi:10.1016/j. jcrs.2012.08.036 [PubMed]. 5. Ho JD, Tsai CY, Liou SW. Accuracy of corneal astigmatism estimation by
neglecting the posterior corneal surface measurement. Am J Ophthalmol. 2009;147(5):788–95. doi:10.1016/j.ajo.2008.12.020. 795.e1-2 [PubMed]. Competing interests p
g
The authors declare that they have no competing interest. Acknowledgements The authors would like to acknowledge all members of the Department of
Ophthalmology, Faculty of Medicine, University of Tsukuba, Hitachi General
Hospital and Seinan Medical Center hospital. The authors have no public or private financial support to disclose. None of the authors has a financial or proprietary interest in any material or
method mentioned herein. 8. Dubbelman M, Sicam VA, Van der Heijde GL. The shape of the anterior and
posterior surface of the aging human cornea. Vision Res. 2006;46(6–7):
993–1001 [PubMed]. 9. Ho JD, Tsai CY, Tsai RJ, Kuo LL, Tsai IL, Liou SW. Validity of the
keratometric index: evaluation by the Pentacam rotating Scheimpflug
camera. J Cataract Refract Surg. 2008;34(1):137–45. doi:10.1016/j.jcrs.2007. 09.033 [PubMed]. 10. Ho JD, Liou SW, Tsai RJ, Tsai CY. Effects of aging on anterior and
posterior corneal astigmatism. Cornea. 2010;29(6):632–7. doi:10.1097/ICO. 0b013e3181c2965f [PubMed]. Funding
None. 11. Ueno Y, Hiraoka T, Miyazaki M, Ito M, Oshika T. Corneal thickness profile and
posterior corneal astigmatism in normal corneas. Ophthalmology. 2015;
122(6):1072–8. doi:10.1016/j.ophtha.2015.01.021 [PubMed]. 10.
Ho JD, Liou SW, Tsai RJ, Tsai CY. Effects of aging on anterior and
posterior corneal astigmatism. Cornea. 2010;29(6):632–7. doi:10.1097/ICO.
0b013e3181c2965f [PubMed]. 9.
Ho JD, Tsai CY, Tsai RJ, Kuo LL, Tsai IL, Liou SW. Validity of the
keratometric index: evaluation by the Pentacam rotating Scheimpflug
camera. J Cataract Refract Surg. 2008;34(1):137–45. doi:10.1016/j.jcrs.2007.
09.033 [PubMed]. AS-OCT: Anterior segment optical coherence tomography; ATR: Against-
the-rule; D: Diopter; IOL: Intraocular lens; SD: Standard deviation; WTR: With-
the-rule g
The authors would like to acknowledge all members of the Department of
Ophthalmology, Faculty of Medicine, University of Tsukuba, Hitachi General
Hospital and Seinan Medical Center hospital.
The authors have no public or private financial support to disclose.
None of the authors has a financial or proprietary interest in any material or
method mentioned herein. Availability of data and materials
Data can be shared upon request. Availability of data and materials
Data can be shared upon request. Availability of data and materials
Data can be shared upon request. Page 9 of 9 Sano et al. BMC Ophthalmology (2016) 16:212 Sano et al. BMC Ophthalmology (2016) 16:212 12. Koch DD, Jenkins RB, Weikert MP, Yeu E, Wang L. Correcting astigmatism
with toric intraocular lenses: effect of posterior corneal astigmatism. J
Cataract Refract Surg. 2013;39(12):1803–9. doi:10.1016/j.jcrs.2013.06.027
[PubMed]. 13. Szalai E, Berta A, Hassan Z, Módis Jr L. Reliability and repeatability of swept-
source Fourier-domain optical coherence tomography and Scheimpflug
imaging in keratoconus. J Cataract Refract Surg. 2012;38(3):485–94. doi:10. 1016/j.jcrs.2011.10.027 [PubMed]. 14. Nakagawa T, Maeda N, Higashiura R, Hori Y, Inoue T, Nishida K. Corneal
topographic analysis in patients with keratoconus using 3-dimensional
anterior segment optical coherence tomography. J Cataract Refract Surg. 2011;37(10):1871–8. doi:10.1016/j.jcrs.2011.05.027 [PubMed]. 15. Yamaguchi T, Ohnuma K, Tomida D, Konomi K, Satake Y, Negishi K, Tsubota
K, Shimazaki J. The contribution of the posterior surface to the corneal
aberrations in eyes after keratoplasty. Invest Ophthalmol Vis Sci. 2011;52(9):
6222–9. doi:10.1167/iovs.11-7647 [PubMed]. 16. Holladay JT, Moran JR, Kezirian GM. Analysis of aggregate surgically induced
refractive change, prediction error, and intraocular astigmatism. J Cataract
Refract Surg. 2001;27(1):61–79 [PubMed]. 17. Visser N, Berendschot TT, Bauer NJ, Nuijts RM. Vector analysis of corneal and
refractive astigmatism changes following toric pseudophakic and toric
phakic IOL implantation. Invest Ophthalmol Vis Sci. 2012;53(4):1865–73. doi:10.1167/iovs.11-8868 [PubMed]. 18. Tang M, Chen A, Li Y, Huang D. Corneal power measurement with Fourier-
domain optical coherence tomography. J Cataract Refract Surg. 2010;36(12):
2115–22. doi:10.1016/j.jcrs.2010.07.018 [PubMed]. 19. Srivannaboon S. Internal astigmatism and its correlation to corneal and
refractive astigmatism. J Med Assoc Thai. 2003;86(2):166–71 [PubMed]. • We accept pre-submission inquiries
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Statistical Considerations in Demonstrating CMC Analytical Similarity for a Biosimilar Product
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Statistics in biopharmaceutical research
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WAGTTBAS
What A Great Time To Be A Statistician! 2015 Duke Industry Stat Symposium 1 This presentation reflects the views of the authors and should
not be construed to represent FDA’s views or policies. 2 2 OUTLINES 2015 Duke Industry Stat Symposium 3 y
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confidence interval in clinical trial, a few years ago was
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difference on hypothesis test and general confidence
interval estimation. • However, it was never clearly emphasized on the
difference on hypothesis test and general confidence
interval estimation. • Furthermore, due to the recent development of statistical
testing in drug development, the duality may lead to some
of the difficulties in constructing test-based confidence
interval to be consistent the with significance test.
• We illustrate the problems with a few examples. • Furthermore, due to the recent development of statistical
testing in drug development, the duality may lead to some
of the difficulties in constructing test-based confidence
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We will also reject H0 if the lower confidence level is greater than
0. That the lower confidence limit can be derived from the test
statistic and its sampling distributions. That the lower confidence limit can be derived from the test
statistic and its sampling distributions. In this case, decisions made with both significance test and
confidence interval are consistent although they may be deriv
independently. In this case, decisions made with both significance test and
confidence interval are consistent although they may be derived
independently. 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 7 III. Comparing Mean Difference of Binary Outcomes
Assuming
),
(
~
i
i
P
B
X
i = T, R represent the binary outcome of test and
reference products. Let us consider significance test, non-inferiority and equivalence tests
under the setting separately. Significance test: Significance test:
When comparing two proportions, the significance testing
hypotheses are,
0
:
0
R
T
P
P
H
versus
0
:
R
T
A
P
P
H
Let
R
T
P
P
, the unbiased estimate of is
R
T
P
P
ˆ
ˆ
ˆ
. The asymptotic
test of the hypotheses is a score test in the form that
)
ˆ
ˆ
(
ˆ
ˆ
R
T
R
T
P
P
e
P
P
Z
(III.1)
)
(
e
is the standard error of estimation.
2015 Duke Industry Stat Symposium
8 Significance test:
When comparing two proportions, the significance testing
hypotheses are,
0
:
0
R
T
P
P
H
versus
0
:
R
T
A
P
P
H
Let
R
T
P
P
, the unbiased estimate of is
R
T
P
P
ˆ
ˆ
ˆ
. The asymptotic
test of the hypotheses is a score test in the form that
)
ˆ
ˆ
(
ˆ
ˆ
R
T
R
T
P
P
e
P
P
Z
(III.1)
)
(
e
is the standard error of estimation. 2015 Duke Industry Stat Symposium
8 8 est is monotone, i.e. if
2
1
the Z value of
1
the Z
, the type I error rate of Z reaches its maximum at =
ng distribution of the statistic is derived from
0
|
Z
.
y, the standard error is derived with restriction to = 0.
0
|)
R
P
] =
n
P
P
/)
1(
2
(III.2)
2
/)
R
P
. When using this significance test, we reject the
s if Z > Z(1 -α/2) asmptotically.
stimate the standard error
)
ˆ
ˆ
(
R
T
P
P
e
without
to null hypothesis, we have
)
ˆ
ˆ
(
R
T
P
P
e
=
P
P
)
1(
Since the test is monotone, i.e. if
2
1
the Z value of
1
the Z
value of
2
, the type I error rate of Z reaches its maximum at =
0. The sampling distribution of the statistic is derived from
0
|
Z
. Accordingly, the standard error is derived with restriction to = 0. It leads to with
2
/)
(
R
T
P
P
P
. When using this significance test, we reject the
null hypothesis if Z > Z(1 -α/2) asmptotically. When we estimate the standard error
)
ˆ
ˆ
(
R
T
P
P
e
without
restriction to null hypothesis, we have
)
ˆ
ˆ
(
R
T
P
P
e
=
P
P
P
P
R
R
T
T
)
1(
)
1(
(III 3) n 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 9 It can be shown that
n
P
P
P
P
R
R
T
T
)
1(
)
1(
n
P
P
/)
1(
2
. That means
restricted standard error is at least as large as the unrestricted
standard error. The conventional confidence interval of is
)
2
/
1(
ˆ
(
Z
n
P
P
P
P
R
R
T
T
)
1(
)
1(
,
)
2
/
1(
ˆ
Z
n
P
P
P
P
R
R
T
T
)
1(
)
1(
). Using conventional confidence interval, one may claim
superiority if
)
2
/
1(
ˆ
Z
n
P
P
P
P
R
R
T
T
)
1(
)
1(
> 0. It is inconsistent to
the significance test constructed under null hypothesis. It can be shown that
n
P
P
P
P
R
R
T
T
)
1(
)
1(
n
P
P
/)
1(
2
. That means
restricted standard error is at least as large as the unrestricted
standard error. est is monotone, i.e. if
2
1
the Z value of
1
the Z
, the type I error rate of Z reaches its maximum at =
ng distribution of the statistic is derived from
0
|
Z
.
y, the standard error is derived with restriction to = 0.
0
|)
R
P
] =
n
P
P
/)
1(
2
(III.2)
2
/)
R
P
. When using this significance test, we reject the
s if Z > Z(1 -α/2) asmptotically.
stimate the standard error
)
ˆ
ˆ
(
R
T
P
P
e
without
to null hypothesis, we have
)
ˆ
ˆ
(
R
T
P
P
e
=
P
P
)
1(
It is also pointed out the
other two test statistics with small to moderate sample sizes, the
type I error rate is not controlled. With a continuity correction, the asymptotic test based on (III.1)
becomes
)
ˆ
ˆ
(
/
1
ˆ
ˆ
R
T
R
T
P
P
e
n
P
P
Z
With a continuity correction, the asymptotic test based on (III.1)
becomes
)
ˆ
ˆ
(
/
1
ˆ
ˆ
R
T
R
T
P
P
e
n
P
P
Z
The continuity correction adjusted confidence interval is then,
(
)
2
/
1(
/
1
ˆ
(
Z
n
n
P
P
P
P
R
R
T
T
)
1(
)
1(
,
)
2
/
1(
/
1
ˆ
Z
n
n
P
P
P
P
R
R
T
T
)
1(
)
1(
)
It was pointed out by Farrington and Manning (1990, SIM ) that
all three statistics converge to the standard normal distribution. But they argued that test statistic using (II.2) is both theoretically
correct and convergent to N(0,1) faster. It is also pointed out the
other two test statistics with small to moderate sample sizes, the
type I error rate is not controlled. ecomes
)
(
R
T
P
P
e
e continuity correction adjusted confidence interval is then,
)
2
/
1(
/
Z
n
n
P
P
P
P
R
R
T
T
)
1(
)
1(
,
)
2
/
1(
/
1
ˆ
Z
n
n
P
P
P
P
R
R
T
T
)
1(
)
1(
)
was pointed out by Farrington and Manning (1990, SIM ) that
three statistics converge to the standard normal distribution. ut they argued that test statistic using (II.2) is both theoretically
rrect and convergent to N(0,1) faster. It is also pointed out the
her two test statistics with small to moderate sample sizes, the
pe I error rate is not controlled. est is monotone, i.e. if
2
1
the Z value of
1
the Z
, the type I error rate of Z reaches its maximum at =
ng distribution of the statistic is derived from
0
|
Z
.
y, the standard error is derived with restriction to = 0.
0
|)
R
P
] =
n
P
P
/)
1(
2
(III.2)
2
/)
R
P
. When using this significance test, we reject the
s if Z > Z(1 -α/2) asmptotically.
stimate the standard error
)
ˆ
ˆ
(
R
T
P
P
e
without
to null hypothesis, we have
)
ˆ
ˆ
(
R
T
P
P
e
=
P
P
)
1(
The conventional confidence interval of is
)
2
/
1(
ˆ
(
Z
n
P
P
P
P
R
R
T
T
)
1(
)
1(
,
)
2
/
1(
ˆ
Z
n
P
P
P
P
R
R
T
T
)
1(
)
1(
). Using conventional confidence interval, one may claim
superiority if
)
2
/
1(
ˆ
Z
n
P
P
P
P
R
R
T
T
)
1(
)
1(
> 0. It is inconsistent to
the significance test constructed under null hypothesis. standard error. The conventional confidence interval of is
)
2
/
1(
ˆ
(
Z
n
P
P
P
P
R
R
T
T
)
1(
)
1(
,
)
2
/
1(
ˆ
Z
n
P
P
P
P
R
R
T
T
)
1(
)
1(
). Using conventional confidence interval, one may claim
superiority if
)
2
/
1(
ˆ
Z
n
P
P
P
P
R
R
T
T
)
1(
)
1(
> 0. It is inconsistent to
the significance test constructed under null hypothesis 2015 Duke Industry Stat Symposium 10 With a continuity correction, the asymptotic test based on (III.1)
becomes
)
ˆ
ˆ
(
/
1
ˆ
ˆ
R
T
R
T
P
P
e
n
P
P
Z
The continuity correction adjusted confidence interval is then,
(
)
2
/
1(
/
1
ˆ
(
Z
n
n
P
P
P
P
R
R
T
T
)
1(
)
1(
,
)
2
/
1(
/
1
ˆ
Z
n
n
P
P
P
P
R
R
T
T
)
1(
)
1(
)
It was pointed out by Farrington and Manning (1990, SIM ) that
all three statistics converge to the standard normal distribution. But they argued that test statistic using (II.2) is both theoretically
correct and convergent to N(0,1) faster. est is monotone, i.e. if
2
1
the Z value of
1
the Z
, the type I error rate of Z reaches its maximum at =
ng distribution of the statistic is derived from
0
|
Z
.
y, the standard error is derived with restriction to = 0.
0
|)
R
P
] =
n
P
P
/)
1(
2
(III.2)
2
/)
R
P
. When using this significance test, we reject the
s if Z > Z(1 -α/2) asmptotically.
stimate the standard error
)
ˆ
ˆ
(
R
T
P
P
e
without
to null hypothesis, we have
)
ˆ
ˆ
(
R
T
P
P
e
=
P
P
)
1(
The continuity correction adjusted confidence interval is then,
(
)
2
/
1(
/
1
ˆ
(
Z
n
n
P
P
P
P
R
R
T
T
)
1(
)
1(
,
)
2
/
1(
/
1
ˆ
Z
n
n
P
P
P
P
R
R
T
T
)
1(
)
1(
) 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 11 Non-inferiority and equivalence tests inferiority and equivalence tests
When comparing two proportions, the non-inferiority hypotheses
R
T
P
P
H :
0
versus
R
T
A
P
P
H :
re δ > 0 is a non-inferiority constant margin.
asymptotic test of the hypotheses is a score test in the form that
)
ˆ
ˆ
(
ˆ
ˆ
R
T
R
T
P
P
e
P
P
Z
(III.4)
)
ˆ
ˆ
(
1
ˆ
ˆ
R
T
R
T
P
P
e
n
P
P
Z
with continuity correction,
re
)
(
e
is the standard error of estimation. The sampling distribution
e statistic is derived from
|
Z
. Accordingly, the standard error is
ed as the maximum likelihood estimate restricted to = - δ. It can
hown as (Farrington and Manning, 1990)
n
P
P
P
P
P
P
e
R
R
T
T
R
T
/
)]
~
1(
~
)
~
1(
~
[
|)
ˆ
ˆ
(
2015 Duke Industry Stat Symposium
12 y
q
hen comparing two proportions, the non-inferiority hypotheses When comparing two proportions, the non-inferiority hypotheses
re, with a =2
b =-[2+
T
p +
R
p + 3δ]
c =δ2 +δ(2
T
p +2) +
T
p +
R
p
d = -
T
p δ(1+δ) where
T
p and
R
p are the sample proportions of test and reference
respectively,
T
P~ =
R
P~ + δ. Again without restriction, we have
)
ˆ
ˆ
(
R
T
P
P
e
=
n
P
P
P
P
R
R
T
T
)
1
(
)
1
(
are,
R
T
P
P
H :
0
versus
R
T
A
P
P
H :
where δ > 0 is a non-inferiority constant margin. The asymptotic test of the hypotheses is a score test
R
T
P
P
H :
0
versus
R
T
A
P
P
H :
R
T
P
P
H :
0
versus
R
T
A
P
P
H :
where δ > 0 is a non-inferiority constant margin. The asymptotic test of the hypotheses is a score test in the fo where δ > 0 is a non-inferiority constant margin. y
g
ptotic test of the hypotheses is a score test in the form that
)
ˆ
ˆ
(
ˆ
R
T
P
P
P
(III.4) y
g
The asymptotic test of the hypotheses is a score test in the form that mptotic test of the hypotheses is a score test in the form that
ˆ
ˆ
P
P
)
ˆ
ˆ
(
ˆ
ˆ
R
T
R
T
P
P
e
P
P
Z
)
ˆ
ˆ
(
1
ˆ
ˆ
R
T
R
T
P
P
e
n
P
P
Z
w 12 where
T
P~ and
R
P~ are the maximum likelihood estimates of PT and PR
restricted to H0. For testing against H0,
T
P~ and
R
P~ are shown to be the
solutions in (δ, 1) of the following equation aX3 + bX2 + cX +d =0
with
a =2
b =-[2+
T
p +
R
p + 3δ]
c =δ2 +δ(2
T
p +2) +
T
p +
R
p
d = -
T
p δ(1+δ) nfidence decision is not different from the one for superiority test
we compare its lower limit with – δ. This confidence decision is not different from the one for superiority test
except we compare its lower limit with – δ. 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 13 test
ence test consists of two one-sided hypotheses
R
P
versus
R
T
A
P
P
H
:
1
R
P
versus
R
T
A
P
P
H
:
2
ting both null hypotheses, one shows that
R
P
istic corresponds to testing the second one-sided
s
)
ˆ
ˆ
R
R
P
P
(III.5)
)
ˆ
ˆ
1
ˆ
R
T
R
P
P
n
P
with continuity correction.
d error is derived as the maximum likelihood estimate
= δ. It can be derived as
n
P
P
P
P
R
R
T
T
/
)]
~
1(
~
)
~
1(
~
[
|)
2015 Duke Industry Stat Symposium
14 The equivalence test consists of two one-sided hypotheses
R
T
P
P
H :
01
versus
R
T
A
P
P
H
:
1
R
T
P
P
H
:
02
versus
R
T
A
P
P
H
:
2 When rejecting both null hypotheses, one shows that
R
T
P
P The test statistic corresponds to testing the second one-sided
hypotheses is hypotheses is )
ˆ
ˆ
(
ˆ
ˆ
R
T
R
T
P
P
e
P
P
Z
(III.5) 14 where
TP~ and
R
P~
are the maximum likelihood estimates of PT and PR
restricted to H02. For testing against H0,
TP~ and
RP~ are shown to be the
solutions in (δ, 1) of the following equation
3
2 with On the other hand, using the confidence interval, the decision of
equivalence is derived with the unrestricted maximum likelihood
estimate )
ˆ
ˆ
(
R
T
P
P
e
=
n
P
P
P
P
R
R
T
T
)
1
(
)
1
(
15 The inconsistency applies to any distribution(such as Bernoulli and
Poisson) of which the variance is a function is linearly dependent
to the mean 2015 Duke Industry Stat Symposium 16 lternative Comparisons of Normal Outcomes
st for exchangeability hypotheses involving both mean and
y of significance test and confidence interval decision rules
istent.
for a probability hypothesis of non-inferiority such as
)
1(5.0
)
Pr(
:
vs.
)
1(5.0
)
P
L
X
X
H
P
L
X
R
T
a
R
ecified margin and P a pre-specified percentage.
rmality assumption,
)
2,
(
~
2
R
T
R
T
N
X
X
, Tsong and
nd Dong and Tsong (2015) showed the one-sided tolerance
)
of
R
T
X
X
with significance level 1-α/2 and coverage
5(1+P). One reject the null hypothesis if
P
L > L. It is an IV. Alternative Comparisons of Normal Outcomes
VI.1 Considering test for exchangeability hypotheses involving both mean and
variance, the duality of significance test and confidence interval decision rules
may not be as consistent. For example, for a probability hypothesis of non-inferiority suc For example, for a probability hypothesis of non-inferiority such as
)
1(5.0
)
Pr(
:
vs. )
1(5.0
)
Pr(
:
0
P
L
X
X
H
P
L
X
X
H
R
T
a
R
T
where L is a pre-specified margin and P a pre-specified percentage. p
p
y yp
y
5.0
)
Pr(
:
vs. )
1(5.0
)
Pr(
:
0
L
X
X
H
P
L
X
X
H
R
T
a
R
T
where L is a pre-specified margin and P a pre-specified percentage. 0
)
Pr(
:
vs. )
1(5.0
)
Pr(
:
0
L
X
X
H
P
L
X
X
H
R
T
a
R
T
where L is a pre-specified margin and P a pre-specified percentage. Under the normality assumption,
)
2,
(
~
2
R
T
R
T
N
X
X
, Tsong and
Shen (2007) and Dong and Tsong (2015) showed the one-sided tolerance
interval
)
,
(
P
L
of
R
T
X
X
with significance level 1-α/2 and coverage
percentage 0.5(1+P). One reject the null hypothesis if
P
L > L. It is an
exact test. 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 17 However , for an equivalence hypotheses •Corresponding to the two one-sided tests, •Corresponding to the two one-sided tests, •Corresponding to the two one-sided tests, p
g
,
2015 Duke Industry Stat Symposium
18
Test based confidence interval is then
)
,
(
P
L
∩
)
,
(
P
U
. On the other hand, if we use a regular two-sided tolerance interval with
1 – α confidence level and P coverage, we are considering a tolerance
interval
)
,
(
kS
X
X
kS
X
X
R
T
R
T
with k determined by the sample size n, α and P for two-sided tolerance
interval. In this case, the regular confidence interval provides a different decision
rule that significance test. Test based confidence interval is then
)
,
(
P
L
∩
)
,
(
P
U
. On the other hand, if we use a regular two-sided tolerance interval with
1 – α confidence level and P coverage, we are considering a tolerance
interval 18 2015 Duke Industry Stat Symposium n the other hand, if we use a regular two-sided tolerance
terval with 1 – α confidence level and P coverage, we are
onsidering a tolerance interval
)
,
(
kS
X
X
kS
X
X
R
T
R
T
ith k determined by the sample size n, α and P.
ne may reject the null hypothesis if
L
kS
X
X
R
T
.
ther using approximation method or exact method, this
terval provides no assurance that
)
,
(
kS
X
X
R
T
covers
ss that < 0.5(1-P) at 1 - α/2 level.
this case, the regular confidence interval provides a
fferent decision rule from the significance test. On the other hand, if we use a regular two-sided tolerance
interval with 1 – α confidence level and P coverage, we are
considering a tolerance interval )
,
(
kS
X
X
kS
X
X
R
T
R
T
with k determined by the sample size n, α and P. One may reject the null hypothesis if
L
kS
X
X
R
T
. Either using approximation method or exact method, this
interval provides no assurance that
)
,
(
kS
X
X
R
T
covers
less that < 0.5(1-P) at 1 - α/2 level. In this case, the regular confidence interval provides a
different decision rule from the significance test. 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 19 VI.2 Asymptotic Tests for Variance-Adjusted Equivalence w
Normal Endpoints (Chen, Weng, Dong & Tsong, 2015)
Test equivalence hypothesis
•Two one-sided hypotheses VI.2 Asymptotic Tests for Variance-Adjusted Equivalence
Normal Endpoints (Chen, Weng, Dong & Tsong, 2015)
Test equivalence hypothesis
Two one sided hypotheses VI.2 Asymptotic Tests for Variance-Adjusted Equivalence wit
Normal Endpoints (Chen, Weng, Dong & Tsong, 2015)
Test equivalence hypothesis VI.2 Asymptotic Tests for Variance-Adjusted Equivalence with
Normal Endpoints (Chen, Weng, Dong & Tsong, 2015)
Test equivalence hypothesis
•Two one-sided hypotheses symptotic Tests for Variance-Adjusted Equivalence with
al Endpoints (Chen, Weng, Dong & Tsong, 2015)
equivalence hypothesis
one-sided hypotheses 2015 Duke Industry Stat Symposium 20 – Unconstrained maximum likelihood estimates
– Unconstrained uniformly minimum variance
unbiased estimates (Ahn and Fessler, 2003) – Constrained maximum likelihood estimates
(Farrington and Manning, 1990; Ng, Gu, and Tang, 2007; Stucke and
Kieser, 2013) – Constrained maximum likelihood estimates
(Farrington and Manning, 1990; Ng, Gu, and Tang, 2007; Stucke and
Kieser, 2013) 2015 Duke Industry Stat Symposium 21 • Under , if , the log-likelihood
function
• Under , if , the log-likelihood
function
22
2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 22 Statistical inference for
– Based on
– Variance estimates of zL
– estimates , including MLE, UMVUE, constrained MLE
– Test statistic
– P-value =
– Reject if p-value <
Similar test procedure for
23
2015 Duke Industry Stat Symposium • Statistical inference for
– Based on
– Variance estimates of zL
– estimates , including MLE, UMVUE, constrained MLE
– Test statistic
– P-value =
– Reject if p-value <
Similar test procedure for
23
2015 Duke Industry Stat Symposium 23 Type I error rate comparison based on simulation
Set cL=1.5, cU=1.5, effect size=1.5,
Equal and unequal sample size
Generate
Repeat times for each parameter
configuration
Significance level for each one-sided test
24
2015 Duke Industry Stat Symposium Type I error rate comparison based on simulation 24 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 25 2015 Duke Industry Stat Symposium 26 27 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 28 Power comparisons based on simulation
• Set cL=1.5 and cU=1.5
• Equal and unequal sample size
•
• Effect size = -2.0(0.1)2.0
• Generate
• Repeat times for each parameter
configuration
• Significance level for each one-sided test
2015 Duke Industry Stat Symposium Power comparisons based on simulation
• Set cL=1.5 and cU=1.5
• Equal and unequal sample size
•
• Effect size = -2.0(0.1)2.0
• Generate
• Repeat times for each parameter
configuration
• Significance level for each one-sided test
2015 Duke Industry Stat Symposium Power comparisons based on simulation 29 30
Power comparisons based on simulation
2015 Duke Industry Stat Symposium mparisons based on simulation Power comparisons based on simulation 30
2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 30 31
2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 31 • Bioassays are experiment to measure biological activity
(potency) of a drug as a function of concentration/dose; • Relative Potency : ratio of the conc. of the test product that
produces the same biological response as one unit of the
conc. of the reference product 32
conc. of the reference product
ρ < 1: Test drug produces higher
response (lower conc. can produce
the same response as the ref.);
2015 Duke Industry Stat Symposium VI.3 Statistical Methods for Parallelism Test of Bioassays
(Shao, Dong, Torigoe & Tsong, 2015) • Bioassays are experiment to measure biological activity
(potency) of a drug as a function of concentration/dose; ρ < 1: Test drug produces higher
response (lower conc. can produce
the same response as the ref.); 2015 Duke Industry Stat Symposium 32 Parallel-Line Model
33
• Independent
• Normality
• Homogenous variances of
residuals
Relative potency
2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 33 Equivalence Test based on Slope Ratio • Hypothesis: test if the ratio of slopes is close to 1. S
L
T
H
:
0
S
U
T
S
U
T
S
L
a
H
:
Linearized hypothesis
or Use Wald test with restricted and unrestricted standard error 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 34 Pr (Conclude Parallel | Not Parallel) Pr (Conclude Parallel | Not Parallel) Equivalence Test based on Slope Ratio: Fieller’s Method 35
quivalence Test based on Slope Ratio: Fieller s Method
2
2
2
/
,
~
(0,
)
T
S
T
S
T
S
N
2015 Duke Industry Stat Symposium 3
2
2
2
/
,
~
(0,
)
T
S
T
S
T
S
N
2015 Duke Industry Stat Symposium 35 Simulation Studies • Output of simulation studies:
• Questions to be answered:
– Can those methods control the type I error rate (≤ 5%)? – What is the coverage of CI-based approaches (close to 90%)? – What are the impact of sample size and variance on the Type I
error rate and coverage? – Is the decision rule of Test Stat consistent with CI? Type I Error Rate
Pr (Conclude Parallel | Not Parallel)
Coverage
Pr (CI Covers the True Value of Par.)
2015 Duke Industry Stat Symposium • Output of simulation studies:
• Questions to be answered:
– Can those methods control the type I error rate (≤ 5%)? – What is the coverage of CI-based approaches (close to 90%)? – What are the impact of sample size and variance on the Type I
error rate and coverage? – Is the decision rule of Test Stat consistent with CI? 36
Type I Error Rate
Pr (Conclude Parallel | Not Parallel)
Coverage
Pr (CI Covers the True Value of Par.)
2015 Duke Industry Stat Symposium • Output of simulation studies:
Type I Error Rate
Pr (Conclude Parallel |
Coverage
Pr (CI Covers the True • Output of simulation studies:
Type I Error Rate
Pr (
Coverage
Pr ( • Output of simulation studies: Pr (CI Covers the True Value of Par.) Questions to be answered:
– Can those methods control the type I error rate (≤ 5%)? – What is the coverage of CI-based approaches (close to 90%)? – What are the impact of sample size and variance on the Type I
error rate and coverage? – Can those methods control the type I e – What is the coverage of CI-based appro – What is the coverage of CI-based approaches (close to 90 – What are the impact of sample size and variance on the Typ
error rate and coverage? – Is the decision rule of Test Stat consistent with CI? 2015 Duke Industry Stat Symposium 36 Ha : 0.80 < βT / βS < 1.25 2015 Duke Industry Stat Symposium 37 Simulation Studies: Type I Error Rate (5%) 38
ation Studies: Type I Error Rate (5%)
2015 Duke Industry Stat Symposium Simulation Studies: Type I Error Rate (
2015 Duke Industry Stat Symposium 38
ation Studies: Type I Error Rate (5%)
2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 38 Simulation Studies: Coverage (Target = 90%) 39
ulation Studies: Coverage (Target 90%)
2015 Duke Industry Stat Symposium Simulation Studies: Coverage (Target 9
2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 39 Type I Error Rate vs. Coverage • When the coverage of confidence interval can reach (1-2α)
100%, say 90%, the type I error rate may not reach α (5%):
– The definition of coverage is consistent with decision rule of
the significant test, not the equivalence test; • When the coverage of confidence interval can reach (1-2α)
100%, say 90%, the type I error rate may not reach α (5%): – The definition of coverage is consistent with decision rule of
the significant test, not the equivalence test; 40
Fieller
βT/βS
βT
βS
SD.β
Coverage
(%)
RR_low
(%)
RR_Up
(%)
RR_Tost
(%)
N = 5
1.25
5
4
0.5
90.04
79.71
5
1.77
N = 50
1.25
5
4
0.5
90.09
87.4
4.97
0.12
N = 1000
1.25
5
4
0.5
89.93
87.84
4.97
0.02
H0: βT / βS ≠ λL
H0: λL < βT / βS < λU
2015 Duke Industry Stat Symposium eller
βT/βS
βT
βS
SD.β
Coverage
(%)
RR_low
(%)
RR_Up
(%)
RR_Tost
(%)
= 5
1.25
5
4
0.5
90.04
79.71
5
1.77
= 50
1.25
5
4
0.5
90.09
87.4
4.97
0.12
= 1000
1.25
5
4
0.5
89.93
87.84
4.97
0.02
H0: βT / βS ≠ λL
H0: λL < βT / βS < λU H0: λL < βT / βS < λU 2015 Duke Industry Stat Symposium 40 • Fieller’s Method: • Fieller’s Method: Fieller s Method:
– provides a reliable inference for the ratio of slopes
– controls the type I error rate; – provides a reliable inference for the ratio of slopes – controls the type I error rate; – However, this method is solvable only when both slopes are
significant. – However, this method is solvable only when both slopes are
significant. • A confidence interval with a 90% two-sided coverage may
not assure a type error rate of 5% for equivalence test. • A confidence interval with a 90% two-sided coverage ma
not assure a type error rate of 5% for equivalence test. 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 41 Conclusion and recommendation In many situations the standard error estimated under null hypothesis
is different from the one estimated without restriction. It leads to the
failing of consistency in decision making using a significance test and a
traditional confidence interval in various situations. Therefore, in various situations, estimation using regular confidence
interval should be done after significance testing in order to maintain the
consistency of decision making. 2015 Duke Industry Stat Symposium 42 May I answer any question? 2015 Duke Industry Stat Symposium 43
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https://openalex.org/W2908766845
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https://www.scielo.br/j/lajss/a/VTGCDZkmjH8kLqFxbD5kBtd/?lang=en&format=pdf
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Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete
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Latin American Journal of Solids and Structures
| 2,019
|
cc-by
| 5,993
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THEMATIC SECTION - CILAMCE 2018: IBERO-LATIN AMERICAN
CONGRESS ON COMPUTATIONAL METHODS IN ENGINEERING Multi-scale representation of plastic deformation in fiber-reinforced
materials: application to reinforced concrete Multi-scale representation of plastic deformation in fiber-reinforced
materials: application to reinforced concrete Tea Rukavinaa,b* Keywords
bond slip da Keywords
bond-slip, damage, plasticity, multi-scale, reinforced concrete. Abstract Here we present a multi-scale model to carry out the computation of brittle composite materials reinforced
with fibers, and we show its application to standard reinforced concrete. The computation is carried out
within an operator-split framework on the macro-scale, which allows for different failure mechanisms to
develop in separate phases, as both the concrete and the bond-slip exhibit non-linear behavior. The computations on the micro-scale are performed for each constituent separately. The reinforcement is
taken to be linear elastic, and the bond-slip is handled as a plastic deformation. The standard elastoplastic
procedure is used to compute the bond stresses, combined with the X-FEM methodology to give the global
representation of slip. The crack development in concrete, on the other hand, is described with a damage
model with exponential softening, where ED-FEM is used to represent localized failure. A numerical example
is shown to test the developed methodology. Ivica Kozarb niversité de Technologie de Compiègne, Sorbonne Universités, Laboratoire Roberval de Mécanique, Centre de Rech
200 Compiègne, France. E-mail: tea.rukavina@utc.fr, adnan.ibrahimbegovic@utc.fr a Université de Technologie de Compiègne, Sorbonne Universités, Laboratoire Roberval de Mécanique, Centre de Recherche Royallieu,
60200 Compiègne, France. E-mail: tea.rukavina@utc.fr, adnan.ibrahimbegovic@utc.fr
b University of Rijeka, Faculty of Civil Engineering, Radmile Matejcic 3, 51000 Rijeka, Croatia. E-mail: ivica.kozar@uniri.hr niversity of Rijeka, Faculty of Civil Engineering, Radmile Matejcic 3, 51000 Rijeka, Croatia. E-mail: ivica.kozar@uniri. *Corresponding author Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Received: October 25, 2018. In Revised Form: October 30, 2018. Accepted: October 31, 2018. Available online: November 01, 2018
http://dx.doi.org/10.1590/1679-78255341 1 INTRODUCTION (2010), where a Drucker-Prager non-associative plasticity model is used, but still the
issue with introducing a special element persists. That is why we have decided to simplify the handling of bond-slip, but without sacrificing the model capabilities. Building on top of the work of Ibrahimbegovic et al. (2010), we are proposing a novel and efficient way to take into
account the slip between the reinforcing bar and the surrounding concrete. Instead of having a special finite element
for every constituent (i.e. a CST element for concrete, a truss bar for steel, and a Q4 element for bond-slip), we have
encapsulated all three ingredients in a single finite element, without the need to worry about the nodal connections
between the concrete, steel, and interface part. In the next chapter, the formulation of the model is presented, with its specific micro−macro treatment of the
interaction of three different constituents, and after that we show the results of the numerical simulation of a
reinforced concrete specimen. 1 INTRODUCTION To model fiber-reinforced composite materials, one has to take into account the behavior of each of the
constituents, and their interaction. In the case of fiber-reinforced concrete (FRC), we are dealing with concrete, steel
fibers, and the interface between them. We can consider standard reinforced concrete as a special case of such a
material, with its specific features. First of all, there are not more than a few reinforcement bars embedded in concrete
(compared to a large number of randomly oriented short fibres in FRC), that is usually straight, larger in diameter, and
anchored at its ends. This facilitates the numerical implementation, as the steel reinforcement can lie along the finite
element’s edge, and the anchorage prevents the pull-out of the bar. One of the most important choices to make when dealing with the interaction of two different materials (steel
and concrete in this case), is the treatment of the interface between them. Many authors have opted for the insertion
of a special interface element, i.e. Ožbolt et al. (2002), Kohnehpooshi and Jaafar (2017), while others have opted for a
layered description of reinforced concrete, where each layer can represent a different material, i.e. Jukić et al. (2014)
or Šćulac at al. (2014). An example of the utilisation of a special interface element can be found in the work of
Dominguez et al. (2005), where a degenerated Q4 element is used to handle the relative displacements between the
steel and concrete. Despite the fact that such a description of slip can provide many benefits, it can be gruesome to Received: October 25, 2018. In Revised Form: October 30, 2018. Accepted: October 31, 2018. Available online: November 01, 2018
http://dx.doi.org/10.1590/1679-78255341 1/11 1/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete handle and implement. A simplification regarding the constitutive law implementation in this kind of Q4 element can
be found in Ibrahimbegovic et al. (2010), where a Drucker-Prager non-associative plasticity model is used, but still the
issue with introducing a special element persists. handle and implement. A simplification regarding the constitutive law implementation in this kind of Q4 element can
be found in Ibrahimbegovic et al. 2.1 Displacement field approximation We start by defining the bond-slip as the relative displacement between steel and concrete 𝜶𝑏𝑏𝒅𝑐𝒅𝑠 𝜶𝜶𝑏𝑏𝑏𝑏= 𝒅𝒅𝑐𝑐−𝒅𝒅𝑠𝑠 (1) 𝒅𝑠 where 𝜶𝜶𝑏𝑏𝑏𝑏 represents the slip on the interface along the reinforcement bar, 𝒅𝒅𝑐𝑐 is the concrete displacement, and 𝒅𝒅𝑠𝑠 is
the steel displacement. This kind of description allows us to write the displacement field of a single finite element as
sum of the standard and the enriched part, according to the X-FEM methodology (Fries and Belytschko, 2010) 𝒖𝒙𝑛𝑁𝒅𝑐𝑛𝑁𝜓𝜶𝑏𝑏 𝒖𝒖(𝒙𝒙)|𝛺𝛺𝑒𝑒= ∑
𝑖𝑖=1
𝑛𝑛
𝑁𝑁𝑖𝑖𝒅𝒅𝑖𝑖
𝑐𝑐+ ∑
𝑖𝑖=1
𝑛𝑛
𝑁𝑁𝑖𝑖𝜓𝜓𝜶𝜶𝑖𝑖
𝑏𝑏𝑏𝑏
(2) 𝑁𝑖𝜓𝛺 𝒖𝒖(𝒙𝒙)|𝛺𝛺𝑒𝑒= ∑
𝑖𝑖=1
𝑛𝑛
𝑁𝑁𝑖𝑖𝒅𝒅𝑖𝑖
𝑐𝑐+ ∑
𝑖𝑖=1
𝑛𝑛
𝑁𝑁𝑖𝑖𝜓𝜓𝜶𝜶𝑖𝑖
𝑏𝑏𝑏𝑏 (2) Equation (2) exploits the partition of unity property of standard shape functions 𝑁𝑁𝑖𝑖, that allows for any other
function (in this case the enrichment function 𝜓𝜓) to be reproduced exactly on the element domain 𝛺𝛺𝑒𝑒, (Melenk and
Babuška, 1996). The enrichment function 𝜓𝜓 is equal to the Heaviside function that takes the value one in the elements
that contain the reinforcement bar (that is located at coordinate 𝑦𝑦), and zero in all the other elements 𝜓𝐻𝑦𝑦𝑦 𝜓𝜓= 𝐻𝐻(𝑦𝑦) = ൜ 1, 𝑦𝑦= 𝑦𝑦
0, 𝑦𝑦≠𝑦𝑦
(3) 𝜓𝜓= 𝐻𝐻(𝑦𝑦) = ൜ 1, 𝑦𝑦= 𝑦𝑦
0, 𝑦𝑦≠𝑦𝑦
(3) 𝜓𝜓= 𝐻𝐻(𝑦𝑦) = ൜ 1, 𝑦𝑦= 𝑦𝑦
0, 𝑦𝑦≠𝑦𝑦 (3) Since in our case the fiber coincides with the lower edge of the enriched element, as shown on Figure 1, we can
simplify equation (2) by exploiting the properties of the enrichment function 𝒖𝒙𝑁𝒅𝑁𝛼 ∑
𝑖𝑖=1
3
𝑁𝑁𝑖𝑖𝒅𝒅𝑖𝑖
𝑐𝑐+ ∑
𝑖𝑖=1
2
𝑁𝑁𝑖𝑖𝛼𝛼𝑖𝑖
𝑏𝑏𝑏𝑏
Figure 1: Finite element with enriched degrees of freedom (the reinforcement bar is shown in bold). 𝒖𝒖(𝒙𝒙)|𝛺𝛺𝑒𝑒= ∑
𝑖𝑖=1
3
𝑁𝑁𝑖𝑖𝒅𝒅𝑖𝑖
𝑐𝑐+ ∑
𝑖𝑖=1
2
𝑁𝑁𝑖𝑖𝛼𝛼𝑖𝑖
𝑏𝑏𝑏𝑏 (4) Figure 1: Finite element with enriched degrees of freedom (the reinforcement bar is shown in bold). 2/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete The multi-scale characteristics of our model can be observed in the treatment of the failure mechanisms on two
levels, macro and micro. 2.1 Displacement field approximation The macro-scale computations are divided in two phases, according to the macro-scale
operator-split procedure: the macro-global phase (where the concrete and the steel are taken into account), and the
macro-local phase (where the redistribution of slip is computed, with the steel bar acting as a coupling term). But, that
is not all, since every constituent is taken care of within a micro-scale computation, which can have its own operator-split
solution procedure, as in the case of concrete and bond-slip, giving rise to micro-local and micro-global computations. All of this is important for ensuring the best possible description of the behavior of each constituent, with its own
peculiar phases related to specific failure mechanisms. Also, it is important to carry out all the computations in a
defined order, based on experimental observations on composite specimens. Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 2.2 Micro-scale computations 3/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete
Tea Rukavina et Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete Since the softening part of the response is controlled by an anisotropic multi-surface damage model, we have to
consider each direction at the discontinuity surface separately. This kind of model can account for the crack opening in
mode I (in the normal direction) and mode II (in the tangential direction). In the following equations the
subscripts 1 and 2 denote the direction, and 𝒏𝒏 and 𝒎𝒎 stand for the normal and tangential vector on the discontinuity,
respectively. The damage functions for the discontinuity are then 𝜙𝒕𝒏𝜎𝑞𝑐 𝜙𝜙ധ1 = 𝒕𝒕⋅𝒏𝒏−൫𝜎𝜎ധ𝑓𝑓−𝑞𝑞ധ𝑐𝑐൯
𝜙𝜙ധ2 = |𝒕𝒕⋅𝒎𝒎| −൬𝜎𝜎ധ𝑠𝑠−
𝜎𝜎ന𝑠𝑠
𝜎𝜎ന𝑓𝑓𝑞𝑞ധ𝑐𝑐൰
(8) 𝒕𝜎𝑓𝜎𝑠𝑞 𝜙𝜙ധ1 = 𝒕𝒕⋅𝒏𝒏−൫𝜎𝜎ധ𝑓𝑓−𝑞𝑞ധ𝑐𝑐൯
𝜙𝜙ധ2 = |𝒕𝒕⋅𝒎𝒎| −൬𝜎𝜎ധ𝑠𝑠−
𝜎𝜎ന𝑠𝑠
𝜎𝜎ന𝑓𝑓𝑞𝑞ധ𝑐𝑐൰ 𝒕 (8) where 𝒕𝒕 is the traction at the discontinuity, 𝜎𝜎ധ𝑓𝑓 is the ultimate stress in tension, and 𝜎𝜎ധ𝑠𝑠 is the ultimate stress in shear. The stress-like softening variable 𝑞𝑞ധ𝑐𝑐 is the same for both directions, and it depends on the chosen softening law, i.e. linear or exponential softening. 𝒕𝜶𝑐 where 𝒕𝒕 is the traction at the discontinuity, 𝜎𝜎ധ𝑓𝑓 is the ultimate stress in tension, and 𝜎𝜎ധ𝑠𝑠 is the ultimate stress in shear. The stress-like softening variable 𝑞𝑞ധ𝑐𝑐 is the same for both directions, and it depends on the chosen softening law, i.e. linear or exponential softening. 𝒕𝜶𝑐 The constitutive equation relating the traction at the discontinuity 𝒕𝒕 and the crack opening 𝜶𝜶𝑐𝑐 is based on a
traction-separation cohesive law 𝒕𝐐𝜶𝑐 (9) 𝒕𝒕= 𝐐𝐐ന−1𝜶𝜶𝑐𝑐 (9) where 𝐐𝐐ന is the damage compliance tensor for the discontinuity. The handling of the displacement jump inside the
element is done through the introduction of incompatible mode functions 𝑴𝑴. The total displacement field of a single
element is then a sum of the standard and the incompatible part 𝒖𝑵𝒅𝑐𝑴𝜶𝑐 (10) 𝒖𝒖= 𝑵𝑵𝒅𝒅𝑐𝑐+ 𝑴𝑴𝜶𝜶𝑐𝑐 from which it follows that the strain can be written as 𝜺𝑩𝒅𝑐𝐆𝜶𝑐 (11) 𝜺𝜺= 𝑩𝑩𝒅𝒅𝑐𝑐+ 𝐆𝐆෩𝜶𝜶𝑐𝑐 where 𝑩𝑩 is the matrix of the shape functions’ derivatives, and 𝑮𝑮 consists of the regular part of the incompatible shape
functions’ derivatives. where 𝑩𝑩 is the matrix of the shape functions’ derivatives, and 𝑮𝑮 consists of the regular part of the incompatible shape
functions’ derivatives. 2.2 Micro-scale computations To be able to couple the three constituents, each one of them has to provide the stress value that is computed
from its own constitutive law and kinematics (Figure 2). Steel computation. Since the steel reinforcement is taken to be linear elastic, its constitutive 𝜎𝑠𝐸𝑠𝜀𝑠 omputation. Since the steel reinforcement is taken to be linear elastic, its constitutive equation is 𝐸𝜀𝑠 (5) (5) 𝜎𝜎𝑠𝑠= 𝐸𝐸𝑠𝑠𝜀𝜀𝑠𝑠 n (5), 𝜎𝜎𝑠𝑠 is the stress, 𝐸𝐸𝑠𝑠 is the Young’s modulus for steel, and 𝜀𝜀𝑠𝑠 is the strain that is defined as 𝑑𝑠 In equation (5), 𝜎𝜎𝑠𝑠 is the stress, 𝐸𝐸𝑠𝑠 is the Young’s modulus for steel, and 𝜀𝜀𝑠𝑠 is the strain that is defined as 𝜀𝑠𝑁𝑑𝑠 In equation (5), 𝜎𝜎𝑠𝑠 is the stress, 𝐸𝐸𝑠𝑠 is the Young’s modulus for steel, and 𝜀𝜀𝑠𝑠 is the strain that is defined as 𝜀𝑠𝑁𝑑𝑠 (6) where the steel displacements are computed according to (1). Figure 2: Constitutive law for each material: (a) damage model for concrete; (b) linear elasticity for steel; (c) elastoplasticity for
bond-slip. Figure 2: Constitutive law for each material: (a) damage model for concrete; (b) linear elasticity for steel; (c) elastoplasticity for
bond-slip. Concrete computation. The chosen material model for concrete is the damage model with isotropic hardening,
that can take into account the formation of micro-cracks in the bulk, combined with a localized failure part that
describes the opening of the macro-crack at the discontinuity. We will give here just a short description of the model,
since all the details regarding the formulation and implementation can be found in the work of Brancherie and
Ibrahimbegovic (2009), or Do et al. (2017) for applications in dynamics. 𝜙 The damage functions 𝜙𝜙 are used to check the admissibility of the stress state, and for the hardening part, this
function is defined as 𝜙𝑐𝝈𝑐𝜎𝑞𝑐 𝜙𝜙ത𝑐𝑐= ||𝝈𝝈𝑐𝑐||𝑫𝑫𝑒𝑒−
1
√𝐸𝐸𝑐𝑐(𝜎𝜎𝑓𝑓−𝑞𝑞ത𝑐𝑐) ≤0 𝑫𝑒𝑫𝑪 (7) 𝐸 (7) where 𝑫𝑫𝑒𝑒 is the undamaged elastic compliance tensor for the bulk material. It is computed as the inverse of the elastic
constitutive matrix: 𝑫𝑫𝑒𝑒= (𝑪𝑪𝑒𝑒)−1. In the above equation, ||𝝈𝝈𝑐𝑐||𝑫𝑫𝑒𝑒 represents the norm of 𝝈𝝈𝑐𝑐in the stress space, 𝐸𝐸𝑐𝑐 is
Young’s modulus for concrete, 𝜎𝜎𝑓𝑓 is the stress at the first cracking, and 𝑞𝑞ത𝑐𝑐 is the stress-like hardening variable that
controls the damage threshold evolution. 2.2 Micro-scale computations The final value of stress in concrete can then be computed from 𝝈𝑐𝑪𝑒𝑒𝜺𝑐 (12) 𝝈𝝈𝑐𝑐= 𝑪𝑪𝑒𝑒𝑒𝑒𝜺𝜺𝑐𝑐 and the value of the elasto-damage modulus 𝑪𝑪𝑒𝑒𝑒𝑒 has to be chosen according to the current phase of the computation,
which is determined from the damage functions described in (7-8). 𝜎𝑏𝑏 and the value of the elasto-damage modulus 𝑪𝑪𝑒𝑒𝑒𝑒 has to be chosen according to the current phase of the computation,
which is determined from the damage functions described in (7-8). 𝜎𝑏𝑏 Bond-slip computation. The main novelty in this work is the description of bond-slip through a one-dimensional
elasto-plastic computation. Since the bond-slip law is given as a relationship between the bond stress 𝜎𝜎𝑏𝑏𝑏𝑏 and the slip
𝛼𝛼𝑏𝑏𝑏𝑏, instead of the plastic strain 𝜀𝜀𝑝𝑝 that is usually employed in this kind of computations, we will have the plastic slip
𝛼𝛼𝑏𝑏𝑏𝑏,𝑝𝑝. The computation is carried out within an operator-split solution procedure (see Ibrahimbegovic, 2009) where the
evolution equations of internal variables are solved in the local phase, on the level of the Gauss numerical integration
point of each element, and the equilibrium equations are solved globally, on the level of the whole structure. We start
by an elastic trial step, in which the plastic multiplier 𝛾𝛾𝑏𝑏𝑏𝑏 is equal to zero, and the values of all internal variables are
frozen (they take the value from the previous time step) 𝛼𝑏𝑏𝑝𝑡𝑡𝑡𝑡𝑡𝛼𝑏𝑏𝑝 𝛼𝛼𝑛𝑛+1
𝑏𝑏𝑏𝑏,𝑝𝑝,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡= 𝛼𝛼𝑛𝑛
𝑏𝑏𝑏𝑏,𝑝𝑝
𝜉𝜉𝑛𝑛+1
𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡= 𝜉𝜉𝑛𝑛
𝑏𝑏𝑏𝑏
𝑞𝑞𝑛𝑛+1
𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡= 𝑞𝑞𝑛𝑛
𝑏𝑏𝑏𝑏= −𝐾𝐾𝑏𝑏𝑏𝑏,ℎ𝜉𝜉𝑛𝑛
𝑏𝑏𝑏𝑏 (13) 𝑞𝑞𝑛𝑛+1
𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡= 𝑞𝑞𝑛𝑛
𝑏𝑏𝑏𝑏= −𝐾𝐾𝑏𝑏𝑏𝑏,ℎ𝜉𝜉𝑛𝑛
𝑏𝑏𝑏𝑏 (13) 4/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete 𝜉𝑞 Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete 𝜉𝑞 Tea Rukavina et al. In the above equations, 𝜉𝜉𝑏𝑏𝑏𝑏 represents the hardening variable, 𝑞𝑞𝑏𝑏𝑏𝑏 is the stress-like hardening variable for bond-slip,
and 𝐾𝐾𝑏𝑏𝑏𝑏,ℎ is the hardening modulus. The trial value of bond stress is then computed as 𝜎𝑏𝑏𝑡𝑡𝑡𝑡𝑡𝐾𝑏𝑏𝛼𝑏𝑏𝛼𝑏𝑏𝑝 𝜎𝜎𝑛𝑛+1
𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡= 𝐾𝐾𝑏𝑏𝑏𝑏(𝛼𝛼𝑛𝑛+1
𝑏𝑏𝑏𝑏
−𝛼𝛼𝑛𝑛
𝑏𝑏𝑏𝑏,𝑝𝑝) (14) where 𝐾𝐾𝑏𝑏𝑏𝑏 is the bond-slip tangent modulus. The term in the brackets represents the elastic slip 𝛼𝛼𝑏𝑏𝑏𝑏,𝑒𝑒𝑒𝑒, since the total
slip can be represented as the sum of the elastic and the plastic part: 𝛼𝛼𝑏𝑏𝑏𝑏= 𝛼𝛼𝑏𝑏𝑏𝑏,𝑒𝑒𝑒𝑒+ 𝛼𝛼𝑏𝑏𝑏𝑏,𝑝𝑝. The final value of stress is then 𝜎𝑏𝑏𝜎𝑏𝑏𝑡𝑡𝑡𝑡𝑡𝐾𝑏𝑏𝛾𝑏𝑏𝜎𝑏𝑏𝑡𝑡𝑡𝑡𝑡 𝜎𝜎𝑛𝑛+1
𝑏𝑏𝑏𝑏
= 𝜎𝜎𝑛𝑛+1
𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡−𝐾𝐾𝑏𝑏𝑏𝑏𝛾𝛾𝑛𝑛+1
𝑏𝑏𝑏𝑏sign(𝜎𝜎𝑛𝑛+1
𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡)
(20) 𝐾𝑏𝑏 𝜎𝜎𝑛𝑛+1
𝑏𝑏𝑏𝑏
= 𝜎𝜎𝑛𝑛+1
𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡−𝐾𝐾𝑏𝑏𝑏𝑏𝛾𝛾𝑛𝑛+1
𝑏𝑏𝑏𝑏sign(𝜎𝜎𝑛𝑛+1
𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡) (20) The elastoplastic tangent modulus in the elastic phase is equal to 𝐾𝐾𝑏𝑏𝑏𝑏, and in the plastic phase, it has the value 𝐶𝑒𝑒𝑏𝑏𝐾𝑏𝑏𝐾𝑏𝑏 The elastoplastic tangent modulus in the elastic phase is equal to 𝐾𝐾𝑏𝑏𝑏𝑏, and in the plastic phase, it ha 𝐶𝐶𝑛𝑛+1
𝑒𝑒𝑒𝑒,𝑏𝑏𝑏𝑏= 𝐾𝐾𝑏𝑏𝑏𝑏𝐾𝐾𝑏𝑏𝑏𝑏,ℎ
𝐾𝐾𝑏𝑏𝑏𝑏+𝐾𝐾𝑏𝑏𝑏𝑏,ℎ
(21)
2.3 Macro-scale computations𝑑 𝐶𝐶𝑛𝑛+1
𝑒𝑒𝑒𝑒,𝑏𝑏𝑏𝑏= 𝐾𝐾𝑏𝑏𝑏𝑏𝐾𝐾𝑏𝑏𝑏𝑏,ℎ
𝐾𝐾𝑏𝑏𝑏𝑏+𝐾𝐾𝑏𝑏𝑏𝑏,ℎ (21) 2.2 Micro-scale computations The total slip is computed
from the nodal values of slip, 𝛼𝛼𝑖𝑖
𝑏𝑏𝑏𝑏, which are obtained from the local iteration of the macro-level computation 𝛼𝑏𝑏𝑁𝑏𝑏𝛼𝑏𝑏 𝛼𝛼𝑛𝑛+1
𝑏𝑏𝑏𝑏
= ∑
𝑖𝑖=1
2
𝑁𝑁𝑖𝑖
𝑏𝑏𝑏𝑏𝛼𝛼𝑖𝑖
𝑏𝑏𝑏𝑏 (15) The bond-slip shape functions 𝑁𝑁𝑖𝑖
𝑏𝑏𝑏𝑏 are actually the product of the standard shape functions for concrete, and the
X-FEM enrichment function 𝜓𝜓, as given in equation (2). For our case it follows 𝑁𝑏𝑏𝑥𝑁𝒙𝜓𝒙𝑁𝑥 𝑁𝑁𝑖𝑖
𝑏𝑏𝑏𝑏(𝑥𝑥) = 𝑁𝑁𝑖𝑖(𝒙𝒙)𝜓𝜓(𝒙𝒙) = 𝑁𝑁𝑖𝑖(𝑥𝑥) (16) since the enrichment function is a Heaviside function. Here, 𝑁𝑁𝑖𝑖(𝑥𝑥) are the linear shape functions for a truss bar. To check if the trial value of stress is admissible, we introduce the yield function 𝜙𝑏𝑏𝑡𝑡𝑡𝑡𝑡𝜎𝑏𝑏𝑡𝑡𝑡𝑡𝑡𝜏𝑞𝑏𝑏𝑡𝑡𝑡𝑡𝑡 since the enrichment function is a Heaviside function. Here, 𝑁𝑁𝑖𝑖(𝑥𝑥) are the linear shape functions for a truss bar. To check if the trial value of stress is admissible, we introduce the yield function 𝜙𝑏𝑏𝑡𝑡𝑡𝑡𝑡𝜎𝑏𝑏𝑡𝑡𝑡𝑡𝑡𝜏𝑞𝑏𝑏𝑡𝑡𝑡𝑡𝑡 𝜙𝜙𝑛𝑛+1
𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡= |𝜎𝜎𝑛𝑛+1
𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡| −(𝜏𝜏𝑦𝑦−𝑞𝑞𝑛𝑛+1
𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡) 𝜏𝑦 (17) where 𝜏𝜏𝑦𝑦 is the limit value of the bond stress. We have two cases to consider: if the trial value of the yield function is
negative or zero, the step is indeed elastic and the trial values are accepted as final. If not, we have to proceed to the
plastic step to correct the value of bond stress due to the plastic slip activation. where 𝜏𝜏𝑦𝑦 is the limit value of the bond stress. We have two cases to consider: if the trial value of the yield function is
negative or zero, the step is indeed elastic and the trial values are accepted as final. If not, we have to proceed to the
plastic step to correct the value of bond stress due to the plastic slip activation. p, we have to compute a new value for the plastic multiplier In the plastic step, we have to compute a new value for the plastic multiplier 𝛾𝑏𝑏𝜙𝑏𝑏𝑡𝑡𝑡𝑡𝑡 𝛾𝛾𝑛𝑛+1
𝑏𝑏𝑏𝑏
=
𝜙𝜙𝑛𝑛+1
𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡
𝐾𝐾𝑏𝑏𝑏𝑏+𝐾𝐾𝑏𝑏𝑏𝑏,ℎ
(18)
which is used to update the internal variables according to𝛼𝛼𝛾𝜎 𝛾𝛾𝑛𝑛+1
𝑏𝑏𝑏𝑏
=
𝜙𝜙𝑛𝑛+1
𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡
𝐾𝐾𝑏𝑏𝑏𝑏+𝐾𝐾𝑏𝑏𝑏𝑏,ℎ
(18) 𝛾𝛾𝑛𝑛+1
𝑏𝑏𝑏𝑏
=
𝜙𝜙𝑛𝑛+1
𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡
𝐾𝐾𝑏𝑏𝑏𝑏+𝐾𝐾𝑏𝑏𝑏𝑏,ℎ (18) which is used to update the internal variables according to 𝛼𝑏𝑏𝑝𝛼𝑏𝑏𝑝𝛾𝑏𝑏𝜎𝑏𝑏𝑡𝑡𝑡𝑡𝑡 which is used to update the internal variables according to 𝛼𝑏𝑏𝑝𝛼𝑏𝑏𝑝𝛾𝑏𝑏𝜎𝑏𝑏𝑡𝑡𝑡𝑡𝑡 𝛼𝛼𝑛𝑛+1
𝑏𝑏𝑏𝑏,𝑝𝑝= 𝛼𝛼𝑛𝑛
𝑏𝑏𝑏𝑏,𝑝𝑝+ 𝛾𝛾𝑛𝑛+1
𝑏𝑏𝑏𝑏sign(𝜎𝜎𝑛𝑛+1
𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡) 𝜉𝑏𝑏𝜉𝑏𝑏𝛾𝑏𝑏 𝜉𝜉𝑛𝑛+1
𝑏𝑏𝑏𝑏
= 𝜉𝜉𝑛𝑛
𝑏𝑏𝑏𝑏+ 𝛾𝛾𝑛𝑛+1
𝑏𝑏𝑏𝑏 (19) The final value of stress is then 𝜎𝑏𝑏𝜎𝑏𝑏𝑡𝑡𝑡𝑡𝑡𝐾𝑏𝑏𝛾𝑏𝑏𝜎𝑏𝑏𝑡𝑡𝑡𝑡𝑡 2.3 Macro-scale computations By inserting the approximation of the virtual displacement field into the weak form of the equilibrium equations,
and after gathering the terms related to the concrete displacements 𝑑𝑑𝑐𝑐, and the ones related to the bond-slip
displacements 𝛼𝛼𝑏𝑏𝑏𝑏, we are left with two systems of equations 𝒓𝑐𝑐𝔸𝑛𝒇𝑐𝑖𝑖𝑖𝑒𝔸𝑛𝑛𝑛𝒇𝑠𝑖𝑖𝑖𝑒𝒇𝑒𝑒𝑒𝟎 𝒓𝒓𝑐𝑐𝑐𝑐= 𝔸𝔸
𝑛𝑛
𝑒𝑒=1𝒇𝒇𝑐𝑐,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒+ 𝔸𝔸
𝑛𝑛𝑛𝑛𝑛𝑛
𝑒𝑒=1𝒇𝒇𝑠𝑠,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒−𝒇𝒇𝑒𝑒𝑒𝑒𝑒𝑒= 𝟎𝟎 5/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 materials: application to reinforced
Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete
Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete Tea Rukavina et al. 𝒓𝒓𝑠𝑠𝑠𝑠𝑠𝑠= 𝔸𝔸
𝑛𝑛𝑛𝑛𝑛𝑛
𝑒𝑒=1 ቀ𝒇𝒇𝑠𝑠,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒+ 𝒇𝒇𝑏𝑏𝑏𝑏,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒ቁ= 𝟎𝟎 𝑛 (22) Here, 𝑛𝑛 denotes the number of elements, and 𝑛𝑛𝑛𝑛𝑛𝑛 the number of enriched elements (the ones containing the
steel bar). The global equation (22a) is solved with a fixed slip, and in the local one (22b) the redistribution of slip is
computed. The external force vector and the internal force vectors for concrete, steel, and bond-slip, are defined in the
following manner 𝒇𝑐𝑖𝑖𝑖𝑒𝑩𝑇𝝈𝑐𝑑𝑑 𝒇𝒇𝑐𝑐,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒= ∫𝛺𝛺𝑒𝑒𝑩𝑩𝑇𝑇𝝈𝝈𝑐𝑐𝑑𝑑𝑑𝑑
𝒇𝒇𝑠𝑠,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒= ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝜎𝜎𝑠𝑠𝐴𝐴𝑠𝑠𝑑𝑑𝑑𝑑
𝒇𝒇𝑏𝑏𝑏𝑏,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒= ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝜎𝜎𝑏𝑏𝑏𝑏𝐴𝐴𝑏𝑏𝑏𝑏𝑑𝑑𝑑𝑑
𝒇𝒇𝑒𝑒𝑒𝑒𝑒𝑒= ∫𝛺𝛺𝑒𝑒𝑵𝑵𝑵𝑵𝑑𝑑𝛺𝛺𝑒𝑒+ ∫𝛤𝛤𝜎𝜎𝑵𝑵𝑵𝑵𝑑𝑑𝛤𝛤𝜎𝜎
(23) 𝑩𝑠𝐴𝑠𝐴 𝒇𝒇𝑐𝑐,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒= ∫𝛺𝛺𝑒𝑒𝑩𝑩𝑇𝑇𝝈𝝈𝑐𝑐𝑑𝑑𝑑𝑑
𝒇𝒇𝑠𝑠,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒= ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝜎𝜎𝑠𝑠𝐴𝐴𝑠𝑠𝑑𝑑𝑑𝑑
𝒇𝒇𝑏𝑏𝑏𝑏,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒= ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝜎𝜎𝑏𝑏𝑏𝑏𝐴𝐴𝑏𝑏𝑏𝑏𝑑𝑑𝑑𝑑
𝒇𝒇𝑒𝑒𝑒𝑒𝑒𝑒= ∫𝛺𝛺𝑒𝑒𝑵𝑵𝑵𝑵𝑑𝑑𝛺𝛺𝑒𝑒+ ∫𝛤𝛤𝜎𝜎𝑵𝑵𝑵𝑵𝑑𝑑𝛤𝛤𝜎𝜎 𝑩𝑠𝐴 𝒇𝒇𝑏𝑏𝑏𝑏,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒= ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝜎𝜎𝑏𝑏𝑏𝑏𝐴𝐴𝑏𝑏𝑏𝑏𝑑𝑑𝑑𝑑 𝒇𝑒𝑒𝑒𝑵𝑵𝑑𝛺𝑒𝑵𝑵𝑑𝛤 (23) In equation (23), 𝑩𝑩𝑠𝑠 is the matrix of derivatives of the shape functions for the steel bar, 𝐴𝐴𝑠𝑠 is the cross-sectional area of
the bar, and 𝐴𝐴𝑏𝑏𝑏𝑏 is the bond-slip area that is computed as the circumference of the bar (it is the part of the steel bar in
contact with concrete). In the external force vector, 𝒃𝒃 represents the volumetric forces, and 𝒕𝒕 is the imposed traction
acting at the boundary 𝛤𝛤𝜎𝜎. 3 NUMERICAL RESULTS The proposed methodology was implemented in FEAP (Finite Element Analysis Program), developed at Berkeley
by professor R. L. Taylor (2014). All the described constituents are implemented into a single finite element that allows
for an efficient computation of their interaction. 𝜙 To test the performance of the developed formulation, we have performed a tension test on a concrete specimen
of dimensions 400x100x100 mm3, reinforced by a steel bar of diameter 𝜙𝜙=16 mm. Since our model is 2D (as shown of
Figure 3.), the area of the bar and the bond-slip area are divided by the thickness of the specimen. Figure 3: Geometry of the reinforced concrete specimen in 2D. Figure 3: Geometry of the reinforced concrete specimen in 2D. Figure 3: Geometry of the reinforced concrete specimen in 2D. We consider the steel bar to be fixed at both ends, as it is anchored in concrete. The material properties used in
the model are listed here: 𝐸𝑐𝜈𝑐𝜎𝑓𝐾𝜎𝑓 • for concrete: 𝐸𝐸𝑐𝑐= 45700MPa, 𝜈𝜈𝑐𝑐= 0.2, 𝜎𝜎ത𝑓𝑓= 3.5MPa, 𝐾𝐾ℎ= 1000MPa, 𝜎𝜎ധ𝑓𝑓= 4MPa, 𝜎𝑓𝑤𝑤𝑤𝑤𝛽𝑐 • for concrete: 𝐸𝐸𝑐𝑐= 45700MPa, 𝜈𝜈𝑐𝑐= 0.2, 𝜎𝜎ത𝑓𝑓= 3.5MPa, 𝐾𝐾ℎ= 1000MPa, 𝜎𝜎ധ𝑓𝑓= 4MPa, 𝜎𝑓𝑤𝑤𝑤𝑤𝛽𝑐 • for concrete: 𝐸𝐸𝑐𝑐= 45700MPa, 𝜈𝜈𝑐𝑐= 0.2, 𝜎𝜎ത𝑓𝑓= 3.5MPa, 𝐾𝐾ℎ= 1000MPa, 𝜎𝜎ധ𝑓𝑓= 4MPa, 𝜎𝜎ധ𝑓𝑓,𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤= 3.8MPa, 𝛽𝛽𝑐𝑐= 20; 𝐸𝑠 • for the steel bar: 𝐸𝐸𝑠𝑠= 210000MPa, 𝐴𝐴𝑠𝑠= 2.01mm2;
• for the bond: 𝐾𝐾𝑏𝑏𝑏𝑏= 30N/mm3, 𝜏𝜏𝑦𝑦= 6MPa, 𝐾𝐾𝑏𝑏𝑏𝑏,ℎ= 0.03N/mm3, 𝐴𝐴𝑏𝑏𝑏𝑏= 0.5mm. 𝜈𝑐𝛽𝑐𝜎 • for the steel bar: 𝐸𝐸𝑠𝑠= 210000MPa, 𝐴𝐴𝑠𝑠= 2.01mm2;
• for the bond: 𝐾𝐾𝑏𝑏𝑏𝑏= 30N/mm3, 𝜏𝜏𝑦𝑦= 6MPa, 𝐾𝐾𝑏𝑏𝑏𝑏,ℎ= 0.03N/mm3, 𝐴𝐴𝑏𝑏𝑏𝑏= 0.5mm. 𝜈𝑐𝛽𝑐𝜎 Among the properties listed above, 𝜈𝜈𝑐𝑐 is the Poisson’s ratio for concrete, and 𝛽𝛽𝑐𝑐 is the parameter that controls the
exponential softening. 𝜎𝜎ധ𝑓𝑓,𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤 is the ultimate stress for a strip of weakened elements in the middle of the specimen
where the crack will appear. Figure 4: Finite element mesh with enriched elements shown in grey. 𝑢 Figure 4: Finite element mesh with enriched elements shown in grey. 𝑢 The specimen is fixed at the left-hand side, and there is an imposed displacement 𝑢𝑢ത= 0.5 mm acting on the right-hand
side. The finite element mesh shown on Figure 4 consists of 800 CST elements, where the triangle sides of each
element have the dimension 10 mm. There are 40 enriched elements along the reinforcement bar. 𝑥 The specimen is fixed at the left-hand side, and there is an imposed displacement 𝑢𝑢ത= 0.5 mm acting on the right-hand
side. 2.3 Macro-scale computations By linearizing (22), we obtain the following system of equations 𝑲𝑐𝑐𝑭𝑐𝑐𝛥𝒅𝑐𝒓𝑐𝑐 ቂ𝑲𝑲𝑐𝑐𝑐𝑐
𝑭𝑭𝑐𝑐𝑐𝑐
𝑭𝑭𝑠𝑠𝑠𝑠𝑠𝑠
𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠ቃቂ𝛥𝛥𝒅𝒅𝑐𝑐
𝛥𝛥𝜶𝜶𝑏𝑏𝑏𝑏ቃ= −ቂ𝒓𝒓𝑐𝑐𝑐𝑐
𝒓𝒓𝑠𝑠𝑠𝑠𝑠𝑠ቃ
(26) ቂ𝑲𝑲𝑐𝑐𝑐𝑐
𝑭𝑭𝑐𝑐𝑐𝑐
𝑭𝑭𝑠𝑠𝑠𝑠𝑠𝑠
𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠ቃቂ𝛥𝛥𝒅𝒅𝑐𝑐
𝛥𝛥𝜶𝜶𝑏𝑏𝑏𝑏ቃ= −ቂ𝒓𝒓𝑐𝑐𝑐𝑐
𝒓𝒓𝑠𝑠𝑠𝑠𝑠𝑠ቃ ቂ𝑲𝑲𝑐𝑐𝑐𝑐
𝑭𝑭𝑐𝑐𝑐𝑐
𝑭𝑭𝑠𝑠𝑠𝑠𝑠𝑠
𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠ቃቂ𝛥𝛥𝒅𝒅𝑐𝑐
𝛥𝛥𝜶𝜶𝑏𝑏𝑏𝑏ቃ= −ቂ𝒓𝒓𝑐𝑐𝑐𝑐
𝒓𝒓𝑠𝑠𝑠𝑠𝑠𝑠ቃ
(26) ቂ𝑲𝑲𝑐𝑐𝑐𝑐
𝑭𝑭𝑐𝑐𝑐𝑐
𝑭𝑭𝑠𝑠𝑠𝑠𝑠𝑠
𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠ቃቂ𝛥𝛥𝒅𝒅𝑐𝑐
𝛥𝛥𝜶𝜶𝑏𝑏𝑏𝑏ቃ= −ቂ𝒓𝒓𝑐𝑐𝑐𝑐
𝒓𝒓𝑠𝑠𝑠𝑠𝑠𝑠ቃ (26) where the tangent stiffness matrices are defined as 𝑲𝑐𝑐𝑩𝑐𝑇𝑪𝑒𝑒𝑩𝑐𝑑𝑑𝑩𝑠𝑇𝐸𝑠𝐴𝑠𝑩𝑠𝑑𝑑 where the tangent stiffness matrices are defined as 𝑲𝑐𝑐𝑩𝑐𝑇𝑪𝑒𝑒𝑩𝑐𝑑𝑑𝑩𝑠𝑇𝐸𝑠𝐴𝑠𝑩𝑠𝑑𝑑 𝑲𝑲𝑐𝑐𝑐𝑐= ∫𝛺𝛺𝑒𝑒𝑩𝑩𝑐𝑐,𝑇𝑇𝑪𝑪𝑒𝑒𝑒𝑒𝑩𝑩𝑐𝑐𝑑𝑑𝑑𝑑+ ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐸𝐸𝑠𝑠𝐴𝐴𝑠𝑠𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑
𝑭𝑭𝑐𝑐𝑐𝑐= 𝑭𝑭𝑠𝑠𝑠𝑠𝑠𝑠= ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐸𝐸𝑠𝑠𝐴𝐴𝑠𝑠𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑
𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠= ∫𝛤𝛤𝑠𝑠
𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐸𝐸𝑠𝑠𝐴𝐴𝑠𝑠𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑+ ∫𝛤𝛤𝑠𝑠
𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐾𝐾𝑏𝑏𝑏𝑏𝐴𝐴𝑏𝑏𝑏𝑏𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑
(25) 𝑲𝑲𝑐𝑐𝑐𝑐= ∫𝛺𝛺𝑒𝑒𝑩𝑩𝑐𝑐,𝑇𝑇𝑪𝑪𝑒𝑒𝑒𝑒𝑩𝑩𝑐𝑐𝑑𝑑𝑑𝑑+ ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐸𝐸𝑠𝑠𝐴𝐴𝑠𝑠𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑
𝑭𝑭𝑐𝑐𝑐𝑐= 𝑭𝑭𝑠𝑠𝑠𝑠𝑠𝑠= ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐸𝐸𝑠𝑠𝐴𝐴𝑠𝑠𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑 𝑯𝑠𝑠𝑠𝑩𝑠𝑇𝐸𝑠𝐴𝑠𝑩𝑠𝑑𝑑𝑩𝑠𝑇𝐾𝑏𝑏𝐴𝑏𝑏𝑩𝑠𝑑𝑑 𝑲𝑲𝑐𝑐𝑐𝑐= ∫𝛺𝛺𝑒𝑒𝑩𝑩𝑐𝑐,𝑇𝑇𝑪𝑪𝑒𝑒𝑒𝑒𝑩𝑩𝑐𝑐𝑑𝑑𝑑𝑑+ ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐸𝐸𝑠𝑠𝐴𝐴𝑠𝑠𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑 𝑭𝑐𝑐𝑭𝑠𝑠𝑠𝛤𝑩𝑠𝑇𝐸𝑠𝐴𝑠𝑩𝑠𝑑𝑑 𝑭𝑭𝑐𝑐𝑐𝑐= 𝑭𝑭𝑠𝑠𝑠𝑠𝑠𝑠= ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐸𝐸𝑠𝑠𝐴𝐴𝑠𝑠𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑 𝑯𝑠𝑠𝑠𝑩𝑠𝑇𝐸𝑠𝐴𝑠𝑩𝑠𝑑𝑑𝑩𝑠𝑇𝐾 𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠= ∫𝛤𝛤𝑠𝑠
𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐸𝐸𝑠𝑠𝐴𝐴𝑠𝑠𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑+ ∫𝛤𝛤𝑠𝑠
𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐾𝐾𝑏𝑏𝑏𝑏𝐴𝐴𝑏𝑏𝑏𝑏𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑 𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠= ∫𝛤𝛤𝑠𝑠
𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐸𝐸𝑠𝑠𝐴𝐴𝑠𝑠𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑+ ∫𝛤𝛤𝑠𝑠
𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐾𝐾𝑏𝑏𝑏𝑏𝐴𝐴𝑏𝑏𝑏𝑏𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑
(25) (25) The system (24) is handled within the macro-scale operator-split solution procedure, that gives us two equations to
solve sequentially 𝑲𝛥𝒅𝑐𝒓𝑐𝑐 𝑲𝑲 𝛥𝛥𝒅𝒅𝑐𝑐= −𝒓𝒓𝑐𝑐𝑐𝑐
𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠𝛥𝛥𝜶𝜶𝑏𝑏𝑏𝑏= −𝒓𝒓𝑠𝑠𝑠𝑠𝑠𝑠
(26) 𝑲 𝑲𝑲 𝛥𝛥𝒅𝒅𝑐𝑐= −𝒓𝒓𝑐𝑐𝑐𝑐
𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠𝛥𝛥𝜶𝜶𝑏𝑏𝑏𝑏= −𝒓𝒓𝑠𝑠𝑠𝑠𝑠𝑠
(26) 𝑲 𝑲𝑲 𝛥𝛥𝒅𝒅𝑐𝑐= −𝒓𝒓𝑐𝑐𝑐𝑐
𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠𝛥𝛥𝜶𝜶𝑏𝑏𝑏𝑏= −𝒓𝒓𝑠𝑠𝑠𝑠𝑠𝑠 𝑲 𝑲𝑲 𝛥𝛥𝒅𝒅𝑐𝑐= −𝒓𝒓𝑐𝑐𝑐𝑐 𝑯𝑠𝑠𝑠𝛥𝜶𝑏𝑏𝒓𝑠𝑠𝑠 𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠𝛥𝛥𝜶𝜶𝑏𝑏𝑏𝑏= −𝒓𝒓𝑠𝑠𝑠𝑠𝑠𝑠 𝑲 (26) where 𝑲𝑲 is the condensed stiffness matrix 𝑲𝑲𝑐𝑐𝑭𝑐𝑐𝑯𝑠𝑠𝑠𝑭𝑠𝑠𝑠 where 𝑲𝑲 is the condensed stiffness matrix 𝑲𝑲𝑐𝑐𝑭𝑐𝑐𝑯𝑠𝑠𝑠𝑭𝑠𝑠𝑠 where 𝑲𝑲 is the condensed stiffness matrix 𝑲𝑲𝑐𝑐𝑭𝑐𝑐𝑯𝑠𝑠𝑠𝑭𝑠𝑠𝑠 𝑲𝑲= 𝑲𝑲𝑐𝑐𝑐𝑐−𝑭𝑭𝑐𝑐𝑐𝑐(𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠)−1𝑭𝑭𝑠𝑠𝑠𝑠𝑠𝑠 𝒅 (27) Equation (26a) is the macro-global equation that gives us the value of the concrete displacement for the fixed
value of slip. With the value of 𝒅𝒅𝑐𝑐 in hand, we proceed to the macro-local equation (26b), where we update the value
of the slip 𝜶𝜶𝑏𝑏𝑏𝑏. 6/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Tea Rukavina et al. Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete
Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
oncrete Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Tea Rukavina et al. 3 NUMERICAL RESULTS The finite element mesh shown on Figure 4 consists of 800 CST elements, where the triangle sides of each
element have the dimension 10 mm. There are 40 enriched elements along the reinforcement bar. 𝑥 The force-displacement diagram shown on Figure 5. plots the reaction in the 𝑥𝑥-direction at the left-hand side
against the imposed displacement on the right-hand side. We can observe several particular phases of the composite
behavior: the linear elastic phase is followed by a hardening phase when micro-cracks start to appear in concrete. After
the ultimate load of about 500 N is reached, the crack develops in the weak zone in the middle of the specimen, so we
enter the softening phase. After a while, the reaction starts ascending, due to the redistribution of stresses and the
reinforcement activation. This resembles the typical diagrams that are found in the literature, which are obtained for a
specimen with a single crack. 7/11 7/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete Figure 5: Force-displacement diagram for the tension test. 𝛼 Figure 5: Force-displacement diagram for the tension test. 𝛼 On Figure 6. we can see the distribution of slip along the reinforcement bar, where the largest slips are taking
place near the crack, and at the ends the slip is zero. On the left of the crack, 𝛼𝛼𝑏𝑏𝑏𝑏 has a positive value, and on the left
side it is negative, because the reinforcement is moving (relatively to the concrete) from the ends to the centre (in
other words, towards the crack). Figure 6: Distribution of slip 𝛼𝛼𝑏𝑏𝑏𝑏 along the reinforcement bar at the end of the analysis (nodal values). Figure 6: Distribution of slip 𝛼𝛼𝑏𝑏𝑏𝑏 along the reinforcement bar at the end of the analysis (nodal values). The bond stress has a very similar distribution (Figure 7a), according to the chosen bond-slip law for the interface. The evolution of bond stress in time gives rise to an interesting plot (Figure 7b), where the dashed lines represent the
elements left of the crack, and the dotted lines represent the ones on the right. Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 3 NUMERICAL RESULTS We can see that the elements on the
left have a positive value of bond-stress, and for the elements on the right the bond stress is negative. The bold solid
line represents the value of bond stress in the cracked element, that is very near to zero, since it is the inflection point
of the curve shown on Figure 7a. The bold dashed and dotted line represent the elements nearest to the crack, where
the bond stress is largest, and where the ultimate value of bond stress, 𝜏𝜏𝑦𝑦, has been reached. 8/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete Tea Rukavina et al. Figure 7: Bond stress: (a) distribution of 𝜎𝜎𝑏𝑏𝑏𝑏 along the reinforcement bar (values at Gauss points); (b) evolution of 𝜎𝜎𝑏𝑏𝑏𝑏 in time for all
enriched elements. Figure 7: Bond stress: (a) distribution of 𝜎𝜎𝑏𝑏𝑏𝑏 along the reinforcement bar (values at Gauss points); (b) evolution of 𝜎𝜎𝑏𝑏𝑏𝑏 in time for all
enriched elements. Figure 8: Plastic slip: (a) distribution of 𝛼𝛼𝑏𝑏𝑏𝑏,𝑝𝑝 along the reinforcement bar at the end of the analysis (values at Gauss points);
(b) evolution in time for the enriched elements that have entered the plastic phase. Figure 8: Plastic slip: (a) distribution of 𝛼𝛼𝑏𝑏𝑏𝑏,𝑝𝑝 along the reinforcement bar at the end of the analysis (values at Gauss points);
(b) evolution in time for the enriched elements that have entered the plastic phase. Related to that is the plastic slip development shown on Figure 8b, where it can be observed that plasticity occurs
only at the end of the analysis, in five elements that are nearest to the crack (two on the left side, and three on the
right). The same can be seen on Figure 8a, where the plastic slip is plotted in Gauss points along the reinforcement bar. The plastic slip is equal to zero in most of the enriched element, since they have not yet entered the plastic phase and
the interface is behaving elastically. Related to that is the plastic slip development shown on Figure 8b, where it can be observed that plasticity occurs
only at the end of the analysis, in five elements that are nearest to the crack (two on the left side, and three on the
right). 3 NUMERICAL RESULTS The same can be seen on Figure 8a, where the plastic slip is plotted in Gauss points along the reinforcement bar. The plastic slip is equal to zero in most of the enriched element, since they have not yet entered the plastic phase and
the interface is behaving elastically. Figure 9: Crack opening in concrete: (a) distribution of 𝛼𝛼𝑐𝑐 along the reinforcement bar at the end of the analysis (values at Gauss
points); (b) evolution of 𝛼𝛼𝑐𝑐 in time for the cracked element in the middle of the specimen. Figure 9: Crack opening in concrete: (a) distribution of 𝛼𝛼𝑐𝑐 along the reinforcement bar at the end of the analysis (values at Gauss
points); (b) evolution of 𝛼𝛼𝑐𝑐 in time for the cracked element in the middle of the specimen. Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 9/11 Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete Figure 9. gives the representation of the crack opening in concrete, that reaches the value of 𝛼𝛼𝑐𝑐=0.47 mm at the
end of the analysis. As it has been already explained, the macro-crack starts to open when the material reaches the
ultimate stress, which happens around time 0.25. There are also two small cracks in the elements right next to the
main crack, but their size is negligible compared to the main crack. Figure 10: Comparison of the evolution in time of 𝛼𝛼𝑐𝑐 and 𝛼𝛼𝑏𝑏𝑏𝑏: the crack opening in concrete in the middle of the element is nearly
equal to the sum of the absolute values of slip in the left and right node of the same element. Figure 10: Comparison of the evolution in time of 𝛼𝛼𝑐𝑐 and 𝛼𝛼𝑏𝑏𝑏𝑏: the crack opening in concrete in the middle of the element is nearly
equal to the sum of the absolute values of slip in the left and right node of the same element. It is interesting to point out that the crack opening in concrete coincides with the slip activation, which can be
compared on Figure 7b and Figure 9b. This is in accordance with our model assumption that there is no slip when the
concrete and the steel have the same displacement. 3 NUMERICAL RESULTS So, the crack opening in concrete, 𝛼𝛼𝑐𝑐, is giving rise to the
difference between the concrete and steel strain field, that is, in turn, activating the bond-slip 𝛼𝛼𝑏𝑏𝑏𝑏. Moreover, we can
test this notion by plotting the crack opening evolution and the slip evolution on the same diagram, as has been done
on Figure 10. We are plotting the values of 𝛼𝛼𝑐𝑐 in the Gauss point of the cracked element (represented by the solid line),
and the values of 𝛼𝛼𝑏𝑏𝑏𝑏 in left and right node of the same element (dashed and dotted line, respectively). While we have
already noted that the two nodes in question have the displacements of opposite sign, it is even more compelling that,
quantitatively, the absolute values of the two slips add up to give nearly the same value as the crack opening at a given
time. The sum of two slips is represented by a dotted line on Figure 10. Editor: Paulo de Mattos Pimenta. Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 4 CONCLUSIONS The methodology presented in this paper allows us to model the failure mechanisms in reinforced concrete
happening on the different scales of the material, by taking into account the behavior of each of the constituents. The
bond-slip is handled within a 1D elastoplastic framework, and we have shown that this simplification does not affect
the model predictivity; hence it represents an improvement compared to previous works based on interface elements. We have given the multi-scale set-up for this kinds of problems, where multi-level operator-split solution
procedures can be performed during the computation. In the numerical example we have shown the relationship
between the crack opening and the bond-slip, which corresponds to the realistic processes happening during the
failure of fiber-reinforced composites. The next step would be to extend the methodology to take into account the complete pull-out of the fiber, in
order to capture the failure mechanisms taking place when the fiber’s ends are not anchored in the surrounding The next step would be to extend the methodology to take into account the complete pull-out of the fiber, in
order to capture the failure mechanisms taking place when the fiber’s ends are not anchored in the surrounding
material. Author’s Contributions: Conceptualization, A Ibrahimbegovic; Investigation, T Rukavina; Methodology, T
Rukavina, A Ibrahimbegovic; Project administration, I Kozar, A Ibrahimbegovic; Software, T Rukavina; Supervision, A
Ibrahimbegovic, I Kozar; Validation, T Rukavina, A Ibrahimbegovic, I Kozar; Visualisation, T Rukavina, Writing - original
draft, T Rukavina; Writing - review & editing, A Ibrahimbegovic, I Kozar, T Rukavina. Editor: Paulo de Mattos Pimenta. 10/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced
concrete Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 References Brancherie, D., Ibrahimbegovic, A. (2009). Novel anisotropic continuum-discrete damage model capable of representing
localized failure of massive structures: Part I: theoretical formulation and numerical implementation, Engineering
Computations 26(1/2): 100–127. Do, X.N., Ibrahimbegovic, A., Brancherie, D. (2017). Dynamics framework for 2D anisotropic continuum-discrete damage
model for progressive localized failure of massive structures, Computers & Structures 183: 14–26. Dominguez, N., Brancherie, D., Davenne, L., Ibrahimbegovic, A. (2005). Prediction of crack pattern distribution in reinforced
concrete by coupling a strong discontinuity model of concrete cracking and a bond-slip of reinforcement model. Engineering
Computations 22(5/6): 558-582. Fries, T.-P., Belytschko, T. (2010). The extended/generalized finite element method: an overview of the method and its
applications, International Journal for Numerical Methods in Engineering 84(3): 253-304. Ibrahimbegovic, A. (2009). Nonlinear solid mechanics: Theoretical formulations and finite element solution methods, Springer,
Dodrecht. Ibrahimbegovic, A., Boulkertous, A., Davenne, L., Brancherie, D. (2010). Modelling of reinforced-concrete structures providing
crack-spacing based on X-FEM, ED-FEM and novel operator split solution procedure. International Journal of Numerical
Methods in Engineering 83(4): 452-481. Jukić, M., Brank, B., Ibrahimbegovic, A. (2014). Failure analysis of reinforced concrete frames by beam finite element that
combines damage, plasticity and embedded discontinuity, Engineering Structures, 75: 507-527. Kohnehpooshi, O., Jaafar, M.S. (2017). Non-linear three dimensional finite elements for composite concrete structures, Latin
American Journal of Solids and Structures, 14: 398-42. Melenk, J.M., Babuška, I. (1996). The partition of unity finite element method: Basic theory and applications, Computer
Methods in Applied Mechanics and Engineering, 139(1-4): 289-314. Ožbolt, J., Lettow, S., Kožar, I. (2002). Discrete bond element for 3D finite element analysis of reinforced concrete structures,
in book Beiträge aus der Befestigungstechnik und dem Stahlbetonbau, Lettow, S., Hoffman, J. (ed.), Ibidem Verlag, Stuttgart. Šćulac, P., Jelenić, G., Škec, L. (2014). Kinematics of layered reinforced-concrete planar beam finite elements with embedded
transversal cracking, International Journal of Solids and Structures, 51: 74-92. Taylor, R. (2014). FEAP - Finite Element Analysis Program, University of California Berkeley, version 8.4. 11/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141
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Spectrum of outcomes following traumatic brain injury—relationship between functional impairment and health-related quality of life
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Acta neurochirurgica
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Abstract Background The outcome following traumatic brain injury
(TBI) is heterogeneous and poorly defined and physical dis-
ability scales like the extended Glasgow Outcome Score
(GOSE) while providing valuation information in terms of
broad categorisation of outcome are unlikely to capture the
full spectrum of deficits. Quality of life questionnaires such
as SF-36 are emerging as potential tools to help characterise
factors important to patients’ recovery. This study assessed the
association between physical disability and subjective health
rating. The relationship is of value as it may help evaluate the
impact of TBI on patients’ lives and facilitate the delivery of
appropriate neuro-rehabilitation services. Results There is a statistically significant difference in the
MCS and PCS scores based on patients’ GOSE scores. The
mean scores of the eight SF-36 domains showed significant
association with GOSE. GLMM demonstrated that GOSE
was the strongest predictor of PCS and MCS. Age was an
important variable in the PCS score while time following trau-
ma was a significant predictor of MCS rating. Conclusions This study highlights that patients’ physical out-
come following TBI is a strong predictor of the subjective
mental and physical health. Nevertheless, there remains tre-
mendous variability in individual SF-36 scores for each
GOSE category, highlighting that additional factors play a role
in determining quality of life. Methods A single-centre retrospective study was undertaken
to assess the relationship between physical outcome as mea-
sured by GOSE and quality of life captured by the SF-36
questionnaire. Cronbach’s alpha was calculated for each of
the eight SF-36 domains to measure internal consistency of
the test. Multivariate analysis of variance was conducted to
look at the association between GOSE and the physical (PCS)
and mental (MCS) component scores on the SF-36. Finally,
we performed a generalised linear mixed model (GLMM) to Keywords Traumatic brain injury . Quality of life . SF-36 . Glasgow Outcome Scale Keywords Traumatic brain injury . Quality of life . SF-36 . Glasgow Outcome Scale Spectrum of outcomes following traumatic brain
injury—relationship between functional impairment
and health-related quality of life Anastasia Tsyben1,2
& Mathew Guilfoyle1,2 & Ivan Timofeev1,2 & Fahim Anwar3 &
Judith Allanson3 & Joanne Outtrim4 & David Menon4 & Peter Hutchinson1,2 &
Adel Helmy1,2 Anastasia Tsyben1,2
& Mathew Guilfoyle1,2 & Ivan Timofeev1,2 & Fahim Anwar3 &
Judith Allanson3 & Joanne Outtrim4 & David Menon4 & Peter Hutchinson1,2 &
Adel Helmy1,2 Received: 8 August 2017 /Accepted: 11 September 2017 /Published online: 7 October 2017
# The Author(s) 2017. This article is an open access publication assess the relative contribution of GOSE score, age at the time
of trauma, sex and TBI duration towards MCS and PCS
rating. Acta Neurochir (2018) 160:107–115
DOI 10.1007/s00701-017-3334-6 ORIGINAL ARTICLE - BRAIN INJURY * Anastasia Tsyben
anastasia.tsyben@cantab.net 4
Division of Anaesthesia, Department of Medicine, Cambridge
Biomedical Campus, Cambridge, UK 1
Department of Neurosurgery, Addenbrooke’s Hospital, Hills Road,
Cambridge CB2 0QQ, UK
2
Division of Neurosurgery, Department of Clinical Neurosciences,
Cambridge Biomedical Campus, Cambridge, UK
3
Department of Neurorehabilitation, Addenbrooke’s Hospital,
Cambridge, UK
4
Division of Anaesthesia, Department of Medicine, Cambridge
Biomedical Campus, Cambridge, UK Sample and measures A series of multivariate ANOVAs were conducted with
GOSE scores as the independent variable and the two sum-
mary scores, MCS and PCS, as the dependent variables. Interaction was also assessed between age of TBI and SF-36
domain scores at the first clinical appointment following
discharge. This was a retrospective study of patients seen in
Addenbrooke’s Hospital, Cambridge, Neurotrauma
Outpatient Clinic between 2005–2013. The majority of indi-
viduals underwent treatment for TBI at Addenbrooke’s, with a
small proportion of patient’s being referred from other centres. Tests of physical function and subjective health status were
administered at each clinic appointment. Statistical analysis Descriptive statistics were used to summarise the demograph-
ic data. Normality of data was tested using the Shapiro-Wilk
test while Levene’s and Bartlett’s tests were used to test ho-
mogeneity of variance. Based on skewed distribution of the
data, non-parametric testing of correlation among MCS, PCS
and GOSE were examined using Spearman rank. The level of
statistical significance was set at p < 0.05. All analyses were
carried out in SPSS 24.0 (IBM SPSS, Chicago, IL, USA). The purpose of this study was to investigate the relation-
ship between the functional score of patients with TBI and
their subjective health status. The GOSE test was used as a
measure of physical function, while the 36-Item Short Form
Survery (SF-36) was used as an assessment of QoL. While
both these assessment tools have been validated in the TBI
population, there has been no definite characterisation of the
relationship between physical disability and subjective per-
ception of handicap [18, 24, 27]. Introduction Traumatic brain injury (TBI) is a leading cause of mortality
and long-term disability among those under 45 and costs an
estimated $75 billion each year in the USA alone [9, 12]. Despite improvements in the acute management of TBI, most
patients are left with a degree of permanent disability affecting
cognitive, psychological and physical function [39]. Commonly used outcome measures such as the functional
independence measure (FIM), extended Glasgow Outcome
Scale (GOSE) and Disability Rating Scale (DRS) while pro-
viding broad categorisation of outcome are limited in their
ability to capture the full spectrum of deficits following brain Acta Neurochir (2018) 160:107–115 108 injury [11]. In particular, such scales struggle to measure neu-
robehavioral disability, which tends to affect a person’s per-
sonality, cognition and character [43]. However, it is these
aspects that often lead to disintegration of interpersonal rela-
tionships, family burden and inability to return to vocation [3,
25]. 40]. In the scale, the higher scores indicate better subjective
health. Two summary scores, physical component summary
(PCS) and mental component summary (MCS), are derived
by taking unweighted means of the corresponding domains. The SF-36 survey was only administered to patients with
adequate communication skills who were able to respond to
the questionnaire. Thus only patients with GOSE score of 3 or
greater were included in the final analysis. Clinic appoint-
ments with missing GOSE or SF-36 scores were excluded
from analysis. In addition, subjects with incorrect or missing
demographic details were also excluded, as these could not be
incorporated into the final statistical analysis. Quality of life (QoL) questionnaires have gained increasing
popularity, allowing for measurements of objective and sub-
jective health indicators. When compared to objective clinical
measures of physical function, QoL questionnaires are supe-
rior at capturing the patients’ internal judgment of health and
factors that may be important to the well being of the individ-
ual. This emphasis on Patient Reported Outcome Measures
(PROM) is drawing increased interest from both patient
groups and funding bodies. Such information in turn may
facilitate the delivery and evaluation of neuro-rehabilitation
services as well as guide future clinical research. In the TBI
population, QoL measures may have the added benefit of
capturing the heterogeneity of outcomes and may delineate
the natural history of this chronic condition. Measure of internal consistency of SF-36 domain scores
and its association with physical disability Cronbach’s alpha was calculated for each of the eight domain
scores for all patients during their first visit. The minimum
threshold for the coefficient was set at 0.7 and preferable
above 0.8 [13, 30]. A domain was considered distinct if its
respective alpha coefficient exceeded inter-domain correlation
of all other scales. Descriptive statistics A total of 513 adults above the age of 16 who were seen in
neurosurgery clinic between 2005–2013 met the inclusion
criteria and were included in the final analysis. Table 1 shows
the demographic characteristics of the study cohort as well as
the disability GOSE score at the first follow-up clinic. Mean
age at the time of traumatic brain injury was 39.7 years (range:
16–91 years; SD = 16.9). The range of clinic follow-up dates
was 0–611 months, with a total of 922 individual visits col-
lected over this time period. The average number of follow-up
clinics for a patient was two, although these were inconsistent Analysis using univariate ANOVAs found that GOSE
scores had statistically significant effect on both PCS (F =
202.9; p < 0.005; partial η2 = 0.53) and MCS (F = 14.9; p <
0.005; partial η2=0.41) scores. Likewise, the mean scores of
the eight domains of SF-36 also showed a significant associ-
ation with GOSE scores (Pillai’s trace = 0.68; F = 17.8, p <
0.005; partial η2 = 0.14). Comparisons of GOSE and PCS and
MCS scores are plotted in Fig. 1. Table 1 Demographic
characteristics of the
study population
n
513
Sex
Female
152 (30%)
Male
361 (70%)
Age at TBI (years)
Median
38
Mean
39.7
SD
16.9
Range
16–91
GOSE (%) at first follow-up
Upper good recovery
12.9
Lower good recovery
11.6
Upper moderate disability
30.8
Lower moderate disability
17.9
Upper severe disability
19.1
Lower severe disability
7.7
GOS, Extended Glasgow Scale. SD,
standard deviation n
513
Sex
Female
152 (30%)
Male
361 (70%)
Age at TBI (years)
Median
38
Mean
39.7
SD
16.9
Range
16–91
GOSE (%) at first follow-up
Upper good recovery
12.9
Lower good recovery
11.6
Upper moderate disability
30.8
Lower moderate disability
17.9
Upper severe disability
19.1
Lower severe disability
7.7
GOS, Extended Glasgow Scale. SD,
standard deviation Contribution of independent variables to subjective QOL
scores pp
The physical disability was assessed using GOSE, an eight-
scale global measure of function, which has been validated in
TBI [13, 30]. The SF-36 questionnaire was used to assess the
patient-reported QoL. The test consists of 36 multiple choice
questions that are grouped into eight domains: PF, physical
functioning; RP, role limitation due to physical problems; BP,
bodily pain; RE, role limitations due to emotional problems;
VT, vitality; GH, general health perception; MH, mental
health; SF, social functioning. The domain scores were calcu-
lated by transforming the raw data into a scale of 0–100 and
using Likert’s method of unweighted summed ratings [26, A generalised mixed model (GLMM) was used to assess the
relatively contribution of GOSE score, age at the time of trau-
ma, sex and TBI duration on MCS and PCS score. Patient
hospital numbers were identified as random effects, while
sex, age at the time of trauma, TBI duration and GOSE were
fixed effects. GLMM was used to account for the variable
time points of questionnaire collections and the unequal sam-
ple size observed for each follow-up visit. R2s were calculated
to determine the goodness of fit of the model. Acta Neurochir (2018) 160:107–115 109 as they were based on the clinical need of the individual
patients. To look at the general spread of responses on MCS and
PCS for each GOSE category, a box and whisker plot was
constructed. While there may be a positive relationship be-
tween GOSE and SF-36 responses, there may be variability
in responses highlighting individual differences in perceived
QOL. Association between GOSE and SF-36 scores Based on the result of the Shapiro-Wilk test of normality, PCS
and MCS results were found to be non-parametric (p < 10−14). The correlation among PCS, MCS and GOSE was significant-
ly positive, indicating that as the GOSE score of disability
improved, the patients’ subjective rating of health also rose. PCS showed the highest positive Spearman rank correlation of
0.67 (p < 0.001) with GOSE. MCS had a correlation of 0.61 (p
< 0.001) with GOSE and 0.71 (p < 0.001) with PCS. Reliability of SF-36 domain score Summary scores for the eight domains of SF-36 at the first
clinic visit as well as intra-class correlations are summarised in
Table 2. The Cronbach’s alpha for the eight SF-36 domains
was 0.90 suggesting high internal consistency of the question-
naire. Individual alpha coefficients ranged from 0.87–0.89
and were substantially greater than the correlations between
domains. Most domains had a strong inter-item correlation
and were worthy of inclusion, resulting in a lower coefficient
if deleted. Temporal variation in GOSE and SF-36 parameters To explore the relative concordance of GOSE and SF36 in
identifying changes (improvement or deterioration) in out-
come, we identified patients who attended the neurotrauma
clinic on at least two occasions. Patients were divided into
cohorts in which there was a change in GOSE category and
the mean change in SF-36 calculated. Relationship among GOSE, SF-36 domain scores, age
at trauma, gender and time since injury The generalised linear mixed model was used to examine the
relationship among GOSE, PCS/MCS, gender, age at the time
of trauma and time since injury. The PCS and MCS scores
both increased as the GOSE rose. The regression coefficient
shows that, all other things equal, for every 1-point rise in
GOSE, the score on PCS would rise from 4.1–41.9 points (p
< 0.0005). However, relative to GOSE 3, patients with a
GOSE 4 score rated 2.6 lower on PCS, although this was
not statistically significant (p = 0.266). This suggests a plateau
effect at lower GOSE scores, such that physical disability no
longer contributes to further declines in GOSE score below 4,
while variables contributing to the MCS (which are likely to
include cognitive, psychological and psychiatric symptoms)
do. In addition, the age at the time of trauma was also statis-
tically significant at p = 0.03, indicating that the older the
patient was at the time of TBI, the worse they would score Acta Neurochir (2018) 160:107–115 110 Table 2 Domain characteristics
including mean and standard
deviation (SD) with reliability
statistics: Cronbach’s alpha
coefficient and inter-domain
correlations
Domain characteristics
Reliability
SF-36 domain
Mean
SD
Alpha
Inter-domain correlation
mean (range)
PF
67.0
30.9
0.89
0.47 (0.38–0.52)
RP
32.5
41.3
0.89
0.53 (0.43–0.61)
BP
59.7
31.2
0.89
0.54 (0.49–0.59)
GH
58.3
25.0
0.89
0.57 (0.50–0.63)
VI
44.7
25.6
0.88
0.59 (0.43–0.72)
SF
52.7
31.5
0.87
0.62 (0.52–0.71)
RE
44.3
45.5
0.89
0.56 (0.46–0.67)
MH
58.8
24.6
0.88
0.57 (0.38–0.72) physical status likely influence patients’ mental and physical
health perception. The mean score on MCS was 51.77 (SD
25.60) and PCS 52.46 (SD 19.46) (Table 3). on the PCS domain of SF-36 by approximately 0.07 points per
year of age. The clinical significance of this small but statisti-
cally significant difference is unclear. A comparison between scores on concurrent clinic visits
demonstrated that both PCS and MCS correlated positively
with GOSE score. If a patient’s GOSE score dropped during
the subsequent visit, their SF-36 score also tended to drop and
vice versa (Table 4 and Fig. 3), suggesting that both functional
impairment and health-related quality of life metrics change
together as patients recover or deteriorate. Similarly, the MCS score increased by 8.4–54.7 points for
every 1 point rise in GOSE group (p < 0.005). Discussion Although GOSE had a high explanatory power on PCS and
MCS, there was great inter-patient variability of scores as
demonstrated in Fig. 2. Within each GOSE score, there is a
large spectrum of physical and mental component scores
underlining that, on an individual basis, factors other than The aim of this study was to characterise the relationship
between physical outcome following TBI and subjective
health scores, as measured by the SF-36 questionnaire. To
our knowledge, this is one of the largest cohorts of patients
cited in the TBI literature, with a total of 513 patients. The
GOSE score had a significant impact on a patient’s PCS and
MCS scores and the SF-36 proved to be a robust metric for
assessing improvement in GOSE. As the GOSE score in-
creased the subjective rating on MCS and PCS also rose by
an average of 33.8 (p < 0.005) points on the MCS and 33.2 (p
< 0.005) points on the PCS. These trends are consistent with
the well-documented finding that poor physical function and
reduced mobility lead to increased pain and decreased inde-
pendence, as well as precluding return to vocation [22]. The
finding of the positive relationship between physical function
and QOL is important because it suggests that managing re-
sidual physical deficits could lead to an increase in subjective
health status. Interestingly, at lower GOSE categories (GOSE
3 and GOSE 4), there appears to be little difference in average
PCS scores; however, the MCS continues to decline. Targeted
rehabilitation has clearly shown that rehabilitation can de-
crease disability and improve the quality of living [1, 2, 8,
19]. Even patients with moderate to severe TBI demonstrate
some continued neuropsychological recovery several years 0.0
25.0
50.0
75.0
100.0
3
4
5
6
7
8
Mean Score (95% CI)
eGOS
MSC
PSC
Fig. 1 Comparison of GOSE to the two summary scores, PCS and MCS
on SF-36. Mean MCS and PCS scores are plotted against GOSE
categories (error bars: 95% confidence interval). All summary measures
showed increasing scores with more favourable GOSE (multivariate
ANOVA all p < 10−93) [3=Lower severe disability 4=Upper severe
disability 5=Lower moderate disability 6=Upper moderate disability
7=Lower good recovery 8=Upper good recovery] 0.0
25.0
50.0
75.0
100.0
3
4
5
6
7
8
Mean Score (95% CI)
eGOS
MSC
PSC Fig. 1 Comparison of GOSE to the two summary scores, PCS and MCS
on SF-36. Relationship among GOSE, SF-36 domain scores, age
at trauma, gender and time since injury Time since
injury showed an increase in MCS score of 0.04 points per
year and this was statistically significant at p = 0.018. With a
significance value greater than 0.05, there is not enough evi-
dence to conclude whether the gender or progression of time
from TBI had an effect on the PCS outcome. Approximately
48% of the variability in the model was explained by the
independent variables included in the analysis for PCS and
39% for MCS. Discussion Mean MCS and PCS scores are plotted against GOSE
categories (error bars: 95% confidence interval). All summary measures
showed increasing scores with more favourable GOSE (multivariate
ANOVA all p < 10−93) [3=Lower severe disability 4=Upper severe
disability 5=Lower moderate disability 6=Upper moderate disability
7=Lower good recovery 8=Upper good recovery] 111 Acta Neurochir (2018) 160:107–115 Fig. 2 Box and whisker plot
showing the spread of MCS and
PCS responses for each GOSE
category. [3=Lower severe
disability 4=Upper severe
disability 5=Lower moderate
disability 6=Upper moderate
disability 7=Lower good recovery
8=Upper good recovery] more on factors such as neuropsychological function rather
than physical disability [32]. after injury—particularly in the domain of cognitive speed,
visuospatial skills and verbal memory [28]. When combined with age, sex and progression over time
since TBI, the model predicted 48% of variability observed in
PCS and 39% in MCS scores. Although the physical function
played a critical role in QOL rating, it was not the sole deter-
minate and other factors not included in the model influence
subjective health. This is in line with Ruff and colleagues who
found that social function and return to vocation depended The relationship between SF-36 and GOSE is consistent
with findings reported by Wilson et al., who found a positive
correlation between GOSE and eight subscales of SF-36, par-
ticularly in the social functioning domain [42]. A meta-
analysis of 49 studies found SF-36 to be the most widely used
and robust tool for assessing outcome following TBI, with
strong internal consistency and interpretability [31]. Table 3 Generalised linear
mixed model of PCS and MCS
scores. Discussion SE, standard error
PCS
MCS
β
SE
CI (95%)
p
β
SE
CI (95%)
p
GOSE
Upper good recovery
41.9
2.5
37.3–46.7
0.000
54.7
2.5
49.8–59.5
0.000
Lower good recovery
27.7
2.5
22.7–32.7
0.000
44.7
3.0
38.9–50.5
0.000
Upper moderate disability
13.2
2.3
8.6–17.8
0.000
25.0
2.6
19.9–30.0
0.000
Lower moderate disability
4.1
2.4
−0.6–8.7
0.049
13.0
2.9
7.3–18.7
0.000
Upper severe disability
−2.6
2.4
−7.3–2.0
0.266
8.4
2.9
2.6–14.2
0.005
Age at trauma
−0.07
0.03
−0.13– (-0.07)
0.03
0.06
0.05
−0.04–0.16
0.24
Gender
Male
0.10
1.1
−2.1–2.3
0.93
−0.42
2.0
−4.4–3.6
0.84
Months since injury
0.02
0.01
−0.01–0.04
0.08
0.04
0.02
0.01–0.08
0.018
R2
0.48
0.39 Acta Neurochir (2018) 160:107–115 112 Table 4 GOSE and PCS/MCS
score changes between the 1st and
2nd clinic appointment Table 4 GOSE and PCS/MCS
score changes between the 1st and
2nd clinic appointment
GOSE
PCS
MCS
Score
change
Number of
subjects
Mean (SD)
Range
Median
Mean (SD)
Range
Median
−4
1
−16.6
−5.8
−3
1
−31.61
−13.36
−2
5
−6.68 (21.8)
−31.3–27.6
−6.7
5.28 (19.2)
−14.9–30.8
−0.08
−1
25
−5.25 (20.8)
−42.7–41.7
−8.3
−6.19 (22.5)
−42.4–48.9
−8.7
0
114
0.87 (15.5)
−41.22–36.9
2.52
1.40 (17.9)
−50.5–57.4
1.25
1
46
13.25 (15.6)
−23.6–52.1
16.85
13.31 (20.4)
−20.16–76.02
8.83
2
16
23.85 (16.1)
−3.6–51.2
25.2
22.08 (17.7)
−14.8–65.2
20.78
3
9
19.70 (29.8)
−24.4–59.7
11.47
20.66 (20.8)
−20.1–52.1
21
4
4
24.02 (36.9)
−18.03–66
24.06
22.73 (22.3)
−8.8–41.7
29
SD, standard deviation SD, standard deviation However, as mentioned by the authors, SF-36 may not be a
sensitive tool for detecting emotional and cognitive distur-
bances. In addition to the previous reported research, this
study highlights that GOSE is the strongest predictor of PCS
and MCS scores. There is also a statistically significant differ-
ence in the MCS and PCS scores based on patients’ GOSE
scores. 36, 41]. In contrast, other studies found reverse results with
subjects having higher MCS than PCS [15, 17, 21, 34]. They
argued that the discrepancy between the mental and physical
scores could result from lack of awareness in patients with
severe TBI. However, in the study by Steinbuechel et al. the
number of patients with severe TBI was double of those with
mild TBI. In terms of distribution of GOSE scores, we had a
relatively equal number of subjects with GOSE below and
above a score of 5. Fig. 3 Box and whisker plot
showing the change in GOSE
score and PCS/MCS score
between the 1st and 2nd clinic
appointment. Only time points
with ten or more subjects are
illustrated Conclusion The aftermath of TBI is heterogeneous, leaving patients with a
spectrum of physical, cognitive and psychological sequelae. This study highlights that patients’ physical outcome follow-
ing TBI is a strong predictor of their subjective mental and
physical health. Nevertheless, this is not the only factor to
predict QOL post trauma, as there is a tremendous variation
in scores observed across the GOSE spectrum. Thus, despite
poor physical function, some patients rate their quality of life
as high and vice versa. Further analysis of data would include
additional factors such education, employment status, family
network and cognitive ability to better model the predictors
and help understand the natural history of recovering follow-
ing TBI. This in turn could facilitate delivery of appropriate
and individualised rehabilitation to ensure the best possible
outcome for patients with TBI. Despite the positive relationship between GOSE and SF-36
domain scores, there is tremendous variability in MCS and
PCS scores within each of the GOSE categories. This is con-
sistent with the findings of Polinder et al., whose meta-
analysis found significant heterogeneity in SF-36 summary
scores [31]. GOSE is a global score, which captures only a
limited component of a subject’s overall health. Factors such
as degree of cognitive ability, educational background and
psychological health have all been found to affect QOL post
TBI. On the other hand, the majority of studies have found no
gender differences in physical and cognitive function follow-
ing discharge [4, 29, 33, 35]. This is in keeping with our
results that show gender to be a non-significant variable in
influencing SF-36 rating. Interpersonal differences in social
and economic status following trauma may also be an impor-
tant contributing factor to outcome. For instance, economic
advantage can allow better provision of home support, both
physical and mental. It could assist patients in paying for
private therapies to enhance psychological and physical
health. In addition, the family network and meaningful inter-
personal relationship play a crucial part in the support and
rehabilitation of patients following TBI. Acknowledgements
The authors would like to thank Helen Seeley and
Liz Corteen for their assistance in data collection. Funding This study was supported by National Institute for Healthcare
Research (NIHR, UK) through the Acute Brain Injury and Repair theme
of the Cambridge NIHR Biomedical Research Center to PH and AH, an
NIHR Senior Investigator Award to DM and an NIHR Research
Professorship to PH. Discussion For instance, targeted re-
habilitation programmes have been found to increase memory
in patients with mild to moderate Alzheimer’s disease [10] and
focused training of motor skills in Parkinson’s patients lead to
increased activity within motor regions of the brain [37]. These studies provide compelling evidence that targeted ther-
apy and rehabilitation in TBI patients, despite their age, may
yield positive benefits on the cognitive function. These in turn
may facilitate patients’ return to vocation and improving their
emotional health. Nevertheless, there are additional challenges
following trauma in the elderly associated with increased co-
morbidities and not only with neuronal changes [6]. As such,
this group of individuals may require additional in-hospital
support and physiotherapy to promote their return to the
community. were not included in the statistical analysis because of incom-
plete information. These factors have been found to predict
outcome following TBI and may explain some of the variabil-
ity seen in the scores of MCS and PCS observed for our
cohort. For example, research has shown that pre-injury em-
ployment status and educational level are strong predictors of
QOL and return to vocation [23]. Likewise, cognitive ability
has been shown to account for 21% to 30% of the variability
observed in the functional ability post trauma [7]. Our patients were not subdivided based on the severity of
the trauma. As mentioned earlier, some studies have found
that subjects with severe TBI lacked awareness and thus
ranked higher on QOL questionnaires. Self-awareness is di-
rectly associated with motivation and change of behavior,
which may facilitate better functional outcomes following
trauma. On the other hand, it is also more likely to result in
depression and other psychological conditions further de-
creasing perceived QOL [14]. Conclusion Authors were also supported by a European Union
Framework Program 7 grant (CENTER-TBI; Grant Agreement No. 602150). AH is supported by MRC (G0802251). This study has several limitations. First, it is a retrospective
single-centre study from a specialised tertiary neurotrauma
clinic in which the population demographics, socioeconomic
background and availability of rehabilitation services may be
skewed and may not necessarily reflect the UK general TBI
population. In addition, there were a limited number of fixed
effects included in the study. Factors such as education, em-
ployment status, relationship status and cultural background Discussion Our cohort of patients had higher average PCS scores (M =
52.46, SD ± 19.46) compared to MCS (M = 51.77, SD ±
25.60). This is in agreement with results of the study by
Steinbuechel et al., Hawthorne et al. and Wilson et al. [20, Age at the time of trauma was a positive predictor of PCS
score but the effect size was small. For every additional year, Fig. 3 Box and whisker plot
showing the change in GOSE
score and PCS/MCS score
between the 1st and 2nd clinic
appointment. Only time points
with ten or more subjects are
illustrated Acta Neurochir (2018) 160:107–115 113 the PCS score decreased by 0.07 points (p < 0.05). On the
other hand the MCS score tended to rise with increasing age,
although this finding was insignificant (B = 0.04, p = 0.36). This is in accordance with other studies, which found that the
proportion of poor outcome following TBI increased with age
[16, 38]. Despite the changes observed in the ageing brain,
including modification in electrical coupling and cell connec-
tions, it retains remarkable ability to respond to stimulation
and improve working memory [5]. For instance, targeted re-
habilitation programmes have been found to increase memory
in patients with mild to moderate Alzheimer’s disease [10] and
focused training of motor skills in Parkinson’s patients lead to
increased activity within motor regions of the brain [37]. These studies provide compelling evidence that targeted ther-
apy and rehabilitation in TBI patients, despite their age, may
yield positive benefits on the cognitive function. These in turn
may facilitate patients’ return to vocation and improving their
emotional health. Nevertheless, there are additional challenges
following trauma in the elderly associated with increased co-
morbidities and not only with neuronal changes [6]. As such,
this group of individuals may require additional in-hospital
support and physiotherapy to promote their return to the
community. the PCS score decreased by 0.07 points (p < 0.05). On the
other hand the MCS score tended to rise with increasing age,
although this finding was insignificant (B = 0.04, p = 0.36). This is in accordance with other studies, which found that the
proportion of poor outcome following TBI increased with age
[16, 38]. Despite the changes observed in the ageing brain,
including modification in electrical coupling and cell connec-
tions, it retains remarkable ability to respond to stimulation
and improve working memory [5]. Compliance with ethical standards Conflict of Interest
All authors certify that they have no affiliations
with or involvement in any organization or entity with any financial
interest (such as honoraria; educational grants; participation in speakers’
bureaus; membership, employment, consultancies, stock ownership, or
other equity interest; and expert testimony or patent-licensing 114 Acta Neurochir (2018) 160:107–115 arrangements), or non-financial interest (such as personal or professional
relationships, affiliations, knowledge or beliefs) in the subject matter or
materials discussed in this manuscript. 11. Edwards SGM, Playford ED, Hobart JC, Thompson AJ (2002)
Comparison of physician outcome measures and patients’ percep-
tion of benefits of inpatient neurorehabilitation. BMJ 324:1493 12. Faul M, Coronado V (2015) Epidemiology of traumatic brain inju-
ry. Handbook of Clin Neurol 127:3–13 Ethical approval
All procedures performed in studies involving hu-
man participants were in accordance with the ethical standards of the
institutional and/or national research committee and with the 1964
Helsinki Declaration and its later amendments or comparable ethical
standards. For this retrospective study formal consent was not required. 13. Fayers P, Hays RD (2005) Assessing quality of life in clinical trials. Oxford University Press, Oxford 14. Fleming JM, Strong J, Ashton R (1998) Cluster analysis of self-
awareness levels in adults with traumatic brain injury and
relationshipto outcome. J Head Trauma Rehabil 13(5):39–51 15. Forslund MV, Roe C, Sigurdardottir S, Andelic N (2013) Predicting
health-related quality of life 2?years after moderate-to-severe trau-
matic brain injury. Acta Neurol Scand 128(4):220–227 Informed consent
Data for the research were collected as part of a
registered audit at Addenbrooke’s Hospital, Cambridge. Informed con-
sent was obtained from all individual participants included in the study. 16. Gómez PA, Lobato RD, Boto GR, De la Lama A, González PJ, de
la Cruz J (2000) Age and outcome after severe head injury. Acta
Neurochir (Wien) 142(4):373–380 Abbreviations: DRS Disability Rating Scale, FIM Functional indepen-
dence measure, GLMM Generalised linear mixed model, GOSE
Extended Glasgow Outcome Score, MCS Mental Component Score
(SF-36), PCS Physical Component Score (SF-36), PROM Patient
Reported Outcome Measures, QoL Quality of life, SF-36 36-Item Short
Form Surverym, TBI Traumatic brain injury 17. Grauwmeijer E, Heijenbrok-Kal MH, Ribbers GM (2014) Health-
related quality of life 3 years after moderate to severe traumatic
brain injury: a prospective cohort study. Arch Phys Med Rehabil
95(7):1268–1276 18. References 22. Johnstone B, Schopp LH, Harper J, Koscuilek J (1999)
Neuropsychological impairments, vocational outcomes, and finan-
cial costs for individuals with traumatic brain injury receiving state
vocational rehabilitation services. J Head Trauma Rehabil 14(3):
220–232 1. Ashley MJ, Krych DK, Lehr RP (1990) Cost/benefit analysis for
post-acute rehabilitation of the traumatically brain-injured patient. J
Insur Med 22(2):156–161 2. Ben-Yishay Y, Silver SM, Piasetsky E, Rattok J (1987)
Relationship between employability and vocational outcome after
intensive holistic cognitive rehabilitation. J Head Trauma Rehabil
2(1):35–48 23. Keyser-Marcus LA, Bricout JC, Wehman P, Campbell LR, Cifu
DX, Englander J, High W, Zafonte RD (2002) Acute predictors of
return to employment after traumatic brain injury: a longitudinal
follow-up. Arch Phys Med Rehabil 83(5):635–641 3. Brooks DN, McKinlay W (1983) Personality and behavioural
change after severe blunt head injury—a relative’s view. J Neurol
Neurosurg Psychiatry 46(4):336–344 24. Levin HS, Boake C, Song J, McCauley S, Contant C, Diaz-Marchan P,
Brundage S, Goodman H, Kotrla KJ (2001) Validity and sensitivity to
change of the extended glasgow outcome scale in mild to moderate
traumatic brain injury. J Neurotrauma 18(6):575–584 4. Brown AW, Moessner AM, Mandrekar J, Diehl NN, Leibson CL,
Malec JF (2011) A survey of very-long-term outcomes after trau-
matic brain injury among members of a population-based incident
cohort. J Neurotrauma 28(2):167–176 25. Lezak MD (1978) Living with the characterologically altered brain
injured patient. J Clin Psychiatry 39(7):592–598 5. Burke SN, Barnes CA (2006) Neural plasticity in the ageing brain. Nat Rev Neurosci 7(1):30–40 26. Likert R (1932) A technique for the measurement of attitudes. Arch
Psychol 22:1–55 6. Callaway DW, Wolfe R (2007) Geriatric trauma. Emerg Med Clin
North Am 25(3):837–860 27. McCauley SR, Wilde EA, Moretti P et al (2013) Neurological out-
come scale for traumatic brain injury: III. Criterion-related validity
and sensitivity to change in the NABIS hypothermia-II clinical trial. J Neurotrauma 30(17):1506–1511 7. Chaytor N, Temkin N, Machamer J, Dikmen S (2007) The ecolog-
ical validity of neuropsychological assessment and the role of de-
pressive symptoms in moderate to severe traumatic brain injury. J
Int Neuropsychol Soc 13(3):377–385 28. Millis SR, Rosenthal M, Novack TA, Sherer M, Nick TG, Kreutzer
JS, High WM, Ricker JH (2001) Long-term neuropsychological
outcome after traumatic brain injury. J Head Trauma Rehabil
16(4):343–355 8. Cope DN, Cole JR, Hali KM, Barkan H (1991) Brain injury: anal-
ysis of outcome in a post-acute rehabilitation system. Compliance with ethical standards Guilfoyle MR, Seeley HM, Corteen E, Harkin C, Richards H,
Menon DK, Hutchinson PJ (2010) Assessing quality of life after
traumatic brain injury: examination of the short form 36 health
survey. J Neurotrauma 27(12):2173–2181 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 appro-
priate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made. 19. Hajfey WJ, Abrams DL (1991) Employment outcomes for partici-
pants in a brain injury work reentry program: preliminary findings. Head Trauma Rehabil 20. Hawthorne G, Gruen RL, Kaye AH (2009) Traumatic brain injury
and long-term quality of life: findings from an Australian study. J
Neurotrauma 26(10):1623–1633 21. Jacobsson LJ, Westerberg M, Lexell J (2010) Health-related
quality-of-life and life satisfaction 6-15 years after traumatic brain
injuries in northern Sweden. Brain Inj 24(9):1075–1086 References Part 1: general
analysis. Brain Inj 5(2):111–125 29. Moore DW, Ashman TA, Cantor JB, Krinick RJ, Spielman LA
(2010) Does gender influence cognitive outcome after traumatic
brain injury? Neuropsychol Rehabil 20(3):340–354 9. Corso P, Finkelstein E, Miller T, Fiebelkorn I, Zaloshnja E (2006)
Incidence and lifetime costs of injuries in the United States. Inj Prev
12(4):212–218 30. Nunnally JH, Bernstein IH (1994) Psychometric theory. McGraw-
Hill, New York 10. Cotelli M, Manenti R, Brambilla M, Petesi M, Rosini S, Ferrari C,
Zanetti O, Miniussi C (2014) Anodal tDCS during face-name asso-
ciations memory training in Alzheimer’s patients. Front Aging
Neurosci 6:38 31. Polinder S, Haagsma JA, van Klaveren D, Steyerberg EW, van
Beeck EF (2015) Health-related quality of life after TBI: a Acta Neurochir (2018) 160:107–115 115 systematic review of study design, instruments, measurement prop-
erties, and outcome. Popul Health Metr 13:4 37. Subramanian L, Hindle JV, Johnston S, Roberts MV, Husain M,
Goebel R, Linden D (2011) Real-time functional magnetic reso-
nance imaging neurofeedback for treatment of Parkinson’s disease. J Neurosci 31(45):16309–16317 32. Ruff RM, Marshall LF, Crouch J et al (1993) Predictors of outcome
following severe head trauma: follow-up data from the traumatic
coma data bank. Brain Inj 7(2):101–111 38. Thompson HJ, McCormick WC, Kagan SH (2006) Traumatic brain
injury in older adults: epidemiology, outcomes, and future implica-
tions. J Am Geriatr Soc 54(10):1590–1595 33. Schonberger M, Ponsford J, Reutens D, Beare R, Clarke D,
O’Sullivan R (2011) The relationship between mood disorders
and MRI findings following traumatic brain injury. Brain Inj
25(6):543–550 39. Thornhill S, Teasdale GM, Murray GD, McEwen J, Roy CW,
Penny KI (2000) Disability in young people and adults one year
after head injury: prospective cohort study. BMJ 320:1631 34. Scholten AC, Haagsma JA, Andriessen TMJC, Vos PE, Steyerberg
EW, van Beeck EF, Polinder S (2015) Health-related quality of life
after mild, moderate and severe traumatic brain injury: patterns and
predictors of suboptimal functioning during the first year after in-
jury. Injury 46(4):616–624 40. Ware JE, Snow KK, Kosinski M, Gandek B (1993) F- 36 health
survey: manual and interpretation guide. Nimrod Press, Boston 41. Wilson L, Marsden-Loftus I, Koskinen S et al (2017) Interpreting
quality of life after brain injury scores: cross-walk with the short
form-36. J Neurotrauma 34(1):59–65 35. References Slewa-Younan S, van den Berg S, Baguley IJ, Nott M, Cameron ID
(2008) Towards an understanding of sex differences in functional
outcome following moderate to severe traumatic brain injury: a
systematic review. J Neurol Neurosurg Psychiatry 79(11):1197–
1201 42. Wilson JT, Pettigrew LE, Teasdale GM (2000) Emotional and cog-
nitive consequences of head injury in relation to the glasgow out-
come scale. J Neurol Neurosurg Psychiatry 69(2):204–20s9 43. Wood RL (2013) Recognising and assessing neurobehavioural dis-
ability after traumatic brain injury. NeuroRehabilitation 32(4):699–
706 36. von Steinbuechel N, Covic A, Polinder S et al (2016) Assessment of
health-related quality of life after TBI: comparison of a disease-
specific (QOLIBRI) with a generic (SF-36) instrument. Behav
Neurol 2016:1–14
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Detecting Tuberculosis in Prisons: Switching Off the Disease at Its Source
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Clinical infectious diseases/Clinical infectious diseases (Online. University of Chicago. Press)
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© The Author(s) 2020. Published by Oxford University Press for the Infectious
Diseases Society of America.
This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits
unrestricted reuse, distribution, and reproduction in any medium, provided the
original work is properly cited. Detecting Tuberculosis in Prisons: Switching Off the Disease at its Source Detecting Tuberculosis in Prisons: Switching Off the Disease at its Source Dr. Marc Woodman MD1, Dr. Louis Grandjean MD PhD1 Dr. Marc Woodman MD1, Dr. Louis Grandjean MD PhD1 1. Department of Infection, Immunity and Inflammation, Institute of
Child Health, University College London, London, UK, WC1N 1EH Corresponding author: l.grandjean@ucl.ac.uk
Phone: 020 7905 2215
Fax: 020 7905 2882
Corresponding author Institute of Child Health, 30 Guilford Street, University College
London, WC1N 1EH Corresponding author: l.grandjean@ucl.ac.uk
Phone: 020 7905 2215
Fax: 020 7905 2882
Corresponding author Institute of Child Health, 30 Guilford Street, University College
London, WC1N 1EH Corresponding author: l.grandjean@ucl.ac.uk © The Author(s) 2020. Published by Oxford University Press for the Infectious
Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits
unrestricted reuse, distribution, and reproduction in any medium, provided the
original work is properly cited. Mycobacterium tuberculosis is estimated to have killed 1 billion people over the last
200 years (1) and remains the worlds' most deadly human pathogen (2). In order to improve upon the current 2% annual decline in tuberculosis incidence
and get anywhere near the 90% by 2035 reduction target of the World Health
Organization (WHO) "End Tuberculosis Strategy" (3), tuberculosis must be stopped
at source. There are few sources of tuberculosis worthier of intervention than tuberculosis
disease arising in prisons. In resource poor settings, the incidence of tuberculosis is
on average 23 times greater inside prisons than in the surrounding community (4). The incidence in the Brazilian prisons in this study was 3,900 per 100,000, one
hundred times that of the general population. Unless tuberculosis disease is
diagnosed and treated within the prison, on release, prisoners are more likely to
return to high risk transmission networks and fuel the epidemic further (5). Prison
settings often compound the problem by facilitating transmission through
overcrowding, inadequate ventilation, incomplete treatment, late case detection
and high prisoner turnover (6). Thus, the case for prison intervention is compelling. It is naïve to think that tuberculosis disease is contained within the walls of the
prison. As a case in point, Sarita Colonia prison in the province of Callao, Peru
according to publicly available figures is overcrowded by 483% (7). Detecting Tuberculosis in Prisons: Switching Off the Disease at its Source In 2018, it was
shown that living in close proximity to the prison significantly increased the
probability of sharing identical tuberculosis pathogen genotypes with those inside
the prison (8). Prison intervention will therefore diminish the incidence of
tuberculosis cases in the surrounding community. Arguably even more concerning;
due to prisoner exchange between countries, new cases of identical transmitted
strains from Sarita Colonia prison in Peru have now emerged in both Florence and
Madrid (9). Therefore, prison intervention may even act to contain international
tuberculosis spread. Although much research time and many publications have focussed on the benefits
of intervening in prisons to diagnose and treat tuberculosis, there remains a lack of
evidence to determine which - of all the options available - is the best employ (10). Santos and co-authors in this edition of Clinical Infectious Diseases present the
results of an ambitious yet well delivered study that helps to address the gaps in this
important field of research. The authors intensively and prospectively screened consenting prisoners in three
Brazilian prisons for tuberculosis using symptom screening, GeneXpert, sputum
culture and chest radiography with Computer-Aided Detection for Tuberculosis
(CAD4TB). They then retrospectively applied four alternative less comprehensive
screening algorithms to the data to determine which was most cost effective per
case detected. They found that 84% of tuberculosis cases were detectable by a
single sputum sample for Xpert MTB/RIF, and that systematic screening with this
method had a cost per case of US$234. By comparison, symptom screening had a
similar cost per case detected at US$235, but missed twice as many cases, while
algorithms involving CXR screening were more expensive and did not increase
overall yield compared to testing with sputum Xpert/MTBRIF alone. This is a clear, well written paper drawn from research undertaken in extremely
difficult field conditions that has generated clinically applicable results. It
demonstrates that the most sensitive and effective algorithm - of those that were
tested - to detect tuberculosis disease in prisons is to apply GeneXpert to any
prisoner who is able to produce sputum. Considering the challenging environment
that the authors were working in, the screening participation rate of 89.9% is
impressive and adds to the generalizability of the study. Only 31% of patients were able to produce a sputum sample, which, as the authors
conclude themselves likely underestimates the true burden of tuberculosis disease. Detecting Tuberculosis in Prisons: Switching Off the Disease at its Source This raises the question of whether interferon gamma release assays and/or
tuberculin skin testing together with chest radiography could have a role in
detecting these cases that were potentially missed. While many studies have
demonstrated the utility of GeneXpert as a point of care test and it is therefore
expected that it would improve screening algorithms wherever it is applied, there
are few studies that examine its use head to head with other algorithms in prisons. Overall, the merit of this paper rests on the quality of its intensive screening strategy
and the fact that this was applied to all prisoners in an extremely high burden
setting. It clearly demonstrates the utility of GeneXpert in prisons - one of the major
sources of new tuberculosis disease - and argues correctly for the implementation of
this test in similar settings worldwide. If the WHO End TB targets are to be achieved
then well-informed, well-funded and widespread scaling-up tuberculosis control in
prisons is a good place to start. Neither author has any potential conflicts to disclose. References 1. Paulson T. Epidemiology: A mortal foe. Nature. 2013 Oct 10;502(7470):S2-3. 2. Gordon SV, Parish T. Microbe Profile: Mycobacterium tuberculosis:
Humanity’s deadly microbial foe. Microbiol Read Engl. 2018;164(4):437–9. 3. Uplekar M, Weil D, Lonnroth K, Jaramillo E, Lienhardt C, Dias HM, et al. WHO’s new end TB strategy. Lancet Lond Engl. 2015 May 2;385(9979):1799–801. 4. Baussano I, Williams BG, Nunn P, Beggiato M, Fedeli U, Scano F. Tuberculosis
Incidence in Prisons: A Systematic Review. PLoS Med [Internet]. 2010 Dec 21 [cited
2020 Jan 31];7(12). Available from:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3006353/ 5. Altice FL, Azbel L, Stone J, Brooks-Pollock E, Smyrnov P, Dvoriak S, et al. The
perfect storm: incarceration and the high-risk environment perpetuating
transmission of HIV, hepatitis C virus, and tuberculosis in Eastern Europe and Central
Asia. Lancet Lond Engl. 2016 Sep 17;388(10050):1228–48. 6. Escombe AR, Oeser CC, Gilman RH, Navincopa M, Ticona E, Pan W, et al. Natural ventilation for the prevention of airborne contagion. PLoS Med. 2007
Feb;4(2):e68. 7. INPE. Informe Estadistico Penetenciario [Internet]. Ministerio de Justicia y
Derechos Humanos; 2016 [cited 2020 Jan 31]. Available from:
https://www.inpe.gob.pe/revistas/estadistica/2016/octubre2016/mobile/index.htm
l 8. Warren JL, Grandjean L, Moore DAJ, Lithgow A, Coronel J, Sheen P, et al. Investigating spillover of multidrug-resistant tuberculosis from a prison: a spatial and
molecular epidemiological analysis. BMC Med. 2018 Aug 3;16(1):122. 9. Perez Garcia L. Detecting Tuberculosis in Prisons: Switching Off the Disease at its Source Tuberculosis en un escenario global: Nuevas estrategias
transnacionales para optimizar la vigilancia de su transmission. XV Congreso
Argentino de Microbiologia CAM 2019; 2019 Sep 26; Buenos Aires. 10. Systematic review on the diagnosis, treatment, care and prevention of
tuberculosis in prison settings [Internet]. European Centre for Disease Prevention
and Control. 2017 [cited 2020 Jan 31]. Available from:
https://www.ecdc.europa.eu/en/publications-data/systematic-review-diagnosis-
treatment-care-and-prevention-tuberculosis-prison
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Developing selective media for quantification of multispecies biofilms following antibiotic treatment
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PloS one
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cc-by
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Developing selective media for quantification
of multispecies biofilms following antibiotic
treatment Eva Vandeplassche, Tom Coenye, Aure´lie Crabbe´*
Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium Eva Vandeplassche, Tom Coenye, Aure´lie Crabbe´*
Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium * Aurelie.Crabbe@UGent.be * Aurelie.Crabbe@UGent.be OPEN ACCESS Citation: Vandeplassche E, Coenye T, Crabbe´ A
(2017) Developing selective media for
quantification of multispecies biofilms following
antibiotic treatment. PLoS ONE 12(11): e0187540. https://doi.org/10.1371/journal.pone.0187540
Editor: Eduard Torrents, Institute for
Bioengineering of Catalonia (IBEC), SPAIN
Received: July 5, 2017
Accepted: October 20, 2017
Published: November 9, 2017
Copyright: © 2017 Vandeplassche 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. Citation: Vandeplassche E, Coenye T, Crabbe´ A
(2017) Developing selective media for
quantification of multispecies biofilms following
antibiotic treatment. PLoS ONE 12(11): e0187540. https://doi.org/10.1371/journal.pone.0187540 Editor: Eduard Torrents, Institute for
Bioengineering of Catalonia (IBEC), SPAIN Copyright: © 2017 Vandeplassche 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. RESEARCH ARTICLE a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 Abstract a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 The lungs of cystic fibrosis (CF) patients are chronically colonized by a polymicrobial biofilm
community, leading to difficult-to-treat infections. To combat these infections, CF patients
are commonly treated with a variety of antibiotics. Understanding the dynamics of polymi-
crobial community composition in response to antibiotic therapy is essential in the search for
novel therapies. Culture-dependent quantification of individual bacteria from defined multi-
species biofilms is frequently carried out by plating on selective media. However, the influ-
ence of the selective agents in these media on quantitative recovery before or after
antibiotic treatment is often unknown. In the present study we developed selective media for
six bacterial species that are frequently co-isolated from the CF lung, i.e. Pseudomonas aer-
uginosa, Staphylococcus aureus, Streptococcus anginosus, Achromobacter xylosoxidans,
Rothia mucilaginosa, and Gemella haemolysans. We show that certain supplementations to
selective media strongly influence quantitative recovery of (un)treated biofilms. Hence, the
developed media were optimized for selectivity and quantitative recovery before or after
treatment with antibiotics of four major classes, i.e. ceftazidime, ciprofloxacin, colistin, or
tobramycin. Finally, in a proof of concept experiment the novel selective media were applied
to determine the community composition of multispecies biofilms before and after treatment
with tobramycin. Introduction Chronic respiratory tract infections are the main cause of morbidity and mortality in cystic
fibrosis (CF) patients [1]. In the CF lung environment, a polymicrobial community is the
source of these continuing infections, which results in an inflammatory response and eventu-
ally leads to lung deterioration [2–5]. Treatment of chronic lung infections in CF patients is
particularly challenging as members of the bacterial community form biofilms [6] which are
highly resistant to antimicrobial therapy [7,8]. The presence of biofilm-related infections has a
significant impact on clinical health outcomes, more specifically on lung function [9]. Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. Funding: This work was funded by an Odysseus
grant from the Research Foundation Flanders (AC,
TC). Interspecies interactions in polymicrobial communities have been shown to influence vari-
ous phenotypic characteristics of individual bacteria, such as antimicrobial susceptibility and Competing interests: The authors have declared
that no competing interests exist. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 1 / 15 Selective media for quantification of multispecies biofilms production of virulence factors [10]. Hence, the importance of studying the role of complex
bacterial communities in disease progression and antibiotic susceptibility is generally acknowl-
edged [10,11]. To further elucidate the role of interspecies interactions in antibiotic suscepti-
bility of defined polymicrobial communities, in vitro studies have provided valuable insights
[12–14]. To this end, quantification of bacteria in multispecies consortia before and after anti-
biotic treatment is performed using culture-dependent and -independent techniques. One of
the most commonly used culture-dependent quantification methods for biofilms is determina-
tion of CFU (colony forming unit) counts on selective media. Most bacteria in the CF lung are
culturable, making this an effective approach for quantification of CF-related biofilms [15,16]. While selective media are available for a broad range of microorganisms, many are focused
solely on isolation from clinical or environmental samples and not on quantitative recovery
[17–20]. Few studies have investigated how the quantitative recovery on selective media com-
pares to that on a general medium [21–23], although this is essential to obtain an accurate pic-
ture of the amounts of particular taxa in bacterial communities. Furthermore, the influence of
antibiotic treatment on the recovery on a selective medium, which is often supplemented with
various antimicrobial substances, has not yet been investigated. Introduction This is important, as prior
antibiotic exposure could increase susceptibility to the selective agent(s) in the medium, and
therefore affect quantitative recovery. The present study aims at developing selective media for six bacterial species commonly
found together in the CF lung in order to allow accurate quantification of individual species in
complex biofilm communities, before and after antibiotic treatment. Furthermore, previously
described selective media were evaluated in the process as well. Our approach for the develop-
ment of a selective medium for each of the species of interest consists of three steps. First, the
selectivity for each species was assessed using planktonic cultures. Second, the quantitative
recovery of single-species biofilms was compared between different media. Third, the single-
species biofilms were treated with antibiotics of four different classes after which the recovery
was evaluated again. Based on results obtained in each step, media composition was optimized
to improve quantitative recovery on the selective medium. Finally, the developed media were
tested in various experiments, to confirm their applicability for quantification of bacteria from
planktonic cultures and from biofilms, before and after antibiotic treatment. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 General media For each of the six bacterial species included, a general medium with a theoretical recovery of
100% was used: Luria Bertani (LB; Lab M) agar for P. aeruginosa and S. aureus, Brain Heart
Infusion (BHI; Lab M) agar for S. anginosus, Nutrient agar (Lab M) for A. xylosoxidans and R. mucilaginosa, and Columbia agar (CA; Lab M) or Columbia Blood Agar (CBA; Columbia
Agar base, Lab M + 5% defibrinated sheep blood, Biotrading) for G. haemolysans. Bacterial strains and culturing conditions Six bacterial species (clinical strains and one type strain; listed in Table 1) that are frequently
co-isolated from CF patients were selected for the development of selective media: Pseudomo-
nas aeruginosa, Staphylococcus aureus, Streptococcus anginosus, Achromobacter xylosoxidans,
Rothia mucilaginosa, and Gemella haemolysans [24–27]. For each species, at least one strain
was included that was isolated from the respiratory tract or, when this was not readily available
in public repositories, a type strain was used. For all strains, liquid cultures were grown in BHI (Brain Heart Infusion) broth (Lab M) at
37˚C with shaking at 250 rpm until stationary phase. For S. anginosus and G. haemolysans, cul-
tures were incubated in microaerophilic conditions (±5% O2, ±15% CO2). When indicated, microaerophilic conditions (±5% O2, ±15% CO2) were obtained using the
CampyGen Compact system (Oxoid, Thermo Fisher Scientific). To create an anaerobic envi-
ronment (<1% O2), AnaeroGen Compact sachets (Oxoid, Thermo Fischer Scientific) or can-
dle jars with the Anaerocult A system (Merck Millipore) were used. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 2 / 15 Selective media for quantification of multispecies biofilms Table 1. Overview of strains used in this study and isolation sites. Species
Strain
Isolation site
Pseudomonas aeruginosa
AA2
Lung; early CF infection [28]
AA44
Lung; late CF infection [28]
PAO1
Wound
Staphylococcus aureus
SP123
Sputum of extubated patient [29]
Mu50
Wound
Streptococcus anginosus
LMG 14696
Human throat
LMG 14502
Human respiratory tract
Achromobacter xylosoxidans
LMG 26680
Human blood
LMG 14980
Sputum—Belgian CF patient
Rothia mucilaginosa
DSM 20746
Bronchial secretion
ATCC 49042
Throat
Gemella haemolysans
LMG 18984
Unknown
LMG 1068
Type strain
https://doi.org/10.1371/journal.pone.0187540.t001 Table 1. Overview of strains used in this study and isolation sites. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 Formation of single- and multispecies biofilms All biofilms were grown in a PVC flat-bottomed 96-well microtiter plate (Thermo Fisher) as
described previously, with modifications [35,36]. Stationary phase liquid cultures were diluted
in BHI supplemented with 2.5% lysed horse blood (horse blood from Biotrading, protocol for
lysed horse blood according to EUCAST). For single-species biofilms, 100 μL of a 2.5 x 107
CFU/mL suspension was transferred to a 96-well plate. For multispecies biofilms, 2.5 x 106
CFU of each strain were added per well to obtain an equal number of cells per species per well. The inoculated 96-well plates were incubated at 37˚C for 24 hours in a microaerophilic envi-
ronment, thereby mimicking the low oxygen conditions in the CF lung [37,38]. Media and chemicals used for development of the selective media Previously described selective media. Previously described selective media were exam-
ined for selectivity and adjusted when necessary. For all media, each selective component was
examined to distinguish which components were essential for selective inhibition. For P. aeru-
ginosa, Pseudomonas Isolation Agar (PIA; BD Diagnostics) is described which uses 25 μg/mL
triclosan (Irgasan, Sigma Aldrich) as a selective agent since most bacterial species are suscepti-
ble while P. aeruginosa is resistant [30,31]. For S. aureus, Mannitol Salt Agar is widely used for
selection, based on a 7.5% NaCl concentration [32,33]. For S. anginosus, several media were
considered: NAS (nalidixic acid sulfamethazine) agar [19], Mitis Salivarius Agar (Sigma-
Aldrich) and Edwards medium (Oxoid, Thermo Fisher Scientific). For A. xylosoxidans,
MCXVAA medium is described [18], based on MacConkey agar supplemented with 5 mg/mL
xylose, 20 μg/mL vancomycin, 20 μg/mL aztreonam and 5 μg/mL amphotericin. For R. mucila-
ginosa, Rothia Mucilaginosa Selective Medium (RMSM) was previously developed, containing
50 μg/mL sodium selenite and 10 μg/mL colistin [22]. Finally, for G. haemolysans, a supple-
mented Edwards medium with 5 μg/mL colistin sulphate and 2.5 μg/mL oxolinic acid was
described [20]. Antimicrobial susceptibility testing. To identify potential selective or discriminating
antibiotic agents, an antibiotic susceptibility screening was performed. Susceptibility of the six
species to 25 antibiotics was assessed using the disk diffusion assay (S1 Table). To this end,
antibiotic disks were placed on inoculated BHI agar, according to the manufacturer’s instruc-
tions. Subsequently, the MIC90 of selected antibiotics (S2 Table) was determined using the
broth microdilution method according to EUCAST guidelines [34]. For all antibiotics, a range
of 0.5 – 256 μg/mL was tested in BHI or nutrient broth, depending on the general medium for
the intended strain. 3 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 Selective media for quantification of multispecies biofilms Selectivity testing The ability of the medium to select for each of the six species was checked by plating. A plank-
tonic culture of each species, grown to stationary phase in BHI medium, was brought to an
OD 590 nm corresponding to approximately 5 x 107 CFU/mL, serially diluted and then plated
on the respective general medium as a positive control (theoretical recovery of 100%) and in
parallel on each of the candidate selective media (detection limit = 102 CFU/mL). The medium
was considered selective if distinct, countable colonies (clear growth) could be observed for
the intended species only. All media were incubated at 37˚C for 16 hours or until colonies
were of sufficient size for counting. https://doi.org/10.1371/journal.pone.0187540.t002 Concentrations in italic show additional concentrations tested when either no effect or complete removal of biofilm growth occurred with the initial
concentrations. Antibiotic treatment of biofilms After 24 h, biofilms were treated with antibiotics as described previously [35]. Briefly, the bio-
films were rinsed with physiological saline solution (0.9% NaCl in milliQ water). Afterwards,
100 μL of medium (BHI + 2.5% lysed horse blood) was added to the control biofilms or 100 μL
of antibiotic solution in the same medium was added to the biofilms. Four antimicrobials from
four major antibiotic classes were selected. An overview of the antibiotics and their concentra-
tions used is shown in Table 2. As a starting point, one set of antibiotic concentrations that
gave a decrease of one to two logs of P. aeruginosa biofilm was used on all strains. Other con-
centrations of antibiotics were tested when no effect was observed for the other bacterial spe-
cies or in case of complete biofilm killing (detection limit of 102 CFU/mL) with the initial test Table 2. Overview of antibiotic concentrations used for quantitative recovery experiments. Ceftazidime
Ciprofloxacin
Colistin
Tobramycin
Pseudomonas aeruginosa
2000 μg/mL
0.5 μg/mL
500 μg/mL
100 μg/mL
Staphylococcus aureus
2000 μg/mL
0.5 μg/mL
500 μg/mL
100 μg/mL
200 μg/mL
400 μg/mL
Streptococcus anginosus
2000 μg/mL
0.5 μg/mL
500 μg/mL
100 μg/mL
4 μg/mL
Achromobacter xylosoxidans
2000 μg/mL
0.5 μg/mL
500 μg/mL
100 μg/mL
1000 μg/mL
4 μg/mL
Rothia mucilaginosa
2000 μg/mL
0.5 μg/mL
500 μg/mL
100 μg/mL
1000 μg/mL
4 μg/mL
Gemella haemolysans
2000 μg/mL
0.5 μg/mL
500 μg/mL
100 μg/mL
4 μg/mL
25 μg/mL
Concentrations in italic show additional concentrations tested when either no effect or complete removal of biofilm growth occurred with the initial
concentrations. https://doi.org/10.1371/journal.pone.0187540.t002 Overview of antibiotic concentrations used for quantitative recovery experiments. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 4 / 15 Selective media for quantification of multispecies biofilms concentration. All biofilms were incubated at 37˚C under microaerophilic conditions for an
additional 24 hours. concentration. All biofilms were incubated at 37˚C under microaerophilic conditions for an
additional 24 hours. Biofilm quantification and recovery testing Following antibiotic treatment of single or multispecies biofilms, biofilms were rinsed with
physiological saline. To quantify the number of colony forming units, biofilms were homoge-
nized by two rounds of vortexing (900 rpm, 5 min) and sonication (5 min; Branson Ultrasonic
bath), as described previously [35]. The resulting suspensions were serially diluted and plated
on the appropriate general and/or selective media. Plates were incubated at 37˚C overnight (16
hours) or until colonies were of sufficient size for counting. Recovery was considered quantita-
tive when no significant difference was observed between the number of CFU on the general
medium compared to the selective medium. Statistics All experiments were carried out at least in biological triplicate. Biofilm experiments contained
three technical replicates. Statistical analyses were performed in SPSS 23.0. Normality of the
data was checked through the Shapiro-Wilk test. Differences between the means of normally
distributed data were assessed by ANOVA-testing, followed by a Dunnett’s post hoc analysis,
or by an independent samples t-test. When normality was not confirmed, a Kruskal-Wallis
non-parametric test or Mann-Whitney test was performed. Means and standard deviations are
shown in each graph. Statistical significance is assumed when p-values are < 0.05. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 Selective media for quantification of multispecies biofilms Fig 1. Overview of selectivity experiments. The first column shows the intended species and an abbreviated name of the
medium, the second column (O2) shows aerobic (A) or anaerobic (AN) incubation, and the third column (t) shows incubation
times. Strains belonging to the same species showed the same results. Pa: P. aeruginosa AA2, PAO1, and AA44; Sa: S. aureus SP123 and Mu50; Sag: S. anginosus LMG 14696 and LMG 14502; Ax: A. xylosoxidans LMG 26680 and LMG
14980; Rm: R. mucilaginosa DSM 20746 and ATCC 49042; Gh: G. haemolysans LMG 18984 and LMG 1068. Results are
shown for all tested media: Pseudomonas Isolation Agar (PIA), LB + triclosan (LB tricl), LB + 7.5% NaCl (LB NaCl),
nalidixic acid sulphamethazine agar (NAS), Mitis-salivarius agar (MSA), McConkey + 5 mg/mL xylose + 20 μg/mL
vancomycin (McXV), McConkey + 5 mg/mL xylose + 20 μg/mL vancomycin (McXV) + 20 μg/mL aztreonam (McXVA),
McConkey + aztreonam (McA), Rothia mucilaginosa selective medium (RMSM), nutrient agar + 5 μg/mL mupirocin + 10 μg/
mL colistin sulphate (NMC), Edwards medium + 10 μg/mL colistin sulphate (Edwards + col), Columbia blood agar + 32/
6.4 μg/mL co-trimoxazole (CBA + co32), Columbia agar + 32/6.4 μg/mL co-trimoxazole (CA + co32). https://doi org/10 1371/journal pone 0187540 g001 Fig 1. Overview of selectivity experiments. The first column shows the intended species and an abbreviated name of the
medium, the second column (O2) shows aerobic (A) or anaerobic (AN) incubation, and the third column (t) shows incubation
times. Strains belonging to the same species showed the same results. Pa: P. aeruginosa AA2, PAO1, and AA44; Sa: S. aureus SP123 and Mu50; Sag: S. anginosus LMG 14696 and LMG 14502; Ax: A. xylosoxidans LMG 26680 and LMG
14980; Rm: R. mucilaginosa DSM 20746 and ATCC 49042; Gh: G. haemolysans LMG 18984 and LMG 1068. Results are Fig 1. Overview of selectivity experiments. The first column shows the intended species and an abbreviated name of the
medium, the second column (O2) shows aerobic (A) or anaerobic (AN) incubation, and the third column (t) shows incubation
times. Strains belonging to the same species showed the same results. Pa: P. aeruginosa AA2, PAO1, and AA44; Sa: S. aureus SP123 and Mu50; Sag: S. anginosus LMG 14696 and LMG 14502; Ax: A. xylosoxidans LMG 26680 and LMG
14980; Rm: R. mucilaginosa DSM 20746 and ATCC 49042; Gh: G. Assessment of selectivity and quantitative recovery of selective media The results of the selectivity testing using planktonic cultures on previously described and
modified selective media are summarized in Fig 1. Following a positive outcome for selectivity,
the selective media were evaluated for quantitative recovery of cells from untreated biofilms
(Fig 2). P. aeruginosa. We confirmed selectivity of Pseudomonas Isolation Agar (PIA, containing
25 μg/mL triclosan). In order to reduce the concentration of the selective agent, lower triclosan
concentrations (5 – 0.31 μg/mL in LB agar) were tested. All tested concentrations of triclosan
inhibited growth of all the other bacteria while allowing growth of P. aeruginosa after aerobic
overnight (16h) incubation. At concentrations of 1.25 μg/mL triclosan or higher, S. anginosus
showed growth of small colonies after 16h incubation which did not interfere with quantifica-
tion of P. aeruginosa. It is important to notice that longer incubation times (24-48h) lead to
interference of S. anginosus and A. xylosoxidans growth for quantification of P. aeruginosa,
highlighting the importance of selecting appropriate incubation times. Subsequently, recovery
of a single-species biofilm was tested on triclosan-supplemented LB agar. Addition of 5 μg/mL
triclosan resulted in a significant decrease of P. aeruginosa recovery (p < 0.05), while addition
of 2.5 and 1.25 μg/mL triclosan did not affect recovery. Hence, LB agar with 1.25 μg/mL triclo-
san (LB tricl 1.25) was used for further experiments. S. aureus. Mannitol Salt Agar contains 7.5% NaCl, and we evaluated LB agar supple-
mented with the same amount of NaCl (LB NaCl). Aerobic overnight incubation showed that
only S. aureus was able to grow on this medium, confirming its selectivity. Furthermore, recov-
ery of biofilm cells on LB NaCl showed no difference in CFU counts compared to LB agar. S. anginosus. NAS agar and Mitis Salivarius Agar combined with anaerobic incubation
were examined for selectivity but both media also supported growth of S. aureus and R. 5 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 haemolysans LMG 18984 and LMG 1068. Results are
shown for all tested media: Pseudomonas Isolation Agar (PIA), LB + triclosan (LB tricl), LB + 7.5% NaCl (LB NaCl),
nalidixic acid sulphamethazine agar (NAS), Mitis-salivarius agar (MSA), McConkey + 5 mg/mL xylose + 20 μg/mL
vancomycin (McXV), McConkey + 5 mg/mL xylose + 20 μg/mL vancomycin (McXV) + 20 μg/mL aztreonam (McXVA),
McConkey + aztreonam (McA), Rothia mucilaginosa selective medium (RMSM), nutrient agar + 5 μg/mL mupirocin + 10 μg/
mL colistin sulphate (NMC), Edwards medium + 10 μg/mL colistin sulphate (Edwards + col), Columbia blood agar + 32/
6.4 μg/mL co-trimoxazole (CBA + co32), Columbia agar + 32/6.4 μg/mL co-trimoxazole (CA + co32). https://doi.org/10.1371/journal.pone.0187540.g001 https://doi.org/10.1371/journal.pone.0187540.g001 mucilaginosa in these conditions. Since 1.25 μg/mL triclosan permitted growth of S. anginosus,
LB supplemented with 1.25, 0.625 and 0.31 μg/mL triclosan was tested. Incubation took place
under anaerobic conditions to inhibit P. aeruginosa growth, as P. aeruginosa cannot grow
anaerobically without an alternative terminal electron acceptor [39,40]. This approach allowed
growth of S. anginosus only. In addition, LB with triclosan (LB 1.25, 0.625, 0.31) also allowed 6 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 Selective media for quantification of multispecies biofilms Fig 2. Quantification of untreated biofilms of the six bacterial species on candidate selective media compared to a general medium. (A) P. aeruginosa AA2 (B) S. aureus SP 123 (C) S. anginosus LMG 14696 (D) A. xylosoxidans LMG 26680 (E) R. mucilaginosa DSM 20476 (F) G. haemolysans
LMG 18984. PIA = Pseudomonas Isolation Agar; LB tricl = LB agar supplemented with triclosan in various concentrations (μg/mL); LB NaCl = LB
supplemented with 7.5% NaCl; McA5 = McConkey agar supplemented with 5 μg/mL aztreonam; NMC = nutrient agar supplemented with 5 μg/mL
mupirocin and 10 μg/mL colistin sulphate; CA + co32 = Columbia agar with 32/6.4 μg/mL co-trimoxazole. Graphs show mean recovery and standard
deviations. * p < 0.05, n 3. Fig 2. Quantification of untreated biofilms of the six bacterial species on candidate selective media compared to a general medium. (A) P. aeruginosa AA2 (B) S. aureus SP 123 (C) S. anginosus LMG 14696 (D) A. xylosoxidans LMG 26680 (E) R. mucilaginosa DSM 20476 (F) G. haemolysans
LMG 18984. PIA = Pseudomonas Isolation Agar; LB tricl = LB agar supplemented with triclosan in various concentrations (μg/mL); LB NaCl = LB
supplemented with 7.5% NaCl; McA5 = McConkey agar supplemented with 5 μg/mL aztreonam; NMC = nutrient agar supplemented with 5 μg/mL
mupirocin and 10 μg/mL colistin sulphate; CA + co32 = Columbia agar with 32/6.4 μg/mL co-trimoxazole. Graphs show mean recovery and standard
deviations. * p < 0.05, n 3. https://doi.org/10.1371/journal.pone.0187540.g002 https://doi.org/10.1371/journal.pone.0187540.g002 quantitative recovery of cells from single-species S. anginosus biofilms. Based on these results,
LB tricl 0.31 (with anaerobic incubation) was selected for further experiments. A. xylosoxidans. MCXVAA (based on MacConkey agar supplemented with 5 μg/mL
xylose, 20 μg/mL vancomycin, 20 μg/mL aztreonam and 5 μg/mL amphotericin) [18], was con-
firmed to be a selective medium for A. xylosoxidans. The selective value of each of these com-
ponents (except for the anti-fungal amphotericin) was subsequently tested. MacConkey agar
inhibited growth of the Gram-positive bacteria, while addition of 20 μg/mL aztreonam inhib-
ited growth of P. aeruginosa. MacConkey agar with 1 μg/ml (or less) aztreonam also supported
growth of P. aeruginosa, while at concentrations of 10 μg/mL or higher, partial growth inhibi-
tion of A. xylosoxidans was observed. MacConkey supplemented with 5 μg/mL aztreonam
(McA5) was tested further and this medium allowed quantitative recovery of cells from
untreated A. xylosoxidans single-species biofilms. R. mucilaginosa. RMSM only inhibited growth of P. aeruginosa, while the other five spe-
cies, including R. mucilaginosa, showed at least partial growth on this medium. Growth inhibi-
tion of P. aeruginosa was due to 10 μg/mL colistin sulphate, while sodium selenite did not
inhibit growth of any of the species investigated. Results from disk diffusion assays suggested
that mupirocin could be a suitable selective agent (S1 Table). Based on MIC values (S2 Table),
5 μg/mL mupirocin was added to nutrient agar, together with 10 μg/mL colistin sulphate. This
medium (NMC) consisting of nutrient agar, mupirocin and colistin sulphate, was selective for
R. mucilaginosa and allowed quantitative recovery of R. mucilaginosa from untreated biofilms. G. haemolysans. We could not confirm selectivity for the previously described supple-
mented Edwards medium [20] as it did not allow growth of G. haemolysans in our experi-
ments. To develop a new selective medium for G. haemolysans, co-trimoxazole was used as
this antibiotic did not inhibit G. haemolysans growth in the disk diffusion assay, while inhibi-
tion zones were observed for all other species (S1 Table). Based on MIC values (S2 Table) for
all strains, 32/6.4 μg/mL co-trimoxazole was added to CBA. However, besides G. haemolysans,
S. aureus also showed growth on CBA agar with co-trimoxazole. Yet, in the absence of blood
complete growth inhibition was observed for all bacteria except for G. haemolysans, which lead
to the use of Columbia agar with 32/6.4 μg/mL co-trimoxazole (CA + co32) for further testing. Our data showed that the CA + co32 medium allowed quantitative recovery of G. haemolysans
from untreated biofilms. Selective media for quantification of multispecies biofilms Selective media for quantification of multispecies biofilms
PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017
7 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 7 / 15 Quantitative recovery of biofilm cells after antibiotic treatment Treated biofilms were plated on the developed selective media and on general media to assess
quantitative recovery following antibiotic treatment. Results are summarised in Fig 3. The
optimized selective media are presented in Table 3. For P. aeruginosa, no significant difference was observed between the recovery of biofilm
cells on LB, LB with 1.25 μg/mL triclosan, or PIA after treatment with ceftazidime, ciprofloxa-
cin, colistin or tobramycin. Thus, LB tricl 1.25 with aerobic overnight incubation was an
appropriate medium for quantitative recovery of P. aeruginosa biofilm cells after antibiotic
treatment. 8 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 Selective media for quantification of multispecies biofilms Fig 3. Quantification of biofilms after antibiotic treatment on selective media compared to a general medium. (A) P. aeruginosa AA2 (B)
S. aureus SP 123 (C) S. anginosus LMG 14696 (D) A. xylosoxidans LMG 26680 (E) R. mucilaginosa DSM 20476 (F) G. haemolysans LMG 18984. PIA = Pseudomonas Isolation Agar; LB tricl = LB agar supplemented with triclosan in various concentrations (μg/mL); LB NaCl = LB supplemented with
7.5% NaCl; McA5 = McConkey agar supplemented with 5 μg/mL aztreonam; NMC = nutrient agar supplemented with 5 μg/mL mupirocin and 10 μg/mL
colistin sulphate; CA + co32 = Columbia agar with 32/6.4 μg/mL co-trimoxazole. Graphs show mean recovery and standard deviations. * p < 0.05, n 3. https://doi.org/10.1371/journal.pone.0187540.g003 Similarly, in terms of recovery of S. aureus biofilms cells, there was no significant difference
between LB and LB + 7.5% NaCl after treatment with ceftazidime, ciprofloxacin, colistin or
tobramycin. After treatment with ceftazidime, ciprofloxacin, colistin, or tobramycin, complete recovery
of S. anginosus biofilm cells was observed on LB supplemented with 1.25 μg/mL, 0.625 μg/mL
or 0.31 μg/mL triclosan (incubated anaerobically) as no differences with recovery on BHI were
observed. For further experiments LB with 0.31 μg/ml triclosan (LB tricl 0.31) was used
together with anaerobic incubation. On McConkey agar with 5 μg/mL aztreonam, no significant difference in A. xylosoxidans
recovery was observed after treatment with ciprofloxacin, colistin or tobramycin, compared to
nutrient agar. However, after treatment with 2000 μg/mL ceftazidime, a significant decrease
(p < 0.05) in recovery on McA5 was noted compared to nutrient agar. Hence, a lower concen-
tration of aztreonam in MacConkey agar (1 μg/mL; McA1) was tested but this did not restore
recovery of the A. xylosoxidans biofilm treated with ceftazidime. To test whether this effect was
dependent on the concentration of the antibiotic, the concentration of ceftazidime was low-
ered to 1000 μg/mL, which did improve biofilm recovery on both tested media (McA1 and
McA5). However, as seen in the previous selectivity experiments, McA1 showed suboptimal
inhibition of P. aeruginosa and furthermore did not lead to increased recovery compared to
McA5. Taken together, McA5 was used as a selective medium for A. xylosoxidans. However, it
remains important to keep in mind that recovery on McA5 is affected when biofilms are
treated with ceftazidime, which varies dependent on the ceftazidime concentration. Selective media for quantification of multispecies biofilms Selective media for quantification of multispecies biofilms PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 9 / 15 Validation of the selective media For further validation of the six novel selective media, an additional strain (another clinical iso-
late or type strain) of each species was tested (S1 and S2 Figs). The developed selective media
allowed quantitative recovery of all the additional strains, with the exception of A. xylosoxidans
LMG 14980 after ceftazidime treatment, and G. haemolysans LMG 1068 for both the untreated
and treated (with ceftazidime and ciprofloxacin) single-species biofilm. Furthermore, a mixed
planktonic culture with a defined composition of the six bacteria (± 5 x 107 CFU/mL of each)
was plated on the six selective media and recovery was compared to the recovery of single-spe-
cies cultures. The results show no significant decrease in recovery of the strains in the mixed
culture compared to the single-species cultures showing that no growth inhibition occurred
and that the mixed culture did not decrease recovery of the individual strains (S3 Fig). It is there-
fore important to consider that highly concentrated supplementations to selective media may
strongly influence quantitative recovery, in particular after preceding antibiotic treatment. For R. mucilaginosa, no significant difference between the recovery on NMC and nutrient
agar was observed after treatment with either ceftazidime, ciprofloxacin, colistin or Table 3. Composition of optimized selective media. Selective medium
Composition
Pseudomonas aeruginosa
LB tricl 1.25
LB agar
1.25 μg/mL triclosan
Staphylococcus aureus
LB NaCl
LB agar
7.5% NaCl
Streptococcus anginosus
LB tricl 0.31 (anaerobic)
LB agar
0.31 μg/mL triclosan
Achromobacter xylosoxidans
McA5
McConkey agar
5 μg/mL aztreonam
Rothia mucilaginosa
NMC
Nutrient agar
5 μg/mL mupirocin
10 μg/mL colistin sulphate
Gemella haemolysans
CA + co32
Columbia agar
32/6.4 μg/mL co-trimoxazole
https://doi.org/10.1371/journal.pone.0187540.t003 Table 3. Composition of optimized selective media. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 10 / 15 Selective media for quantification of multispecies biofilms Fig 4. Proof of concept: Quantification of multispecies biofilms. A multispecies biofilm of the six bacterial
species was grown for 24 hours and subsequently incubated with fresh medium as an untreated control or
treated with 100 μg/mL tobramycin in medium for an additional 24 hours in microaerophilic conditions. Graphs
show mean recovery and standard deviations. The detection limit of 102 CFU/mL is represented by a dashed
line. * p < 0.05, n 3. htt
//d i
/10 1371/j
l
0187540 004 Fig 4. Proof of concept: Quantification of multispecies biofilms. A multispecies biofilm of the six bacterial
species was grown for 24 hours and subsequently incubated with fresh medium as an untreated control or
treated with 100 μg/mL tobramycin in medium for an additional 24 hours in microaerophilic conditions. Graphs
show mean recovery and standard deviations. The detection limit of 102 CFU/mL is represented by a dashed
line. * p < 0.05, n 3. https://doi.org/10.1371/journal.pone.0187540.g004 https://doi.org/10.1371/journal.pone.0187540.g004 tobramycin. The NMC medium was used for quantitative recovery of R. mucilaginosa follow-
ing antibiotic treatment in further experiments. After treatment with ceftazidime, ciprofloxacin, colistin or tobramycin, no significant dif-
ference in recovery of G. haemolysans on the developed selective medium (CA + co32) com-
pared to Columbia agar was observed, confirming the developed selective medium for
quantification after antibiotic treatment. Conclusion In this study, novel selective media were developed that allow quantitative recovery of (un)
treated multispecies biofilms comprised of six bacteria frequently co-isolated from CF samples. We observed that incubation conditions, including incubation time and oxygen concentration,
were essential to enable selective recovery. Furthermore, evaluation of the developed selective
media for quantitative recovery of biofilms before and after antibiotic treatment revealed that
certain selective agents profoundly affect recovery. These findings highlight the importance of
keeping antibiotic supplementation in selective media to a minimum and to evaluate and
potentially modify available selective media to ensure optimal recovery. In conclusion, six
selective media were optimized for multispecies biofilm recovery, and we demonstrated that if
a selective medium is to be used for quantitative recovery of untreated or antibiotic-treated
cultures, prior recovery assessment is indispensable to avoid introducing biases for data
interpretation. Supporting information S1 Table. Antibiotic disk diffusion assay. Mean inhibition zones and standard deviations
(SD) are shown in mm per strain, ‘z’ shows that a clearer zone could be observed, yet no com-
plete inhibition occurred. This assay was performed for P. aeruginosa PAO1, AA2, and AA44;
S. aureus SP123; S. anginosus LMG 14696; A. xylosoxidans LMG 26680; R. mucilaginosa DSM
20746; G. haemolysans LMG 18984. (PDF) S2 Table. MIC data overview. Minimal Inhibitory Concentrations (range of 256–0.5 μg/mL)
were determined for gentamicin and mupirocin in nutrient broth, and for co-trimoxazole
(SMT-TMP) in BHI broth. -, not determined. S2 Table. MIC data overview. Minimal Inhibitory Concentrations (range of 256–0.5 μg/mL)
were determined for gentamicin and mupirocin in nutrient broth, and for co-trimoxazole
(SMT-TMP) in BHI broth. -, not determined. (PDF) (PDF)
S1 Fig. Quantification of untreated (left) and treated (right) biofilms of additional strains. (A) P. aeruginosa PAO1 and AA44, (B) S. aureus Mu50, PIA = Pseudomonas Isolation Agar;
LB tricl = LB agar supplemented with triclosan in various concentrations (μg/mL); LB
NaCl = LB supplemented with 7.5% NaCl. Graphs show mean recovery and standard devia-
tions. p < 0.05, n 3. (TIF)
S2 Fig. Quantification of untreated (left) and treated (right) biofilms of additional strains. (C) S. anginosus LMG 14502, (D) A. xylosoxidans LMG 26680, (E) R. mucilaginosa ATCC
49042, and (F) G. haemolysans LMG 18984. LB tricl = LB agar supplemented with triclosan in
various concentrations (μg/mL); McA5 = McConkey agar supplemented with 5 μg/mL aztreo-
nam; NMC = nutrient agar supplemented with 5 μg/mL mupirocin and 10 μg/mL colistin sul-
phate; CA + co32 = Columbia agar with 32/6.4 μg/mL co-trimoxazole. Graphs show mean
recovery and standard deviations. p < 0.05, n 3. (TIF) S1 Fig. Quantification of untreated (left) and treated (right) biofilms of additional strains. (A) P. aeruginosa PAO1 and AA44, (B) S. aureus Mu50, PIA = Pseudomonas Isolation Agar;
LB tricl = LB agar supplemented with triclosan in various concentrations (μg/mL); LB
NaCl = LB supplemented with 7.5% NaCl. Graphs show mean recovery and standard devia-
tions. p < 0.05, n 3. (TIF) S2 Fig. Quantification of untreated (left) and treated (right) biofilms of additional strains. (C) S. anginosus LMG 14502, (D) A. xylosoxidans LMG 26680, (E) R. mucilaginosa ATCC
49042, and (F) G. haemolysans LMG 18984. Proof of concept: Multispecies biofilms Finally, as a proof of concept experiment to show that the media can be used to isolate strains
grown in a biofilm, the six bacterial species were quantified in multispecies biofilms, before
and after treatment with 100 μg/mL tobramycin. Biofilms were grown for 24h and subse-
quently treated for 24h in microaerophilic conditions (Fig 4). The results indicate that in the
multispecies biofilm P. aeruginosa, S. anginosus and G. haemolysans become less abundant
after treatment with tobramycin (p < 0.05), while S. aureus, A. xylosoxidans and R. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 11 / 15 Selective media for quantification of multispecies biofilms mucilaginosa are not affected by the treatment. Furthermore, biofilms were also grown for 48h
and subsequently treated for 24h (S4 Fig). All species, except for G. haemolysans, could be iso-
lated from this multispecies biofilm before and after treatment as well. These findings show
that community composition can be altered by antibiotic treatment. Hence, deciphering the
dynamics of multispecies consortia in response to antibiotic treatment using in vitro tools may
help improve our understanding of how multispecies communities respond to antibiotics in
vivo. Acknowledgments The authors would like to thank Charlotte Rigauts, Petra Rigole, Lisa Ostyn and Inne D’Hondt
for their practical assistance. Supporting information LB tricl = LB agar supplemented with triclosan in
various concentrations (μg/mL); McA5 = McConkey agar supplemented with 5 μg/mL aztreo-
nam; NMC = nutrient agar supplemented with 5 μg/mL mupirocin and 10 μg/mL colistin sul-
phate; CA + co32 = Columbia agar with 32/6.4 μg/mL co-trimoxazole. Graphs show mean
recovery and standard deviations. p < 0.05, n 3. (TIF) 12 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 Selective media for quantification of multispecies biofilms S3 Fig. Validation of the novel selective media. Planktonic mono-species cultures and a
mixed culture were plated on the newly developed selective media and showed equal recovery. Graphs show mean recovery and standard deviations. (TIF) S4 Fig. Proof of concept: Quantification of multispecies biofilms. A multispecies biofilm of
the six bacterial species was grown for 48 hours and subsequently incubated with fresh
medium as an untreated control or treated with 100 μg/mL tobramycin in medium for an
additional 24 hours in microaerophilic conditions. Graphs show mean recovery and standard
deviations. The detection limit of 102 CFU/mL is represented by a dashed line. p < 0.05,
n 3. (TIF) Conceptualization: Aure´lie Crabbe´. Data curation: Eva Vandeplassche, Aure´lie Crabbe´. Formal analysis: Eva Vandeplassche. Formal analysis: Eva Vandeplassche. Funding acquisition: Tom Coenye, Aure´lie Crabbe´. Project administration: Aure´lie Crabbe´. Supervision: Tom Coenye, Aure´lie Crabbe´. Supervision: Tom Coenye, Aure´lie Crabbe´. Writing – original draft: Eva Vandeplassche. Writing – original draft: Eva Vandeplassche. Writing – original draft: Eva Vandeplassche. Writing – review & editing: Tom Coenye, Aure´lie Crabbe´. Writing – review & editing: Tom Coenye, Aure´lie Crabbe´. Author Contributions Conceptualization: Aure´lie Crabbe´. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 References Insights into cystic fibrosis polymicrobial consortia: the role of
species interactions in biofilm development, phenotype, and response to in-use antibiotics. Front Micro-
biol. 2017; 7:1–11. 15. Sibley CD, Grinwis ME, Field TR, Eshaghurshan CS, Faria MM, Dowd SE, et al. Culture enriched
molecular profiling of the cystic fibrosis airway microbiome. PLoS One. 2011; 6(7):25–7. 16. Rabin HR, Surette MG. The cystic fibrosis airway microbiome. Curr Opin Pulm Med. 2012; 18(6):622–7
https://doi.org/10.1097/MCP.0b013e328358d49a PMID: 22965275 17. Sibley CD, Grinwis ME, Field TR, Parkins MD, Norgaard JC, Gregson DB, et al. McKay agar enables
routine quantification of the “Streptococcus milleri” group in cystic fibrosis patients. J Med Microbiol. 2010; 59(5):534–40. 18. Amoureux L, Bador J, Fardeheb S, Mabille C, Couchot C, Massip C, et al. Detection of Achromobacter
xylosoxidans in hospital, domestic, and outdoor environmental samples and comparison with human
clinical isolates. Appl Environ Microbiol. 2013; 79(23):7142–9. https://doi.org/10.1128/AEM.02293-13
PMID: 24038696 19. Waite RD, Wareham DW, Gardiner S, Whiley RA. A simple, semiselective medium for anaerobic isola-
tion of anginosus group Streptococci from patients with chronic lung disease. J Clin Microbiol. 2012; 50
(4):1430–2. https://doi.org/10.1128/JCM.06184-11 PMID: 22238446 20. Sawant A a, Pillai SR, Jayarao BM. Evaluation of five selective media for isolation of catalase-negative
gram-positive cocci from bulk tank milk. J Dairy Sci. 2002; 85(5):1127–32. https://doi.org/10.3168/jds. S0022-0302(02)74174-X PMID: 12086047 21. Takada K, Hirasawa M. A novel selective medium for isolation of Streptococcus mutans. J Microbiol
Methods. 2005; 60(2):189–93. https://doi.org/10.1016/j.mimet.2004.09.009 PMID: 15590093 22. Kobayashi T, Uchibori S, Tsuzukibashi O, Goto H, Aida M. A selective medium for Rothia mucilaginosa
and its distribution in oral cavities. J Microbiol Methods. 2012; 91(3):364–5. https://doi.org/10.1016/j. mimet.2012.09.011 PMID: 22995714 23. Ho P-L, Ho L-Y, Yau C-Y, Tong M-K, Chow K-H. A novel selective medium for isolation of Bacteroides
fragilis from clinical specimens. J Clin Microbiol. 2016; 55(2):1–22. 24. Klepac-Ceraj V, Lemon KP, Martin TR, Allgaier M, Kembel SW, Knapp AA, et al. Relationship between
cystic fibrosis respiratory tract bacterial communities and age, genotype, antibiotics and Pseudomonas
aeruginosa. Environ Microbiol. 2010; 12(5):1293–303. https://doi.org/10.1111/j.1462-2920.2010. 02173.x PMID: 20192960 25. Cox MJ, Allgaier M, Taylor B, Baek MS, Huang YJ, Daly RA, et al. Airway microbiota and pathogen
abundance in age-stratified cystic fibrosis patients. PLoS One. 2010; 5(6):e11044. https://doi.org/10. 1371/journal.pone.0011044 PMID: 20585638 26. Zhao J, Schloss PD, Kalikin LM, Carmody LA, Foster BK, Petrosino JF, et al. Decade-long bacterial
community dynamics in cystic fibrosis airways. Proc Natl Acad Sci U S A. 2012; 109(15):5809–14. References 1. Lyczak JB, Cannon CL, Pier GB. Lung infections associated with cystic fibrosis. Clin Microbiol Rev. 2002; 15(2):194–222. https://doi.org/10.1128/CMR.15.2.194-222.2002 PMID: 11932230 2. Sibley CDSD Surette MGSG. The polymicrobial nature of airway infections in cystic fibrosis: Cangene
Gold Medal Lecture. Can J Microbiol. 2011; 57(2):69–77. https://doi.org/10.1139/w10-105 PMID:
21326348 3. Caverly LJ, Zhao J, LiPuma JJ. Cystic fibrosis lung microbiome: opportunities to reconsider manage-
ment of airway infection. Pediatr Pulmonol. 2015; 50(S40):31–8. 4. de Koff EM, Groot KM de W, Bogaert D. Development of the respiratory tract microbiota in cystic fibro-
sis. Curr Opin Pulm Med. 2016; 22(6):623–8. 5. Huang YJ, LiPuma JJ. The microbiome in cystic fibrosis. Clin Chest Med. 2016; 37(1):59–67. https://
doi.org/10.1016/j.ccm.2015.10.003 PMID: 26857768 6. Høiby N, Bjarnsholt T, Moser C, Jensen PØ, Kolpen M, Qvist T, et al. Diagnosis of biofilm infections in
cystic fibrosis patients. APMIS. 2017; 125(4):339–43. https://doi.org/10.1111/apm.12689 PMID:
28407432 6. Høiby N, Bjarnsholt T, Moser C, Jensen PØ, Kolpen M, Qvist T, et al. Diagnosis of biofilm infections in
cystic fibrosis patients. APMIS. 2017; 125(4):339–43. https://doi.org/10.1111/apm.12689 PMID:
28407432 13 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 Selective media for quantification of multispecies biofilms 7. Van Acker H, Coenye T. The role of efflux and physiological adaptation in biofilm tolerance and resis-
tance. J Biol Chem. 2016; 291(24):12565–72. https://doi.org/10.1074/jbc.R115.707257 PMID:
27129224 8. Hall CW, Mah T-F. Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in patho-
genic bacteria. FEMS Microbiol Rev. 2017; 41(3):1–26. 9. Ciofu O, Tolker-Nielsen T, Østrup P, Wang H, Høiby N. Antimicrobial resistance, respiratory tract infec-
tions and role of biofilms in lung infections in cystic fibrosis patients. Adv Drug Deliv Rev. 2015; 85:7–
23. https://doi.org/10.1016/j.addr.2014.11.017 PMID: 25477303 10. Røder HL, Sørensen SJ, Burmølle M. Studying bacterial multispecies biofilms: where to start? Trends
Microbiol. 2016; 24(6):503–13. https://doi.org/10.1016/j.tim.2016.02.019 PMID: 27004827 11. Filkins LM, O’Toole GA. Cystic fibrosis lung infections: polymicrobial, complex, and hard to treat. PLoS
Pathog. 2015; 11(12):1–8. 12. O’Connell HA, Kottkamp GS, Eppelbaum JL, Stubblefield BA, Gilbert SE, Gilbert ES. Influences of bio-
film structure and antibiotic resistance mechanisms on indirect pathogenicity in a model polymicrobial
biofilm. Appl Environ Microbiol. 2006; 72(7):5013–9. https://doi.org/10.1128/AEM.02474-05 PMID:
16820500 13. Lopes SP, Ceri H, Azevedo NF, Pereira MO. Antibiotic resistance of mixed biofilms in cystic fibrosis:
impact of emerging microorganisms on treatment of infection. Int J Antimicrob Agents. 2012; 40
(3):260–3. https://doi.org/10.1016/j.ijantimicag.2012.04.020 PMID: 22770521 14. Magalhães AP, Lopes SP, Pereira MO. References https://doi.org/10.1073/pnas.1120577109 PMID: 22451929 27. Mahenthiralingam E. Emerging cystic fibrosis pathogens and the microbiome. Paediatr Respir Rev. 2014; 15(S1):13–5. 14 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 Selective media for quantification of multispecies biofilms 28. De Soyza A, Hall AJ, Mahenthiralingam E, Drevinek P, Kaca W, Drulis-Kawa Z, et al. Developing an
international Pseudomonas aeruginosa reference panel. Microbiologyopen. 2013; 2(6):1010–23. https://doi.org/10.1002/mbo3.141 PMID: 24214409 29. Vandecandelaere I, Matthijs N, Nelis HJ, Depuydt P, Coenye T. The presence of antibiotic-resistant
nosocomial pathogens in endotracheal tube biofilms and corresponding surveillance cultures. Pathog
Dis. 2013; 69(2):142–8. https://doi.org/10.1111/2049-632X.12100 PMID: 24115610 30. Zhu L, Lin J, Ma J, Cronan JE, Wang H. Triclosan resistance of Pseudomonas aeruginosa PAO1 is due
to FabV, a triclosan-resistant enoyl-acyl carrier protein reductase. Antimicrob Agents Chemother. 2010;
54(2):689–98. https://doi.org/10.1128/AAC.01152-09 PMID: 19933806 31. Chuanchuen R, Karkhoff-Schweizer RR, Schweizer HP. High-level triclosan resistance in Pseudomo-
nas aeruginosa is solely a result of efflux. Am J Infect Control. 2003; 31(2):124–7. PMID: 12665747 32. Parfentjev IA, Catelli AR. Tolerance of Staphylococcus aureus to sodium chloride. J Bacteriol. 1964; 88
(1):1–3. 33. Bradshaw D. Methicillin-resistant Staphylococcus aureus: evaluation of detection techniques on labora-
tory-passaged organisms. Br J Biomed Sci. 1999; 56(3):170–6. PMID: 10824324 34. EUCAST. Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by agar
dilution. Clin Microbiol Infect. 2000; 6(9):509–15. PMID: 11168187 35. Van den Driessche F, Rigole P, Brackman G, Coenye T. Optimization of resazurin-based viability stain-
ing for quantification of microbial biofilms. J Microbiol Methods. 2014; 98(1):31–4. 36. Tavernier S, Coenye T. Quantification of Pseudomonas aeruginosa in multispecies biofilms using PMA-
qPCR. PeerJ. 2015; 3:e787. https://doi.org/10.7717/peerj.787 PMID: 25755923 37. Worlitzsch D, Tarran R, Ulrich M, Schwab U, Cekici A, Meyer KC, et al. Effects of reduced mucus oxy-
gen concentration in airway Pseudomonas infections of cystic fibrosis patients. J Clin Invest. 2002; 109
(3):317–25. https://doi.org/10.1172/JCI13870 PMID: 11827991 38. Cowley ES, Kopf SH, LaRiviere A, Ziebis W, Newman DK. Pediatric cystic fibrosis sputum can be
chemically dynamic, anoxic, and extremely reduced due to hydrogen sulfide formation. MBio. 2015; 6
(4):e00767. https://doi.org/10.1128/mBio.00767-15 PMID: 26220964 39. Wauven C V., Pierard A, Kley-Raymann M, Haas D. Pseudomonas aeruginosa mutants affected in
anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminase path-
way. J Bacteriol. 1984; 160(3):918–34. 40. Sun Yoon S, Hennigan RF, Hilliard GM, Ochsner UA, Parvatiyar K, Kamani MC, et al. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540
November 9, 2017 References Pseudomonas
aeruginosa anaerobic respiration in biofilms: relationships to cystic fibrosis pathogenesis. Dev Cell. 2002; 3(4):593–603. PMID: 12408810 15 / 15
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Diagnosis-specific sickness absence among injured working-aged pedestrians: a sequence analysis
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BMC public health
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Diagnosis‑specific sickness absence
among injured working‑aged pedestrians:
a sequence analysis Linnea Kjeldgård1*, Helena Stigson1,2,3, Eva L. Bergsten1,4, Kristin Farrants1 and Emilie Friberg BMC Public Health BMC Public Health Kjeldgård et al. BMC Public Health (2023) 23:367
https://doi.org/10.1186/s12889-023-15259-w Open Access Abstract Keywords Sick leave, Disability pension, Pedestrians, Fall accidents, Traffic injury, Population-base 3 Folksam Research, Folksam Insurance Group, Stockholm, Sweden
4 Faculty of Health and Occupational Studies, Department
of Occupational Health Sciences and Psychology, University of Gävle, 801
76 Gävle, Sweden 3 Folksam Research, Folksam Insurance Group, Stockholm, Sweden
4 Faculty of Health and Occupational Studies, Department
of Occupational Health Sciences and Psychology, University of Gävle, 801
76 Gävle, Sweden *Correspondence:
Linnea Kjeldgård
linnea.kjeldgard@ki.se
1 Division of Insurance Medicine, Department of Clinical Neuroscience,
Karolinska Institutet, 171 77 Stockholm, Sweden
2 Division of Vehicle Safety, Mechanics and Maritime Sciences, Chalmers
University of Technology, Gothenburg, Sweden Linnea Kjeldgård
linnea.kjeldgard@ki.se
1 Division of Insurance Medicine, Department of Clinical Neuroscience,
Karolinska Institutet, 171 77 Stockholm, Sweden
2 Division of Vehicle Safety, Mechanics and Maritime Sciences, Chalmers
University of Technology, Gothenburg, Sweden 3 Folksam Research, Folksam Insurance Group, Stockholm, Sweden
4 Faculty of Health and Occupational Studies, Department
of Occupational Health Sciences and Psychology, University of Gävle, 801
76 Gävle, Sweden Abstract Background The knowledge about the long-term consequences in terms of sickness absence (SA) among pedestri-
ans injured in a traffic-related accident, including falls, is scarce. Therefore, the aim was to explore diagnosis-specific
patterns of SA during a four-year period and their association with different sociodemographic and occupational
factors among all individuals of working ages who were injured as a pedestrian. Methods A nationwide register-based study, including all individuals aged 20–59 and living in Sweden, who in
2014–2016 had in- or specialized outpatient healthcare after a new traffic-related accident as a pedestrian. Diagnosis-
specific SA (> 14 days) was assessed weekly from one year before the accident up until three years after the accident. Sequence analysis was used to identify patterns (sequences) of SA, and cluster analysis to form clusters of individuals
with similar sequences. Odds ratios (ORs) with 95% confidence intervals (CIs) for association of the different factors
and cluster memberships were estimated by multinomial logistic regression. Results In total, 11,432 pedestrians received healthcare due to a traffic-related accident. Eight clusters of SA patterns
were identified. The largest cluster was characterized by no SA, three clusters had different SA patterns due to injury
diagnoses (immediate, episodic, and later). One cluster had SA both due to injury and other diagnoses. Two clusters had
SA due to other diagnoses (short-term and long-term) and one cluster mainly consisted of individuals with disability
pension (DP). Compared to the cluster “No SA”, all other clusters were associated with older age, no university education,
having been hospitalized, and working in health and social care. The clusters “Immediate SA”, “Episodic SA” and “Both SA
due to injury and other diagnoses” were also associated with higher odds of pedestrians who sustained a fracture. Conclusions This nationwide study of the working-aged pedestrians observed diverging patterns of SA after their
accident. The largest cluster of pedestrians had no SA, and the other seven clusters had different patterns of SA in
terms of diagnosis (injury and other diagnoses) and timing of SA. Differences were found between all clusters regard-
ing sociodemographic and occupational factors. This information can contribute to the understanding of long-term
consequences of road traffic accidents. © 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
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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. Background W02.4, W03.4, W04.4, W05.4, W10.4, W15.4, W17.4,
W18.4, W19.4, W51.4) during two years before the acci-
dent, were included. The date of the accident, denoted as
T0, refers to the first date of the in- or specialized outpa-
tient healthcare visit/hospitalization, as the actual date of
their accident/fall is not included in the registers. g
Walking as physical activity and active transportation is
good for public health and accordingly encouraged by
different stakeholders [1–3]. However, walking in a road
traffic environment (i.e. being a pedestrian) also involves
some risk. Pedestrians can slip, trip, stumble, or collide
with other road users. Globally, about a fifth of all fatali-
ties within the road transport system are represented by
pedestrians [4, 5]. The UN’s global sustainable develop-
ment goals strive both for more sustainable and safer
transportation [5, 6]. Likewise, the Swedish Vision Zero
has the long-term vision that no one should die or suf-
fer injuries leading to long-term consequences within the
road transport system [7].fif The SA-status of the study population was assessed
weekly during a period of four years; one year before and
three years after the accident date, T0. Individuals who
died (n = 213) or emigrated (n = 58) during the follow-
up period were excluded. In addition, those with dis-
ability pension (DP) (full- or part-time) during all of the
209 weeks of the four-year study period were excluded
(n = 1167). This resulted in a study population of 11,432
injured pedestrians. Being injured in a road traffic accident may affect the
individual’s work ability and lead to sickness absence (SA)
or disability pension (DP). We have previously shown that
many (20%) injured pedestrians had a new SA spell in
connection to the accident [8]. However, the knowledge
of long-term SA and the patterns of SA among injured
pedestrians is limited. In addition, diverse work require-
ments for different types of work (e.g. occupational sec-
tors and type of occupation) could impact the injured
pedestrian’s need of SA or DP. However, the impact of
the occupational factors in relation to SA and DP among
pedestrians involved in a traffic-related accident have, to
the best of our knowledge, not yet been explored. Background Microdata from several nationwide registers were used
and linked at the individual level, using the unique personal
identity number assigned to all residents in Sweden [10].h - The in- and specialized outpatient registers, from the
National Board of Health and Welfare, were used to iden-
tify the study population as well as for medical informa-
tion related to the injury.h - The Cause of Death Register, from the National Board
of Health and Welfare, was used to identify those who
had died within three years from the accident date.h - The Longitudinal Integration Database for Health
Insurance and Labour Market Studies (LISA), from Sta-
tistics Sweden, was used to identify the source popula-
tion, all individuals living in Sweden 31 December the
year before the accident, and information for the study
population regarding sociodemographic factors (sex, age,
educational level, country of birth, type of living area,
and marital status) and occupational factors (occupa-
tional sector, private/public, and type of occupation) also
measured 31 December the year before the accident. To get a broader understanding of the long-term con-
sequences in terms of SA among pedestrians injured in
traffic-related accidents, including falls within the road
transport system, the aim of this study was to explore
patterns of diagnosis-specific (i.e. injury diagnoses or
other diagnoses) SA during a four-years period and their
association with different background factors including
occupational factors among all individuals of working
ages who were injured as a pedestrian. - Micro-data for Analyses of the Social Insurance
(MiDAS), from the Swedish Social Insurance Agency, was
used for information on the dates and diagnoses of SA
and DP. Open Access T Kjeldgård et al. BMC Public Health (2023) 23:367 Kjeldgård et al. BMC Public Health (2023) 23:367 Page 2 of 16 Methods using a modified version of the Barell matrix [11], into
categories of injured body region and type of injury. Most of the pedestrian had only one injury diagnosis
recorded, but for those individuals who had several, the
main diagnosis was prioritized before any of the second-
ary diagnoses. Some pedestrians had up to three visits/
hospitalizations at T0. In these cases, the injuries requir-
ing inpatient healthcare were prioritized over the injuries
treated in outpatient healthcare. In addition, an injury
with ICD10: S00-S99 was prioritized over an injury with
ICD10: T00-T88.h All individuals living in Sweden, ≥ 16 years old, and
with income from work, unemployment, or parental-
leave benefits can apply for SA benefits from the Social
Insurance Agency if having a disease or injury that leads
to reduced work capacity [17]. The first day of a SA spell
is an unreimbursed qualifying day (more days for self-
employed). A physician’s certificate is required after
day 7. For employees, day 2–14 are reimbursed by the
employer [17]. For others, e.g., unemployed, the Social
Insurance Agency administers the benefits from the sec-
ond day of SA, with information on shorter SA spells
available for these individuals. Therefore, in order not to
introduce a bias, only information on SA spells > 14 days
was used. All individuals aged 19–64 can be granted
DP if disease or injury leads to long-term or permanent
work incapacity. Both SA and DP can be granted for full-
or part-time (100, 75, 50, 25%) of ordinary work hours. Accordingly, someone on part-time DP can at the same
time have part-time SA. For young individuals (19–
30 years old) DP can be time limited. In general, it is very
uncommon but possible to go from DP to non-DP.i The injured body region was categorized into the fol-
lowing six groups: head, face, and neck (reference group);
vertebral column and spinal cord; torso; upper extremi-
ties; lower extremities; and “other and unspecified”. The
type of injury was categorized into six groups: fracture;
dislocation; sprains and strains; internal (brain, spinal
cord, and other internal organs); external (open wounds,
contusions, and superficial injuries) (reference group);
and “other and unspecified”. Similar categorizations have
been used in recent studies on injuries among different
groups of road traffic users [8, 12–15]. fi
Healthcare at T0 was also used to categorize inpatient
healthcare: no (only specialized outpatient healthcare)
(reference group); yes. Methods If someone had both specialized
outpatient healthcare and inpatient healthcare they were
categorized as inpatient healthcare.h Weekly states of SA were defined for the study popula-
tion. To do this, the four years of follow-up were divided
into 209 weeks, 52 weeks prior (W−52) through 156 weeks
after (W+156) the week of the accident W0, defined as
T0 ± 3 days. For each week, individuals were assigned a
state (one out of four non overlapping states) based on
their SA situation during that week: No SA or DP (no SA
or DP during the week); SA due to injury diagnosis (any
SA due to an injury diagnosis (ICD10: S00-T98) during
the week, and no DP); SA due to other diagnoses (any SA
due to other diagnoses than injuries, no DP, and no SA
due to an injury diagnosis during the week); and DP (any
DP, regardless of extent or diagnosis, during the week). The sociodemographic factors were categorized as:
sex (women; men (reference group)), age group (20–24;
25–34 (reference group); 35–44; 45–54; 55–59 years),
level of education (elementary school (≤ 9 years includ-
ing missing); high school (9–12 years); university/col-
lege (> 12 years) (reference group)), country of birth
(Sweden (reference group); not Sweden), type of living
area (cities (reference group); town and suburbs; rural
areas), marital status (married (reference group); not
married). These have all been demonstrated to be com-
mon risk factors for SA in general [16] and after a traffic
related injury [8, 12–15]. Several occupational factors were also included. Occupational sector was categorized according to the
Swedish Standard of Industrial Classification SNI 2007
(Manufacturing, agriculture, forestry & fishing (G01,
G02, G03); Construction (G04); Trade, transport, hotels
& restaurants (G05, G06, G07); Finance, communica-
tion & cultural service (G08, G09, G10, G11, G12, G15)
(reference group); Education (G13); Health & social
care (G14); Not in work/Unknown (G99)), Private/pub-
lic employer (private (reference group); public; Not in
work/Unknown), and also type of occupation, according
to the Swedish Standard for Occupational Classification
SSYK (white collar (reference group); blue collar; Not in
work/Unknown). Methods A prospective cohort study was conducted, includ-
ing all pedestrians, 20–59 years of age (which is to say
within working ages throughout the follow-up) who had
at least one hospitalization or visit to specialized outpa-
tient healthcare due to an injury (International Statistical
Classification of Diseases and Related Health Problems;
ICD-10 [9]: S00-T88) sustained in a new traffic-related
accident (including falls and bumping into other pedes-
trians) (ICD-10: V01-V09, W00.4, W01.4, W02.4, W03.4,
W04.4, W05.4, W10.4, W15.4, W17.4, W18.4, W19.4,
W51.4) between 1 January 2014 and 31 December 2016
(n = 12,870) and living in Sweden. Individuals who were
living in Sweden the 31 December the year before the
accident and did not have any traffic related in- or out-
patient healthcare (ICD-10: V01-V99, W00.4, W01.4, Reference groups for the below factors were chosen
based on size of the groups and expected proportions
with SA, with larger groups or groups expected to have
lower proportions of SA being used as reference groups.h The type of accident was categorized into the follow-
ing six groups: collision with pedestrian/bicyclist (V01,
W03.4, W04.4, W51.4); collision with motor vehicle (V02-
V06, V09.0, V09.2); unspecified (V09.1, V09.3, V09.9,
W19.4); fall: snow and ice, street and highway (W00.4);
fall: slipping, tripping, and stumbling, street and highway
(W01.4) (reference group); and fall: other, street and high-
way (W02.4, W05.4, W10.4, W15.4, W17.4, W18.4).h The main diagnosis and the secondary diagnoses for
the healthcare visit/hospitalization were categorized Kjeldgård et al. BMC Public Health (2023) 23:367 Kjeldgård et al. BMC Public Health (2023) 23:367 Kjeldgård et al. BMC Public Health (2023) 23:367 Page 3 of 16 Lastly, season was based on T0 and categorized as win-
ter (December, January, February), spring (Mars, April,
May), summer (June, July, August) (reference group),
autumn (September, October, November). using a modified version of the Barell matrix [11], into
categories of injured body region and type of injury. Most of the pedestrian had only one injury diagnosis
recorded, but for those individuals who had several, the
main diagnosis was prioritized before any of the second-
ary diagnoses. Some pedestrians had up to three visits/
hospitalizations at T0. In these cases, the injuries requir-
ing inpatient healthcare were prioritized over the injuries
treated in outpatient healthcare. In addition, an injury
with ICD10: S00-S99 was prioritized over an injury with
ICD10: T00-T88. Resultsh All clusters other than “No SA” were associated with
older age, no university education, having been hospital-
ized, and working in health and social care. There were 11,432 pedestrians with in- or specialized out-
patient healthcare due to a new traffic accident including
fall accidents 2014–2016 aged 20–59 years (Table 1). The
median age was 45 years among women and 39 years
among men. The most common type of accident was fall:
slipping, tripping, and stumbling, followed by fall: snow
and ice. The most common types of injuries were, frac-
tures, external injuries, and sprains and strains injuries. A quarter of the injured pedestrians worked in Finance,
communication & cultural service. Among women, 29%
worked in Health & social care and 14% worked in Trade,
transport, hotels & restaurants; the corresponding num-
bers in men were 5%, and 24% respectively. The cluster “Immediate SA” was associated with the
types of accidents collision with motor vehicle and Fall:
slipping tripping and stumbling (OR (95% CI): 1.54
(1.26–1.87), and 1.25 (1.05–1.48) respectively). Whereas
the clusters “Long-term or later SA” and “Both SA due to
injury and other diagnoses” were associated with injuries
sustained in a collision with pedestrian/bicyclists (1.60
(1.03—2.48), and 1.70 (1.18—2.45) respectively). Fractures, dislocations, sprains and strains, and inter-
nal injuries had high OR for the clusters “Immediate
SA”, “Episodic SA”, and “Both SA due to injury and other
diagnoses”. Diverse patterns of SA during the study period were
observed among the pedestrians (4358 unique sequences). By far the most common sequence observed, for both
women and men, was to have no SA or DP during all
four years (W−52 to W+156) (38.8% for women and 52.5%
for men) (Fig. 1). Several of the most common sequences
included no SA prior to the accident and then SA of various
durations due to injury diagnoses starting from the week of
the accident (W0). Another common sequence observed
among the individuals was to have SA due to other diag-
nosis (i.e. not injury) during the entire study period (0.6%
among women and 0.2% among men) (Fig. 1). Upper extremities had high OR for all cluster but “Dis-
ability pension”. Lower extremities had high OR for all
cluster but “Disability pension” and “Long-term or later
SA”. Injuries to the torso were associated with the clusters
“Episodic SA”, “Both SA due to injury and other diagno-
ses”, and “Other diagnoses short-term SA”. Resultsh Injuries to the
vertebral column and spinal cord were associated with
the clusters “Episodic SA”, “Long-term or later SA”, and
“Other diagnoses short-term SA”. Regarding occupational factors, blue-collar work
was associated with all clusters but “Disability pension”
compared to the cluster “No SA” and working in Health
& social care was associated with all clusters compared
to “No SA”. Working in Construction had high ORs for
being in the clusters “Immediate SA” (1.62 (1.24–2.13))
and “Episodic SA” (2.35 (1.51–3.65)), whereas working in
Education had high ORs for being in the clusters “Both
SA due to injury and other diagnoses” (1.48 (1.08–2.02))
and “Other diagnoses short-term SA” (1.45 (1.16–1.80)). Cluster analysis was then used to form groups of indi-
viduals who had similar sequences. The cluster partition
quality measures for the different numbers of clusters
are presented in the appendix (Table A.1). Eight clus-
ters were identified: “No SA” (including 47% of the study
population), “Immediate SA” (18%), “Episodic SA” (4%),
“Long-term or later SA” (3%), “Both SA due to injury and
other diagnoses” (7%), “Other diagnoses short-term SA”
(17%), “Other diagnoses long-term SA” (2%), and “Disa-
bility pension” (3%). In the description of the clusters, SA
is referred to SA due to an injury diagnosis unless other-
wise stated. The eight clusters are illustrated using den-
sity plots in Fig. 2 and using index plots in Fig. 3. Sensitivity analyses including the 1167 individuals
who had DP during the entire follow-up did not alter the
results substantially (data not shown). Statistical analyses Descriptive statistics of the study population were con-
ducted including stratified by sex. i
The patterns of SA states during a four-year period
(from 1 year before and through 3 years after W0 (W−52
to W+156)) were analyzed using sequence analysis with
TraMineR in R [18]. Thereafter, cluster analysis with
optimal matching spell algorithm[19] was used to iden-
tify different clusters of individuals who had similar
sequences of SA-states. A cluster tree and several meas-
ures of cluster partition quality [18] were used to choose
the number of clusters (Table A.1). Density plots and
index plots for each cluster are shown. Kjeldgård et al. BMC Public Health (2023) 23:367 Kjeldgård et al. BMC Public Health (2023) 23:367 Page 4 of 16 Page 4 of 16 Multinomial logistic regression models were used to
analyze the association between sociodemographic and
occupational factors, type of accident, type of health-
care, type of injury, injured body region, and SA-clusters. Crude and adjusted odds ratios (OR) and 95% confidence
intervals (CI) were calculated. Sensitivity analyses were
conducted including the 1167 individuals who had DP
during the entire follow-up. The statistical analyses were
performed using SAS (version 9.4) and R (version 3.6.1). Almost all of the 5339 individuals in the cluster “No
SA” were not on SA or DP during the entire follow up. In this cluster there were 53% men and 8% were identi-
fied through the in-patient healthcare. The most com-
mon type of injuries among individuals belonging to this
cluster were external injuries (41%) and fractures (29%). Characteristics of the clusters and adjusted ORs for the
other clusters compared to the cluster “No SA” are pre-
sented in Table 3. Descriptive statistics of all factors of
the clusters and unadjusted and adjusted ORs can be
found in the appendix (Table A.2 and Table A.3). Discussion Characteristics of each of the eight identified clusters
are summarised in Table 2 in terms of sequences, soci-
odemographic characteristics, occupational characteris-
tics and injury related characteristics. In this nationwide register study exploring diagnosis-
specific patterns of SA among injured pedestrians
there were in total 11,432 working-aged pedestrians Kjeldgård et al. Discussion BMC Public Health (2023) 23:367 Page 6 of 16 1 column percent
2 row percent
Table 1 (continued)
All
Women
Men
n
%1
n
%1
n
%1
Summer
2329
20.37
1135
18.27
1194
22.87
Autumn
2565
22.44
1402
22.57
1163
22.28
Year of accident
2014
3393
29.68
1788
28.78
1605
30.75
2015
3956
34.60
2172
34.96
1784
34.18
2016
4083
35.72
2252
36.25
1831
35.08
Occupational sector
Manufacturing, agriculture, forestry & fishing
1143
10.00
309
4.97
834
15.98
Construction
586
5.13
63
1.01
523
10.02
Trade, transport, hotels & restaurants
2160
18.89
886
14.26
1274
24.41
Finance, communication & cultural service
2897
25.34
1568
25.24
1329
25.46
Education
1035
9.05
836
13.46
199
3.81
Health & social care
2076
18.16
1792
28.85
284
5.44
Not in work/Unknown
1535
13.43
758
12.20
777
14.89
Private/Public
Private sector
6118
53.52
2651
42.68
3467
66.42
Public sector
2946
25.77
2390
38.47
556
10.65
Not in work/Unknown
2368
20.71
1171
18.85
1197
22.93
Type of occupation
White collar
4539
39.70
3219
51.82
1320
25.29
Blue collar
3295
28.82
1295
20.85
2000
38.31
Not in work/Unknown
3598
31.47
1698
27.33
1900
36.40 [8]. This could partly be explained by that the winter
seasons during 2010 were colder and with more snow-
fall than during the rather mild winters when the pre-
sent study was conducted in 2014–2016 [22–24]. This
difference in proportion of accidents related to snow
and ice highlights the importance of road environment
maintenance during the winter season to reduce the
risk for these types of accidents, which is also called for
by authors of other studies [25, 26]. that received in- or specialized outpatient healthcare
due to a traffic-related accident. Of them, 71% were
due to falls and a third of the falls related to snow and
ice. Eight different clusters of SA were identified with
different patterns of SA due to an injury diagnosis or
whether due to other diagnoses. Compared to the clus-
ter “No SA” all the other clusters were associated with
older age, no university education, having been hos-
pitalized, and working in health and social care. The
clusters “Immediate SA”, “Episodic SA” and “Both SA
due to injury and other diagnoses” were also associ-
ated with e.g. higher odds of individuals who sustained
a fracture. The present study showed a rather diverse popula-
tion is involved in pedestrian road traffic accidents. Discussion BMC Public Health (2023) 23:367 Page 5 of 16 Table 1 Characteristics of all pedestrians aged 20–59 with a road traffic injury (including falls) in 2014–2016, by sex
All
Women
Men
n
%1
n
%1
n
%1
All
11,432
6212
54.342
5220
45.662
Age group, years
20–24
1737
15.19
752
12.11
985
18.87
25–34
2306
20.17
1093
17.59
1213
23.24
35–44
2243
19.62
1156
18.61
1087
20.82
45–54
3144
27.50
1867
30.05
1277
24.46
55–59
2002
17.51
1344
21.64
658
12.61
Level of education
Elementary school
1778
15.55
770
12.40
1008
19.31
High school
5796
50.70
2925
47.09
2871
55.00
University/College
3858
33.75
2517
40.52
1341
25.69
Country of birth
Sweden
9200
80.48
4983
80.22
4217
80.79
Not Sweden
2232
19.52
1229
19.78
1003
19.21
Type of living area
Cities
4752
41.57
2585
41.61
2167
41.51
Towns and suburbs
4723
41.31
2597
41.81
2126
40.73
Rural areas
1957
17.12
1030
16.58
927
17.76
Married
No
7466
65.31
3755
60.45
3711
71.09
Yes
3966
34.69
2457
39.55
1509
28.91
Type of accident
Collision with pedestrian/bicyclist
591
5.17
286
4.60
305
5.84
Collision with motor vehicle
1595
13.95
734
11.82
861
16.49
Unspecified
1166
10.20
547
8.81
619
11.86
Fall: snow and ice
2529
22.12
1582
25.47
947
18.14
Fall: slipping, tripping, and stumbling
4136
36.18
2491
40.10
1645
31.51
Fall: other
1415
12.38
572
9.21
843
16.15
Inpatient healthcare
No
9923
86.80
5442
87.60
4481
85.84
Yes
1509
13.20
770
12.40
739
14.16
Type of injury
Fracture
4587
40.12
2648
42.63
1939
37.15
Dislocation
366
3.20
145
2.33
221
4.23
Sprains and strains
1724
15.08
932
15.00
792
15.17
Internal
794
6.95
404
6.50
390
7.47
External
3749
32.79
1961
31.57
1788
34.25
Other and unspecified
212
1.85
122
1.96
90
1.72
Injured body region
Head, face and neck
2345
20.51
1146
18.45
1199
22.97
Vertebral column and spinal cord
215
1.88
107
1.72
108
2.07
Torso
630
5.51
295
4.75
335
6.42
Upper extremities
4005
35.03
2308
37.15
1697
32.51
Lower extremities
4185
36.61
2330
37.51
1855
35.54
Other and unspecified
52
0.45
26
0.42
26
0.50
Season
Winter
4133
36.15
2444
39.34
1689
32.36
Spring
2405
21.04
1231
19.82
1174
22.49 Table 1 Characteristics of all pedestrians aged 20–59 with a road traffic injury (including falls) in 2014–2016, by sex Kjeldgård et al. Discussion Higher proportions of older women yet more younger
men were injured pedestrians. Women were more
often injured in falls due to slipping, tripping, and
stumbling as well as falls due to snow and ice, while
men were more often injured in falls due to other rea-
sons and collisions with a motor vehicle. In addition,
more women were working in Health & social care and
more men were working in Trade, transport, hotels &
restaurants. Moreover, a higher proportion of women
had university/college education than men. These dif-
ferences among the injured pedestrians are important
to consider, as suggest different measures might be The most common injuries were fractures and exter-
nal injuries, which has also been reported for pedes-
trians in previous studies [1, 8, 20, 21]. Injuries to the
lower and upper extremities and to the head, face and
neck were the most common type of injured body
region, also in line with previous studies [1, 8, 20, 21]. In contrast, the proportion of injuries from falls due to
snow and ice was lower in the present study compared
to a previous study conducted during 2010, 22% com-
pared to 36% of all pedestrian accidents, respectively Kjeldgård et al. BMC Public Health (2023) 23:367 Page 7 of 16 Fig. 1 The 20 most common sequences of sickness absence states/week from one year before until three years after (W-52 to W+156) the week of
the pedestrian accident (W0) (marked as week 0 in the figure) for women and men Fig. 1 The 20 most common sequences of sickness absence states/week from one year before until three years after (W-52 to W+156) the week of
the pedestrian accident (W0) (marked as week 0 in the figure) for women and men accidents [27–30]. Sickness absence and DP have so
far only been studied for pedestrians in our previous
study, a cross sectional study from Sweden, examin-
ing the risk of new SA in connection to the accident,
but with no follow-up or information on the duration
of SA [8]. That study found that 20% had a new SA in
connection to the accident and that 18% had ongoing
SA or full-time DP at the time of the accident [8]. Even
though those that were on DP throughout follow-up
were excluded in the present study, the eight clusters of
SA and DP could be comparable to our cross-sectional
study [8]. Discussion Furthermore, the present study provides a
much clearer picture of the SA and DP development
during the years after the accident. Separating SA diag-
noses into injury diagnoses and other diagnoses also
helps in understanding what SA is due to the accident
and what could be due to other circumstances. The dis-
tinction between the cluster “Episodic SA” and “Both
SA due to injury diagnoses and other diagnoses” could
not have been made without the possibility to identify
SA with injury diagnoses. The pedestrians in the clus-
ter “Episodic SA” had a SA spell due to injury diagno-
sis in connection to the accident and then one more SA
spell due to an injury diagnosis, while the pedestrians called for in order to reduce the number of individu-
als involved in a pedestrian accident for women and
men. For example, personal devices such as reflectors,
to prevent collisions with other road traffic groups, and
anti-slip shoes to prevent falls due to snow and ice, or
related to the traffic environment such as gritting/salt-
ing slippery roads or improving the separation between
road user groups. Another established difference is also
related to the possibility to return to work in different
types of occupations. Rather few studies have reported on long-term conse-
quences of being involved in an accident as a pedestrian. The long-term consequences can be investigated from
several different perspectives, the present study inves-
tigated the long-term consequences in terms of SA and
DP. A study from Sweden reported the long-term conse-
quences, in terms of risk of Permanent Medical Impair-
ment (PMI), showing that 25% of the pedestrian falls
and 20% of the pedestrian collisions led to a permanent
reduction with a PMI of above 1% [21].h The long-term consequences of pedestrian injuries in
terms of SA and DP to the best of our knowledge has
not been previously studied. There are only a few stud-
ies investigating SA and DP in relation to road traffic Kjeldgård et al. BMC Public Health (2023) 23:367 Page 8 of 16 Fig. 2 Density plots of sickness absence (SA) states/week during the year before through three years after (W−52 to W+156) the week of the
pedestrian accident (marked with 0 in the figure), for the eight identified clusters. The number of individuals in each cluster are stated in each
cluster heading Fig. Discussion 2 Density plots of sickness absence (SA) states/week during the year before through three years after (W−52 to W+156) the week of the
pedestrian accident (marked with 0 in the figure), for the eight identified clusters. The number of individuals in each cluster are stated in each
cluster heading Fig. 2 Density plots of sickness absence (SA) states/week during the year before through three years after (W−52 to W+156) the week of the
pedestrian accident (marked with 0 in the figure), for the eight identified clusters. The number of individuals in each cluster are stated in each
cluster heading Fig. 2 Density plots of sickness absence (SA) states/week during the year before through three years after (W−52 to W+156) the week of the
pedestrian accident (marked with 0 in the figure), for the eight identified clusters. The number of individuals in each cluster are stated in each
cluster heading Fig. 2 Density plots of sickness absence (SA) states/week during the year before through three years after (W−52 to W+156) the week of the
pedestrian accident (marked with 0 in the figure), for the eight identified clusters. The number of individuals in each cluster are stated in each
cluster heading It could be in relation to late effects of the accident
(e.g. injuries, musculoskeletal disease, thrombosis,
pneumonia or PTSD) but also a new accident (lead-
ing to injury) or even another health issue not related
to the accident. In relation to other road user groups,
a previous study on bicyclists investigated SA and DP in the cluster “Both SA due to injury diagnoses and
other diagnoses” had first SA due to an injury diagno-
ses at the time of the accident and then had later SA
due to other diagnoses. The SA spell that occurred later
during follow-up does not necessarily need to be con-
nected to the accident, especially for the latter cluster. Kjeldgård et al. BMC Public Health (2023) 23:367 Page 9 of 16 Fig. 3 Index plots of sickness absence (SA) states/week during the year before through three years after (W−52 to W+156) the week of the pedestrian
accident (marked with 0 in the figure), for the eight identified clusters. The number of individuals in each cluster are stated in each cluster heading
and every line represents one individual. The plots are ordered by the first state in the sequences Fig. Discussion 3 Index plots of sickness absence (SA) states/week during the year before through three years after (W−52 to W+156) the week of the pedestrian
accident (marked with 0 in the figure), for the eight identified clusters. The number of individuals in each cluster are stated in each cluster heading
and every line represents one individual. The plots are ordered by the first state in the sequences Fig. 3 Index plots of sickness absence (SA) states/week during the year before through three years after (W−52 to W+156) the week of the pedestrian
accident (marked with 0 in the figure), for the eight identified clusters. The number of individuals in each cluster are stated in each cluster heading
and every line represents one individual. The plots are ordered by the first state in the sequences traffic accidents in terms of SA and DP, future studies
are needed, especially those with a comparison to the
general population. after a bicycle crash but did not make distinctions in
SA diagnoses [15]. In addition, other studies investi-
gating SA after a road traffic accident did not separate
the analyses for different road user groups or did not
differentiate different SA diagnoses in the analysis [27–
30]. To elucidate the long-term consequences of road g
The relationship between occupational factors and
SA after a pedestrian accident has also not been inves-
tigated in detail. However, an Australian study found Page 10 of 16 Kjeldgård et al. BMC Public Health (2023) 23:367 Table 2 Summary of sociodemographic, injury, and occupational characteristics in the eight identified clusters of SA sequences
Cluster
1. No SA
2. Immediate SA
3. Episodic SA
4. Long-term or
later SA
5. Both SA due to
injury and other
diagnoses
6. Other
diagnoses short-
term SA
7. Other
diagnoses long-
term SA
8. Discussion Disability pension
N
5339 individuals
2046 individuals
445 individuals
369 individuals
796 individuals
1901 individuals
233 individuals
303 individuals
Characterisation of
SA sequences
No SA or DP during
the entire study
period
SA due to injury
diagnoses in
connection to the
accident
Two or more SA
spells due to injury
diagnoses, one at
the time of the acci-
dent and one prior
to or later during
the study period
One or several SA
spells due to injury
diagnoses later dur-
ing follow-up
One SA spell due to
an injury diagnosis
starting in connec-
tion to the accident
and also one or sev-
eral SA spells due
to other diagnoses
during the study
period
One or several
short-term SA spells
due to other diag-
noses spread out
during the study
period
Long-term SA due
to other diagnoses
DP
Characterisation of
sociodemographic
differences
More young men
More individuals
not born in Sweden,
living in cities, and
with higher levels of
education
More women. More
individuals born in
Sweden, married,
and living in towns
or rural areas
More older women
More often married,
and living in towns
or rural areas
More older men
More often married,
born in Sweden,
and with lower
levels of education
More older women
More often married,
born in Sweden,
and living in towns
or rural areas
More women
More often born in
Sweden
More older women
More often born
in Sweden, living
in cities, and with
lower levels of
education
More often either
younger and older
individuals
More often unmar-
ried, living in towns
or rural areas, and
with lower levels of
education
Characterisation of
occupation differ-
ences
More often not in
work/unknown
work
More often working
in Construction,
Trade, transport,
hotels & restaurants,
and Health & social
care
More often working
in Construction, and
Health & social care. More often blue-
collar worker
More often working
in Health & social
care. More often
blue-collar worker
More often working
in Education, and
Health & social care. More often white-
collar worker
More often working
in Education, and
Health & social care
More often work-
ing in Health &
social care. More
often not in work/
unknown work
More often working
in Health & social
care. Discussion More often not
in work/unknown
work
Characterisation of
the injuries
More often external
injuries and injuries
to head, face and
neck
More often fractures
and injuries to
upper and lower
extremities
More often fractures
and injuries to
upper and lower
extremities
More often external
injuries and injuries
to vertebral column
& spinal cord
More often fractures
and injuries to
upper and lower
extremities
More often injuries
to torso and
vertebral column &
spinal cord
More often internal
injuries and injuries to
head, face and neck Kjeldgård et al. BMC Public Health (2023) 23:367 Page 11 of 16 Table 3 Distributions and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for different factors in each of the eight clusters of sickness absence (SA) sequences
spanning 1 year before to 3 years after the pedestrian accident (W−52 to W+156) among 11,432 individuals aged 20–59 years injured in a traffic-related accident in 2014–2016,
using the cluster “No SA” as the reference
1. No SA
2. Immediate SA
3. Episodic SA
4. Long-term or later SA
5. Both SA due to injury and
other diagnoses
6. Other diagnoses short-
term SA
7. Other diagnoses long-
term SA
8. Discussion BMC Public Health (2023) 23:367 Page 12 of 16 Table 3 (continued)
1. No SA
2. Immediate SA
3. Episodic SA
4. Long-term or later SA
5. Both SA due to injury and
other diagnoses
6. Other diagnoses short-
term SA
7. Other diagnoses long-
term SA
8. Discussion Disability pension
n (%)
Adj OR1
(95% CI)
n (%)
Adj OR1 (95%
CI)
n (%)
Adj OR1 (95%
CI)
n (%)
Adj OR1 (95%
CI)
n (%)
Adj OR1 (95%
CI)
n (%)
Adj OR1 (95%
CI)
n (%)
Adj OR1 (95%
CI)
All
5339 (46.7)
2046 (17.9)
445 (3.89)
369 (3.23)
796 (6.96)
1901 (16.63)
233 (2.04)
303 (2.65)
Type of accident
Colli-
sion with
pedestrian/
bicyclist
307 (5.75)
89 (4.35)
1.28
(0.96–1.70)
21 (4.72)
1.36 (0.83–2.24)
28 (7.59)
1.60 (1.03–2.48)
44 (5.53)
1.70 (1.18–2.45)
79 (4.16)
0.78 (0.60–1.03)
10 (4.29)
0.86 (0.44–1.69)
13 (4.29)
0.87 (0.47–1.60)
Collision
with motor
vehicle
777 (14.55)
286 (13.98)
1.54
(1.26–1.87)
57 (12.81)
1.39 (0.98–1.98)
59 (15.99)
1.23 (0.87–1.73)
79 (9.92)
1.12 (0.84–1.50)
256 (13.47)
0.95 (0.79–1.14)
33 (14.16)
0.87 (0.56–1.35)
48 (15.84)
0.91 (0.62–1.33)
Unspeci-
fied
587 (10.99)
163 (7.97)
1.06
(0.85–1.32)
42 (9.44)
1.23 (0.85–1.79)
35 (9.49)
0.93 (0.63–1.39)
61 (7.66)
1.03 (0.76–1.41)
220 (11.57)
1.08 (0.90–1.30)
25 (10.73)
0.93 (0.59–1.49)
33 (10.89)
0.93 (0.61–1.42)
Fall: snow
and ice
1005
(18.82)
600 (29.33)
1.25
(1.05–1.48)
122 (27.42)
1.20 (0.89–1.61)
78 (21.14)
1.08 (0.77–1.50)
249 (31.28)
1.25 (1.00–1.57)
370 (19.46)
0.93 (0.79–1.11)
47 (20.17)
0.79 (0.52–1.19)
58 (19.14)
0.85 (0.58–1.24)
Fall: slip-
ping, trip-
ping, and
stumbling
1938
(36.30)
670 (32.75)
ref
150 (33.71)
ref
130 (35.23)
ref
279 (35.05)
ref
753 (39.61)
ref
102
(43.78)
ref
114 (37.62)
ref
Fall: other
725 (13.58)
238 (11.63)
1.06
(0.87–1.29)
53 (11.91)
1.12 (0.79–1.58)
39 (10.57)
0.86 (0.59–1.25)
84 (10.55)
1.10 (0.83–1.45)
223 (11.73)
0.97 (0.81–1.16)
16 (6.87)
0.57 (0.33–0.99)
37 (12.21)
0.93 (0.62–1.40)
Type of injury
Fracture
1549
(29.01)
1477
(72.19)
6.81
(5.71–8.13)
290 (65.17)
4.91 (3.57–6.75)
109 (29.54)
0.91 (0.68–1.23)
503 (63.19)
4.65 (3.63–5.96)
464 (24.41)
0.73 (0.63–0.85)
86 (36.91)
0.94 (0.66–1.35)
109 (35.97)
1.11 (0.80–1.54)
Disloca-
tion
177 (3.32)
73 (3.57)
2.98
(2.13–4.15)
16 (3.60)
2.37 (1.29–4.35)
13 (3.52)
0.94 (0.51–1.74)
29 (3.64)
2.60 (1.63–4.16)
48 (2.52)
0.75 (0.53–1.06)
-2
-2
-2
-2
Sprains
and strains
890 (16.67)
182 (8.90)
1.45
(1.15–1.83)
57 (12.81)
1.69 (1.13–2.52)
70 (18.97)
1.12 (0.79–1.58)
119 (14.95)
1.90 (1.41–2.57)
353 (18.57)
0.93 (0.78–1.10)
23 (9.87)
0.62 (0.37–1.03)
30 (9.90)
0.90 (0.56–1.43)
Internal
392 (7.34)
82 (4.01)
3.08
(2.17–4.37)
17 (3.82)
3.04 (1.52–6.08)
33 (8.94)
1.32 (0.82–2.11)
34 (4.27)
3.10 (1.86–5.17)
172 (9.05)
1.27 (1.01–1.61)
20 (8.58)
1.57 (0.86–2.87)
44 (14.52)
1.93 (1.23–3.03)
External
2213
(41.45)
220 (10.75)
ref
58 (13.03)
ref
136 (36.86)
ref
103 (12.94)
ref
820 (43.14)
ref
88 (37.77)
ref
ref
ref
Other and
unspeci-
fied
118 (2.21)
12 (0.59)
1.44
(0.76–2.75)
-2
-2
-2
-2
-2
-2
44 (2.31)
1.15 (0.79–1.67)
9 (3.86)
2.38 (1.09–5.18)
-2
-2 Kjeldgård et al. Discussion Disability pension
Injured body region
Head, face
and neck
1394
(26.11)
155 (7.58)
ref
32 (7.19)
ref
83 (22.49)
ref
61 (7.66)
ref
474 (24.93)
ref
49 (21.03)
ref
97 (32.01)
ref
Vertebral
column
and spinal
cord
89 (1.67)
35 (1.71)
1.53
(0.92–2.55)
10 (2.25)
2.74 (1.16–6.46)
15 (4.07)
3.13 (1.59–6.15)
13 (1.63)
1.79 (0.86–3.70)
41 (2.16)
1.77 (1.16–2.72)
-2
-2
-2
-2
Torso
312 (5.84)
68 (3.32)
1.41
(0.97–2.04)
18 (4.04)
2.11 (1.08–4.12)
22 (5.96)
1.27 (0.75–2.13)
31 (3.89)
1.98 (1.18–3.33)
147 (7.73)
1.64 (1.28–2.09)
15 (6.44)
1.65 (0.87–3.15)
17 (5.61)
0.90 (0.50–1.62)
Upper
extremities
1654
(30.98)
938 (45.85)
3.26
(2.48–4.28)
206 (46.29)
4.44 (2.60–7.58)
129 (34.96)
1.74 (1.20–2.52)
344 (43.22)
3.27 (2.18–4.92)
557 (29.30)
1.24 (1.03–1.49)
84 (36.05)
2.01 (1.25–3.26)
93 (30.69)
1.21 (0.81–1.81)
Lower
extremities
1856
(34.76)
847 (41.40)
3.26
(2.49–4.28)
179 (40.22)
4.10 (2.41–6.99)
119 (32.25)
1.32 (0.90–1.92)
347 (43.59)
3.65 (2.44–5.48)
674 (35.46)
1.26 (1.05–1.52)
76 (32.62)
1.64 (1.01–2.65)
87 (28.71)
0.93 (0.62–1.41)
Other and
unspeci-
fied
34 (0.64)
-2
-2
-2
-2
-2
-2
-2
-2
-2
-2
-2
-2
-2
-2
Occupational sector
Manu-
facturing,
agriculture,
forestry &
fishing
556 (10.41)
230 (11.24)
0.97
(0.78–1.20)
48 (10.79)
1.11 (0.74–1.65)
49 (13.28)
1.10 (0.74–1.63)
77 (9.67)
0.96 (0.70–1.31)
165 (8.68)
0.85 (0.68–1.06)
-2
-2
12 (3.96)
0.52 (0.27–1.01)
Construc-
tion
240 (4.50)
147 (7.18)
1.62
(1.24–2.13)
40 (8.99)
2.35 (1.51–3.65)
29 (7.86)
1.46 (0.91–2.35)
38 (4.77)
1.38 (0.92–2.09)
79 (4.16)
1.04 (0.77–1.39)
-2
-2
-2
-2
Trade,
transport,
hotels &
restaurants
993 (18.60)
427 (20.87)
1.20
(1.00–1.44)
85 (19.10)
1.25 (0.89–1.75)
63 (17.07)
0.86 (0.60–1.24)
150 (18.84)
1.22 (0.94–1.59)
386 (20.31)
1.12 (0.94–1.32)
34 (14.59)
1.17 (0.72–1.91)
22 (7.26)
0.51 (0.30–0.87)
Finance,
commu-
nication
& cultural
service
1447
(27.10)
520 (25.42)
ref
95 (21.35)
ref
85 (23.04)
ref
178 (22.36)
ref
475 (24.99)
ref
45 (19.31)
ref
52 (17.16)
ref
Education
409 (7.66)
210 (10.26)
1.19
(0.94–1.51)
44 (9.89)
1.20 (0.79–1.83)
33 (8.94)
1.41 (0.89–2.25)
96 (12.06)
1.48 (1.08–2.02)
215 (11.31)
1.45 (1.16–1.80)
21 (9.01)
1.45 (0.80–2.61)
-2
-2
Health &
social care
656 (12.29)
456 (22.29)
1.85
(1.53–2.25)
124 (27.87)
2.45 (1.76–3.41)
75 (20.33)
1.97 (1.36–2.86)
234 (29.40)
2.38 (1.85–3.07)
443 (23.30)
1.73 (1.44–2.07)
46 (19.74)
1.99 (1.24–3.22)
42 (13.86)
1.75 (1.10–2.79)
Not in
work/
Unknown
1038
(19.44)
56 (2.74)
0.45
(0.31–0.65)
9 (2.02)
0.32 (0.14–0.72)
35 (9.49)
0.99 (0.57–1.73)
23 (2.89)
0.48 (0.27–0.84)
138 (7.26)
0.61 (0.47–0.81)
75 (32.19)
2.94 (1.57–5.52)
161 (53.14)
1.63 (1.06–2.52) Kjeldgård et al. Discussion To elucidate this, further studies are
also needed to investigate different jobs, physical work-
load, and other work demands in more detail. not only those involving a motor vehicle. Several
recent studies stress that pedestrian injuries should be
included in the traditional definition of traffic accidents
[1, 21, 33]. The present study further strengthens this
argument as 71% of the pedestrian accident were due to
fall with no other road user involved. The large number
of included pedestrians allowed for more detailed anal-
yses investigating important factors for the subsequent
patterns of SA. One limitation is that no data from primary health-
care (to also capture the impact of minor injuries) was
included, on the other hand, all injuries from traffic
accidents severe enough to require in- or outpatient
healthcare were included. which are those more likely
to require SA. Another limitation is that only informa-
tion from one injury diagnosis per pedestrian is taken
into consideration, however, the majority had only one
such injury (82%). Another limitation is that only indi-
viduals that survived the entire follow-up period were
included in the study and that those with DP during
the entire study period were excluded. These require-
ments might potentially lead to an underestimation of
the total number of injuries and the shorter-term con-
sequences, there were however few that died or emi-
grated during follow-up (during the three years after
the accident). The focus here was the consequences in
terms of SA and DP and as such the included individu-
als should be at risk of receiving SA or DP. In the present study, the proportion of missing infor-
mation on occupation was higher in the three clusters
“No SA”, “Other diagnoses Long-term SA”, and “DP”. This could be due to that the individuals in the clus-
ter “No SA” to a lower extent have a job and hence are
not eligible to SA benefits (e.g. no income from work,
unemployment and parental leave) the individuals in
this cluster are also slightly younger than in the other
clusters (e.g. still studying or not yet having begun their
first job). The high proportion of missing in the other
two clusters could be owing to the high proportion of
these individuals were already on long-term SA and DP
the year before the accident, when the information on
occupation was assessed. Conclusions
h This nationwide register study of the 11,432 working-
aged pedestrians that received in- or specialized out-
patient healthcare due to a traffic-related accident
observed diverse patterns of SA due to injury diagno-
ses and SA due to other diagnoses following the acci-
dent. Almost half of the pedestrians had no SA during
the entire follow up. Compared to this cluster of SA
sequences, all other clusters were associated with older
age, no university education, having been hospitalized,
and working in health and social care. The three clus-
ters of sequences characterised by SA due to an injury
diagnosis in direct connection to the accident were
associated with women, fractures, lower and upper
extremities. A rather divergent population with more
older women and more younger men was observed. The distribution of type of injuries as well as occupa-
tional sectors were also distinctly different for women
and men. These observed differences can be utilized
to improve understanding of long-term consequences
and prevention of road-traffic accidents among
pedestrians. Discussion BMC Public Health (2023) 23:367 Page 13 of 16 1 Adjusted for: Sex, Age group, Level of education, Country of birth, Type of living area, Married, Type of accident, Inpatient healthcare, Type of injury, Injured body region, Season, Year of accident, Occupational sector,
Private/Public, and Type of occupation
1. No SA
2. Immediate SA
3. Episodic SA
4. Long-term or later SA
5. Both SA due to injury and
other diagnoses
6. Other diagnoses short-
term SA
7. Other diagnoses long-
term SA
8. Disability pension
Type of occupation
White
collar
1974
(36.97)
919 (44.92)
ref
181 (40.67)
ref
133 (36.04)
ref
377 (47.36)
ref
828 (43.56)
ref
72 (30.90)
ref
55 (18.15)
ref
Blue collar
1314
(24.61)
733 (35.83)
1.93
(1.63–2.27)
175 (39.33)
2.51 (1.88–3.34)
141 (38.21)
1.86 (1.37–2.52)
277 (34.80)
2.03 (1.62–2.53)
555 (29.20)
1.35 (1.15–1.58)
51 (21.89)
1.99 (1.29–3.07)
49 (16.17)
1.48 (0.94–2.33)
Not in
work/
Unknown
2051
(38.42)
394 (19.26)
1.05
(0.87–1.26)
89 (20.00)
1.42 (1.02–1.96)
95 (25.75)
1.17 (0.83–1.66)
142 (17.84)
1.05 (0.81–1.35)
518 (27.25)
1.23 (1.04–1.46)
110
(47.21)
2.48 (1.62–3.78)
199 (65.68)
2.13 (1.38–3.29) Kjeldgård et al. BMC Public Health (2023) 23:367 Page 14 of 16 Page 14 of 16 that individuals working as plant and machine opera-
tors and drivers had a longer duration of work dis-
ability after a road traffic accident [28]. In our study,
working in Health & social care was associated with all
clusters of SA sequences compared to the cluster “No
SA”. Accordingly suggesting an association between
this occupational sector and SA following an injury
as a pedestrian. In addition, working in Construction
was associated with the clusters “Immediate SA” and
“Episodic SA” and working in Education was associ-
ated with the clusters “Both SA due to injury and other
diagnoses” and “Other diagnoses long-term”. This may
be explained by the difference in workload and vari-
ous demands the different occupational sectors have. Individuals in some occupations are more likely to have
reduced work capacity in relation to an injury, e.g. due
to work requirements of being physical active and/
or requiring mobility to complete work tasks. Accord-
ingly, sustaining a fracture to the lower extremities
in an occupation where you are required to walk and
stand up more often lead to SA. It would be beneficial
to pay attention to the physical workload of different
jobs in interventions to reduce SA and DP following a
pedestrian injury. Discussion The proportion of individuals
in each occupational sector in this study corresponds
well to those of the general population in Sweden[31],
with the exception of women working in Health &
social care where a higher proportion were observed
among the injured pedestrians. Funding
O Open access funding provided by Karolinska Institute. The study was finan-
cially supported by AFA insurance, and we utilised data from the REWHARD
consortium supported by the Swedish Research Council (VR; grant number
2017–00624). 13. Kjeldgård L, Ohlin M, Elrud R, Stigson H, Alexanderson K, Friberg E. Bicycle
crashes and sickness absence - a population-based Swedish register study
of all individuals of working ages. BMC Public Health. 2019;19(1):943. 14. Ohlin M, Kjeldgård L, Elrud R, Stigson H, Alexanderson K, Friberg E. Dura-
tion of sickness absence following a bicycle crash, by injury type and
injured body region: A nationwide register-based study. J Transp Health. 2018;9:275–81. Strengths and limitations One of the main strengths of this study is the use of
high-quality nationwide register data, with total popu-
lation coverage, several years of follow-up, and that the
results were not hampered by recall bias [32]. Another
strength is that all pedestrian injuries are included, Page 15 of 16 Kjeldgård et al. BMC Public Health (2023) 23:367 Kjeldgård et al. BMC Public Health Abbreviations
DP
Disability pension
SA
Sickness absence
OR
Odds ratio
CI
Confidence interval Abbreviations
DP
Disability pension
SA
Sickness absence
OR
Odds ratio
CI
Confidence interval Consent for publication
Not applicable. 22. Winter 2010-2011 in numbers (In Swedish: Vintern 2010-2011 i siffror)
[https://www.smhi.se/data/vintern-2010-2011-i-siffror-1.15194] 22. Winter 2010-2011 in numbers (In Swedish: Vintern 2010-2011 i siffror)
[https://www.smhi.se/data/vintern-2010-2011-i-siffror-1.15194] Not applicable. 23. The snow situation in winter 2015–2016 (In Swedish: Snösituationen
vintern 2015–2016) [https://www.smhi.se/klimat/klimatet-da-och-nu/
arets-vatten/snosituationen-vintern-2015-2016-1.114940] 23. The snow situation in winter 2015–2016 (In Swedish: Snösituationen
vintern 2015–2016) [https://www.smhi.se/klimat/klimatet-da-och-nu/
arets-vatten/snosituationen-vintern-2015-2016-1.114940] Declarations 18. Gabadinho A, Ritschard G, Muller NS, Studer M. Analyzing and Visualizing
State Sequences in R with TraMineR. J Stat Softw. 2011;40(4):1–37. Availability of data and materials The data cannot be made publicly available, according to privacy regula-
tions. According to the General Data Protection Regulation, the Swedish
law SFS 2018:218, the Swedish Data Protection Act, the Swedish Ethical
Review Act, and the Public Access to Information and Secrecy Act, data
can only be made available, after legal review, for researchers who meet
the criteria for access to this type of sensitive and confidential data. Read-
ers may contact professor Kristina Alexanderson (kristina.alexanderson@
ki.se) regarding the data. 15. Kjeldgård L, Stigson H, Alexanderson K, Friberg E. Sequence analysis of
sickness absence and disability pension in the year before and the three
years following a bicycle crash; a nationwide longitudinal cohort study of
6353 injured individuals. BMC Public Health. 2020;20(1):1710. 16. Allebeck P, Mastekaasa A. Swedish Council on Technology Assessment in
Health Care (SBU). Chapter 5. Risk factors for sick leave - general studies. Scand J Public Health Suppl. 2004;63:49–108. 17. Social Insurance in Figures 2016. Swedish Social Insurance Agency; 2016. Acknowledgements
Not applicable 10. Ludvigsson JF, Otterblad-Olausson P, Pettersson BU, Ekbom A. The Swed-
ish personal identity number: possibilities and pitfalls in healthcare and
medical research. Eur J Epidemiol. 2009;24(11):659–67. Authors’ contributions All authors (LK, HS, ELB, KF, and EF) contributed to the design of the study. LK
performed the data management and the statistical analyses. LK wrote the
first draft of the manuscript and revised it with input from all other authors
(HS, ELB, KF, and EF). All authors (LK, HS, ELB, KF, and EF) read and approved the
final version of the manuscript. 11. Barell V, Aharonson-Daniel L, Fingerhut LA, Mackenzie EJ, Ziv A, Boyko V,
Abargel A, Avitzour M, Heruti R. An introduction to the Barell body region
by nature of injury diagnosis matrix. Inj Prev. 2002;8(2):91–6. 12. Elrud R, Stigson H, Ohlin M, Alexanderson K, Kjeldgård L, Friberg E. Sickness Absence among Passenger Car Occupants following a Crash. In: IRCOBI Conference Proceedings. 2017: IRC-17–18, s 79–90, ISSN
2235–3151. Received: 17 August 2022 Accepted: 10 February 2023 Received: 17 August 2022 Accepted: 10 February 2023 Received: 17 August 2022 Accepted: 10 February 2023 References The online version contains supplem
org/10.1186/s12889-023-15259-w. The online version contains supplementary material available at https://doi.
org/10.1186/s12889-023-15259-w. The online version contains supplementary material available at https://doi.
org/10.1186/s12889-023-15259-w. 1. Oxley J, O’Hern S, Burtt D, Rossiter B. Falling while walking: A hidden
contributor to pedestrian injury. Accident; analysis and prevention. 2018;114:77–82. 2. Common strategy for safe pedestrian traffic 1.0 (In Swedish: Gemensam
inriktning för säker gångtrafik 1.0) Borlänge, Sweden: Swedish Transport
Administration; 2017. Additional file 1. Table A.1. Measures of cluster partition quality for
four to twelve identified clusters of sequences of SA among pedestrians
injured in aroad traffic accident, the measures for the chosen number of
clusters included in the present study are marked in bold. Table A.2. Dis-
tributions of the different sociodemographic, occupation, and injury
factors in the eight identified clusters of sequences of sickness absence
(SA) status/week over 1 year before and 3 years after the date of a pedes-
trian accident (W−52 to W+156) among 11,432 individuals aged 20–59 years
injured in a traffic-related accident in 2014-2016. Table A.3. Crude and
mutually adjusted odds ratios (ORs) and 95% confidence intervals (CIs)
for different sociodemographic, occupation, and injury factors in each
of the eight identified clusters of sequences of sickness absence (SA)
status/week over 1 year before and 3 years after the date of a pedestrian
accident (W−52to W+156) among 11,432 individuals aged 20–59 years
injured in a traffic-related accident in 2014-2016, using the cluster “No SA”
as the reference. 2. Common strategy for safe pedestrian traffic 1.0 (In Swedish: Gemensam
inriktning för säker gångtrafik 1.0) Borlänge, Sweden: Swedish Transport
Administration; 2017. 3. Physical activity strategy for the WHO European Region 2016–2025. Copenhagen: World Health Organization; 2016.fi Copenhagen: World Health Organization; 2016. 4. Naci H, Chisholm D, Baker TD. Distribution of road traffic deaths by ro
user group: a global comparison. Inj Prev. 2009;15(1):55–9. 5. Global Status Report on Road Safety 2018. Geneva: World Health Organi-
zation; 2018. 6. The 2030 Agenda for Sustainable Development [https://sustainabledeve
lopment.un.org/content/documents/21252030%20Agenda%20for%
20Sustainable%20Development%20web.pdf] 6. The 2030 Agenda for Sustainable Development [https://sustainabledeve
lopment.un.org/content/documents/21252030%20Agenda%20for%
20Sustainable%20Development%20web.pdf] 7. Tingvall C. The zero vision - A road transport system free from serious
health losses. Transportation, Traffic Safety and Health. 1997. pp. 37–57. 7. Tingvall C. The zero vision - A road transport system free from serious
health losses. Transportation, Traffic Safety and Health. 1997. pp. 37–57. 8. Kjeldgård L, Stigson H, Klingegard M, Alexanderson K, Friberg E. Ethics approval and consent to participate 19. Studer M. WeightedCluster Library Manual: A practical guide to creating
typologies of trajectories in the social sciences with R. LIVES Working
papers 2013, 24. The study was conducted in accordance with the World Medical Association
Declaration of Helsinki. Participant consent is generally not required in large
register-based studies in the Nordic countries and was for this study waived
by the Regional Ethical Review Board of Stockholm, Sweden, who approved of
the project. All data were anonymized by the administrative authorities before
delivered to the researchers. 20. European Commission, Traffic Safety Basic Facts on Pedestrians, European
Commission,Directorate General for Transport, June 2018. 21. Amin K, Skyving M, Bonander C, Krafft M, Nilson F. Fall- and collision-
related injuries among pedestrians in road traffic environment – A Swed-
ish national register-based study. Journal of Safety Research 2022. References Sickness
absence and disability pension among injured working-aged pedes-
trians - a population-based Swedish register study. BMC Public Health. 2021;21(1):2279. 8. Kjeldgård L, Stigson H, Klingegard M, Alexanderson K, Friberg E. Sickness
absence and disability pension among injured working-aged pedes-
trians - a population-based Swedish register study. BMC Public Health. 2021;21(1):2279. 9. International Statistical Classification of Diseases and Related Health Prob-
lems, Tenth Revision (ICD-10). Geneva: World Health Organization; 1993. 9. International Statistical Classification of Diseases and Related Health Prob-
lems, Tenth Revision (ICD-10). Geneva: World Health Organization; 1993. Competing interests The authors declare that they have no competing interests. Page 16 of 16 Kjeldgård et al. BMC Public Health (2023) 23:367 Kjeldgård et al. BMC Public Health (2023) 23:367 24. Andersson AK. Winter Road Conditions and Traffic Accidents in Sweden and
UK - Present and Future Climate Scenarios. Göteborgs universitet; 2010. 25. Elvik R, Bjørnskau T. Risk of pedestrian falls in Oslo, Norway: Rela-
tion to age, gender and walking surface condition. J Transp Health. 2019;12:359–70. 26. Schepers P, den Brinker B, Methorst R, Helbich M. Pedestrian falls: A
review of the literature and future research directions. J Safety Res. 2017;62:227–34. 27. Gray SE, Collie A. Work absence following road traffic crash in Victoria,
Australia: A population-based study. Injury. 2019;50(7):1293–9. 28. Berecki-Gisolf J, Collie A, McClure R. Work disability after road traffic injury
in a mixed population with and without hospitalisation. Accident; analy-
sis and prevention. 2013;51:129–34. 29. Rissanen R, Liang Y, Moeller J, Nevriana A, Berg H-Y, Hasselberg M. Trajec-
tories of sickness absence after road traffic injury: a Swedish register-
based cohort study. BMJ Open. 2019;9(7): e031132. 29. Rissanen R, Liang Y, Moeller J, Nevriana A, Berg H-Y, Hasselberg M. Trajec-
tories of sickness absence after road traffic injury: a Swedish register-
based cohort study. BMJ Open. 2019;9(7): e031132. 30. Hansson PG. Sick-leave after road traffic accidents. Scand J Soc Med. 1976;4(2):103–7. 30. Hansson PG. Sick-leave after road traffic accidents. Scand J Soc Med. 1976;4(2):103–7. 31. Employees aged 16–64 in the country by occupation (3-digit SSYK 2012),
industry SNI2007 (rough level), age and gender. New time series. Year
2019 - 2020 (In Swedish: Anställda 16–64 år i riket efter yrke (3-siffrig
SSYK 2012), näringsgren SNI2007 (grov nivå), ålder och kön. Ny tidsserie. År 2019 - 2020 [https://www.statistikdatabasen.scb.se/pxweb/sv/ssd/
START__AM__AM0208__AM0208B/YREG61N/] 32. Ludvigsson JF, Andersson E, Ekbom A, Feychting M, Kim JL, Reuterwall C,
Heurgren M, Olausson PO. External review and validation of the Swedish
national inpatient register. BMC Public Health. 2011;11:450. 33. Methorst R, Schepers P, Christie N, Dijst M, Risser R, Sauter D, van Wee B. ’Pedestrian falls’ as necessary addition to the current definition of traffic
crashes for improved public health policies. J Transp Health. 2017;6:10–2. Kjeldgård et al. BMC Public Health (2023) 23:367 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub-
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https://cronfa.swan.ac.uk/Record/cronfa31775/Download/0031775-17022017135318.pdf
|
English
| null |
Bisphenol A causes reproductive toxicity, decreases <i>dnmt1</i> transcription, and reduces global DNA methylation in breeding zebrafish <i>(Danio rerio)</i>
|
Epigenetics
| 2,016
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cc-by
| 12,349
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Cronfa - Swansea University Open Access Repository This is an author produced version of a paper published in :
E i
ti Cronfa URL for this paper:
http://cronfa.swan.ac.uk/Record/cronfa31775 This article is brought to you by Swansea University. Any person downloading material is agreeing to abide by the
terms of the repository licence. Authors are personally responsible for adhering to publisher restrictions or conditions.
When uploading content they are required to comply with their publisher agreement and the SHERPA RoMEO
database to judge whether or not it is copyright safe to add this version of the paper to this repository.
http://www.swansea.ac.uk/iss/researchsupport/cronfa-support/ Laing, L., Viana, J., Dempster, E., Trznadel, M., Trunkfield, L., Uren Webster, T., van Aerle, R., Paull, G., Wilson, R.,
Mill, J. & Santos, E. (2016). Bisphenol A causes reproductive toxicity, decreasesdnmt1transcription, and reduces
global DNA methylation in breeding zebrafish(Danio rerio). Epigenetics, 11(7), 526-538.
http://dx.doi.org/10.1080/15592294.2016.1182272 Cronfa - Swansea University Open Access Repository
_____________________________________________________________
This is an author produced version of a paper published in :
Epigenetics
Cronfa URL for this paper:
http://cronfa.swan.ac.uk/Record/cronfa31775
_____________________________________________________________
Paper:
Laing, L., Viana, J., Dempster, E., Trznadel, M., Trunkfield, L., Uren Webster, T., van Aerle, R., Paull, G., Wilson, R.,
Mill, J. & Santos, E. (2016). Bisphenol A causes reproductive toxicity, decreasesdnmt1transcription, and reduces
global DNA methylation in breeding zebrafish(Danio rerio). Epigenetics, 11(7), 526-538.
http://dx.doi.org/10.1080/15592294.2016.1182272 Cronfa - Swansea University Open Access Repository Epigenetics ISSN: 1559-2294 (Print) 1559-2308 (Online) Journal homepage: http://www.tandfonline.com/loi/kepi20 Paper: ape
Laing, L., Viana, J., Dempster, E., Trznadel, M., Trunkfield, L., Uren Webster, T., van Aerle, R., Paull, G., Wilson, R.,
Mill, J. & Santos, E. (2016). Bisphenol A causes reproductive toxicity, decreasesdnmt1transcription, and reduces
global DNA methylation in breeding zebrafish(Danio rerio). Epigenetics, 11(7), 526-538. http://dx.doi.org/10.1080/15592294.2016.1182272 p
Laing, L., Viana, J., Dempster, E., Trznadel, M., Trunkfield, L., Uren Webster, T., van Aerle, R., Paull, G., Wilson, R.,
Mill, J. & Santos, E. (2016). Bisphenol A causes reproductive toxicity, decreasesdnmt1transcription, and reduces
global DNA methylation in breeding zebrafish(Danio rerio). Epigenetics, 11(7), 526-538. http://dx.doi.org/10.1080/15592294.2016.1182272 This article is brought to you by Swansea University. Any person downloading material is agreeing to abide by the
terms of the repository licence. Authors are personally responsible for adhering to publisher restrictions or conditions. When uploading content they are required to comply with their publisher agreement and the SHERPA RoMEO
database to judge whether or not it is copyright safe to add this version of the paper to this repository. http://www.swansea.ac.uk/iss/researchsupport/cronfa-support/ Epigenetics Date: 17 February 2017, At: 05:50 CONTACT L. V. Laing
ll292@exeter.ac.uk; E. M. Santos
e.santos@exeter.ac.uk
Supplemental data for this article can be accessed on the publisher’s website.
Published with license by Taylor & Francis Group, LLC © L. V. Laing, J. Viana, E. L. Dempster, M. Trznadel, L. A. Trunkfield, T. M. Uren Webster, R. van Aerle, G. C. Paull, R. J. Wilson, J. Mill, and E. M.
Santos.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribu-
tion, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted. ABSTRACT ARTICLE HISTORY
Received 22 February 2016
Revised 18 April 2016
Accepted 19 April 2016
KEYWORDS
Aquatic; endocrine; meth-
ylation; plasticizers; tele-
ost; vertebrate; waste BS
C
Bisphenol A (BPA) is a commercially important high production chemical widely used in epoxy resins and
polycarbonate plastics, and is ubiquitous in the environment. Previous studies demonstrated that BPA
activates estrogenic signaling pathways associated with adverse effects on reproduction in vertebrates
and that exposure can induce epigenetic changes. We aimed to investigate the reproductive effects of
BPA in a fish model and to document its mechanisms of toxicity. We exposed breeding groups of
zebrafish (Danio rerio) to 0.01, 0.1, and 1 mg/L BPA for 15 d. We observed a significant increase in egg
production, together with a reduced rate of fertilization in fish exposed to 1 mg/L BPA, associated with
significant alterations in the transcription of genes involved in reproductive function and epigenetic
processes in both liver and gonad tissue at concentrations representing hotspots of environmental
contamination (0.1 mg/L) and above. Of note, we observed reduced expression of DNA methyltransferase
1 (dnmt1) at environmentally relevant concentrations of BPA, along with a significant reduction in global
DNA methylation, in testes and ovaries following exposure to 1 mg/L BPA. Our findings demonstrate that
BPA disrupts
reproductive processes
in zebrafish, likely via estrogenic mechanisms, and that
environmentally relevant concentrations of BPA are associated with altered transcription of key enzymes
involved in DNA methylation maintenance. These findings provide evidence of the mechanisms of action
of BPA in a model vertebrate and advocate for its reduction in the environment. Abbreviations: E2, 17b-estradiol; 5Fc, 5-formylcytosine; 5hmC, 5-hydroxymethylcytosine; ar, androgen receptor;
amh, anti-M€ullerian hormone; cyp19a1a, aromatase; BPA, bisphenol A; dnmt3, DNA (cytosine-5)-methyltransferase
3; dnmt1, DNA methyltransferase 1; esr1, estrogen receptor 1; esr2a, estrogen receptor 2a; esr2b, estrogen receptor
2b; ER, estrogen receptor; GSI, gonadosomatic index; HSI, hepatosomatic index; hdac1, histone deacetylase 1; hdac3,
histone deacetylase 3; mecp2, methyl CpG binding protein 2; mbd2, methyl-CpG-binding domain protein 2; mbd3a,
methyl-CpG-binding domain protein 3a; 5mC, methylcytosine; PCA, principal component analysis; rpl8, ribosomal
protein L8; vtg1, vitellogenin 1 Bisphenol A causes reproductive toxicity,
decreases dnmt1 transcription, and reduces global
DNA methylation in breeding zebrafish (Danio
rerio) L. V. Laing, J. Viana, E. L. Dempster, M. Trznadel, L. A. Trunkfield, T. M. Uren
Webster, R. van Aerle, G. C. Paull, R. J. Wilson, J. Mill & E. M. Santos To cite this article: L. V. Laing, J. Viana, E. L. Dempster, M. Trznadel, L. A. Trunkfield,
T. M. Uren Webster, R. van Aerle, G. C. Paull, R. J. Wilson, J. Mill & E. M. Santos (2016)
Bisphenol A causes reproductive toxicity, decreases dnmt1 transcription, and reduces global
DNA methylation in breeding zebrafish (Danio rerio), Epigenetics, 11:7, 526-538, DOI:
10.1080/15592294.2016.1182272 To link to this article: http://dx.doi.org/10.1080/15592294.2016.1182272
© 2016 The Author(s). Published with
license by Taylor & Francis Group, LLC© L. V. Laing, J. Viana, E. L. Dempster, M. Trznadel,
L. A. Trunkfield, T. M. Uren Webster, R. van
Aerle, G. C. Paull, R. J. Wilson, J. Mill, and E. M. Santos. View supplementary material
Accepted author version posted online: 27
Apr 2016. Published online: 27 Apr 2016. Submit your article to this journal
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Citing articles: 4 View citing articles To link to this article: http://dx.doi.org/10.1080/15592294.2016.1182272
© 2016 The Author(s). Published with
license by Taylor & Francis Group, LLC© L. V. Laing, J. Viana, E. L. Dempster, M. Trznadel,
L. A. Trunkfield, T. M. Uren Webster, R. van
Aerle, G. C. Paull, R. J. Wilson, J. Mill, and E. M. Santos. View supplementary material
Accepted author version posted online: 27
Apr 2016. Published online: 27 Apr 2016. Submit your article to this journal
Article views: 1504
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Citing articles: 4 View citing articles Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=kepi20 Date: 17 February 2017, At: 05:50 Download by: [Swansea University] Download by: [Swansea University] EPIGENETICS
2016, VOL. 11, NO. 7, 526–538
http://dx.doi.org/10.1080/15592294.2016.1182272 EPIGENETICS
2016, VOL. 11, NO. 7, 526–538
http://dx.doi.org/10.1080/15592294.2016.1182272 Bisphenol A causes reproductive toxicity, decreases dnmt1 transcription,
and reduces global DNA methylation in breeding zebrafish (Danio rerio) L. V. Lainga, J. Vianab, E. L. Dempsterb, M. Trznadela, L. A. Trunkfielda, T. M. Uren Webstera, R. van Aerlec, G. C. Paulla,
R. J. Wilsona, J. Millb,d, and E. M. Santosa aBiosciences, College of Life & Environmental Sciences, Geoffrey Pope Building, University of Exeter, Exeter, EX4 4QD, United Kingdom; bUniversity of
Exeter Medical School, RILD building, University of Exeter, Exeter, EX2 5DW, United Kingdom; cCentre for Environment, Fisheries and Aquaculture
Science (Cefas), Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB, United Kingdom; dInstitute of Psychiatry, Psychology & Neuroscience (IoPPN),
King’s College London, Denmark Hill, London, SE5 8AF, UK Introduction A study
in Gobiocypris rarus found BPA exposure to be associated with
altered DNA methylation in the 50 flanking region of cyp19a1a
(aromatase), and the effects to be time-dependent.19 In addition,
a significant decrease in the expression of DNA methyltransfer-
ase 1 (dnmt1) in ovarian tissue has been reported, with a signifi-
cant decrease in global DNA methylation.19 BPA has been shown to act as an estrogen receptor (ER) ago-
nist,14,15 able to bind to ERs, resulting in feminizing effects.16,17
A study using the human cell line HepG2, found that BPA
strongly activated estrogen receptor 1 (ESR1; previously known
as ERa) mediated responses, but did not activate ESR2 (previ-
ously known as ERb), while in the cell line HeLa, BPA was found
to activate both ESR1 and ESR2.14 In fish, BPA induced esr1
expression in the livers of male fathead minnows (Pimephales
promelas) exposed for 4 d to 10 mg BPA/L, consistent with an
estrogenic mode-of-action.18 BPA has also been shown to alter
the transcriptional profile of steroidogenic enzyme genes in a
time-dependent
manner,
including
aromatase
(cyp19a1a),
which is responsible for the irreversible conversion of androgens
into estrogens and is a key regulator of estrogen synthesis in the
gonads. This enzyme was significantly upregulated in both the
ovary and testis of Gobiocypris rarus exposed to 15 mg/L BPA
for 7d, followed by suppression after 35 d exposure.19 Given the extensive use and ubiquity of BPA, it is important
to understand the mechanisms mediating its toxic effects and
the impacts these can have on both wild populations and
human health. The present study aims to investigate the effects
of BPA on reproduction in the zebrafish model and identify
epigenetic and transcriptional changes associated with BPA
exposure. We exposed breeding groups of zebrafish to BPA for
15 d to determine if reproduction was affected by the exposure. The concentrations tested included environmentally relevant
concentrations found world-wide (0.01 mg/L) and at point
sources (0.1 mg/L).12,38 The highest concentration tested
(1 mg/L) has only been reported in landfill leachate and is
unlikely to occur in surface waters, but it was included to enable
a mechanistic analysis of BPA toxicity. We quantified the tran-
scription of genes involved in epigenetic signaling and repro-
ductive function, together with global and locus-specific DNA
methylation in exposed fish. Adverse impacts on reproduction have been observed in
several fish models. Introduction ranging from 0.2 to 17.6 mg/g.6 BPA has been detected in the
urine of »95% of adults in the USA and Asia.7,8 It has also
been measured in the serum of adult men and women 9 and in
breast milk, fetal plasma, and placental tissue, raising concerns
about
human
exposures
during
critical
periods
of
development.1,10 Bisphenol A (BPA) is a commercially important high produc-
tion chemical widely used in the production of epoxy resins,
utilized in food and beverage packaging, dental sealants, and as
a monomer component of polycarbonate plastics.1,2 With over
three million tons produced globally per annum, environmen-
tal exposure is common,3 and in the USA BPA was measurable
in 75% of food products tested.4 Human exposure occurs pre-
dominantly via ingestion of contaminated food, caused by
leaching of BPA from linings of canned goods and polycarbon-
ate packaging. BPA has also been detected in drinking water at
concentrations up to 15 ng/L.5 In addition, inhalation is
thought to be a plausible secondary route of exposure,3 with
BPA present in 86% of domestic dust samples at concentrations BPA is moderately water soluble, entering the environment
via direct discharge from BPA production and processing
industries, wastewater treatment plants and leachate from land-
fill sites.11 Its presence is ubiquitous in the aquatic environment
and surface water concentrations have been detected up to the
low mg/L range, with peak concentrations reaching up to
21 mg/L.12 Concentrations in landfill leachate have been
reported to reach up to 17,200 mg/L.1 Due to its ubiquitous EPIGENETICS
527 EPIGENETICS
527 527 nature, the potential for environmental exposure in wildlife
populations, including fish, is very significant. Levels of BPA
reported in fish vary; values of 1-11 ng BPA/g dry weight in
muscle and 2-75 ng BPA/g dry weight in liver have been
reported.13 To date, few studies have investigated the potential for BPA
to induce epigenetic and transcriptional changes in fish. Water chemistry The mean measured concentrations of BPA in the tank water
were between 100 and 139% of the nominal concentrations for
all treatments, and are presented in Supporting Information
Table S1. Introduction A multi-generational study in fathead min-
now showed that BPA reduced gonadal growth in males and
females, reduced hatching in F1 offspring of fish exposed to
640 mg/L and induced the estrogen regulated egg yolk protein,
vitellogenin, a well established biomarker of xenoestrogen
exposure, in the liver of male fish exposed to 640 and 1280 mg/
L BPA.20 Further multigenerational studies have demonstrated
the potential adverse effects associated with exposure to
BPA.21,22 Exposure to BPA in guppies has been associated with
reduced sperm quality,23 and the presence of necrotic cells in
the seminiferous tubules of Xiphophorus helleri was also
reported.24 Together, these studies demonstrate the potential
reproductive consequences following exposure to relatively
high concentrations of BPA in fish. Effects of BPA on morphometric parameters The mean mass and length of male and female fish were
460.0 § 0.008 mg and 36.5 § 0.02 mm, and 480.6 § 0.01 mg
and 35.7 § 0.03 mm, respectively. There were no significant
differences in size or condition factor (mean 0.95 and 1.05 for
males and females, respectively) between treatment groups. g
Evidence also exists supporting the involvement of BPA in
the etiology of a range of human disease phenotypes including
cardiovascular disease,25 altered behavior in children,26 prostate
cancer27 and recurrent miscarriage.28 In addition to the well-
established estrogenic mode-of-action, additional mechanisms
have been proposed, including potential anti-androgenic activ-
ity.29 Low dose effects and non-monotonic dose response
curves have been reported.30,31 More recently, increasing evi-
dence suggests that BPA may alter the epigenetic regulation of
gene expression; for example, altered DNA methylation pat-
terns have been observed both globally (i.e., changes to the total
genomic content of DNA methylation) and at the regulatory
regions of specific genes (i.e., locus-specific) in mammals.32-36
In humans, exposure to BPA in the workplace has been associ-
ated with hypomethylation of LINE-1 in spermatozoa, a
marker of global DNA methylation levels in the genome.37
Understanding the effects of BPA exposure on epigenetic pro-
cesses, and how these alterations perturb expression of genes
that are related to development and reproduction, are impor-
tant to the evaluation of adverse effects associated with BPA
exposure, both in humans and wildlife, particularly for expo-
sures at environmentally relevant concentrations. No alterations in general feeding and swimming behavior
were observed in any spawning group, with the exception of
the mortality of one female in the 0.1 mg/L BPA treatment. The egg output calculations for that group were adjusted
accordingly. Hepatosomatic index (HSI; the ratio of liver
weight to body weight) in males was significantly increased in
fish exposed to 1 mg/L BPA, but no effects of BPA were
observed in females (Supporting Information Fig. S1). There
were no significant differences in the gonadosomatic index
(GSI; the ratio of gonad weight to body weight) of males or
females as a result of the BPA exposure. Effects of bisphenol A on gene transcription Analysis of genes involved in reproductive processes in the liver
revealed that vtg1 and esr2b were significantly upregulated in
males following exposure to 1 mg/L BPA when compared to the
solvent control group (fold-change D 172.90, PD0.009 and fold-
change D 5.40, PD0.014, respectively). In females, esr2b was sig-
nificantly upregulated following exposure to 0.01 mg/L BPA (P D
0.044). For genes involved in epigenetic regulation, the most pro-
nounced changes observed were for dnmt1, which was signifi-
cantly downregulated in the livers of females exposed to 0.01 mg/
L BPA (P D 0.040) and in both males and females exposed to 0.1
(males: P D 0.020; females: P D 0.005) and 1 mg/L BPA (males: P
D 0.020; females: P D 0.005). In addition, changes were also
observed for histone deacetylase 3 (hdac3), methyl-CpG-binding
domain protein 2 (mbd2) and methyl CpG binding protein 2
(mecp2) in males, and for mbd2 in females (Fig. 2A and B; Sup-
porting Information Figs. S2 and 3). In the gonads, BPA exposure was also associated with signif-
icant changes in transcription for genes involved in reproduc-
tive function and on epigenetic pathways (Figs. 2, 3). Principal
component analysis (PCA) for the testis indicated clear separa-
tions between the transcription profiles of fish exposed to the
solvent control and fish exposed 1 mg/L BPA, based on the
data for all genes quantified (Fig. 3). For ovaries, changes were
more pronounced and PCA revealed a separation between fish
exposed to 0.1 and 1 mg/L BPA and the solvent control (Fig. 3). In the testis, the transcript encoding esr2a and cyp19a1a were
significantly downregulated in response to 1 mg/L BPA
(P D 0.002 and 0.018, respectively; Fig. 2; Supporting Information
Fig. S4). There was also a significant association between the con-
centration of BPA and the level of transcription for cyp19a1a (P
D 0.025; Supporting Information Table S4), which decreased
with increasing concentrations of BPA. In addition, for anti-
M€ullerian hormone (amh), BPA affected gene transcription (P
0.05) and a decreasing trend across all concentrations was
observed, but this was not statistically significant (P D 0.094; Sup-
porting Information Fig. S4). Similarly to the testis, in the ovaries
of exposed females, the transcript encoding esr2a was significantly
downregulated following exposure to 1 mg/L BPA (P 0.001). Effects of bisphenol A on gene transcription In
addition, there were similar (but non-significant) trends for other
genes involved in reproductive function including esr1 and ar,
which appeared to decrease with increasing exposure concentra-
tions (Fig. 2; Supporting Information Fig. S5). Figure 1. A) Cumulative number of eggs per female per day in breeding groups
exposed to 0.01, 0.1, and 1 mg/L BPA. Data is presented for a 10 d pre-exposure
followed by a 15 d chemical exposure periods (n D 3 replicate groups per treat-
ment). Statistical comparisons were conducted in R (version 3.0.2), and the lme4
package was used to fit mixed effects linear models, followed by repeated meas-
ures ANOVA and Chi-squared Wald test to determine the effects of the exposure
to BPA compared to the solvent control. B) Mean fertilization success (%) during
the 15 d chemical exposure period (n D 3 replicate groups per treatment). Statisti-
cal analyses were conducted using R (version 3.0.2); the Regression coefficient (R2)
was calculated using linear modeling. Asterisks indicate significant differences
between treatment groups (P < 0.01; P < 0.001). BPA spawned a significantly greater number of eggs per female
when compared to all other treatment groups (P 0.01); this
increased egg production intensified throughout the exposure
period (Fig. 1A). During the pre-exposure, fertilization success
remained consistently high with no significant differences
between groups and an overall mean fertilization rate of 85.6%. During the 15 d exposure, fertilization success in colonies
exposed to 1 mg/L BPA significantly declined (P D 0.001;
Fig. 1B). Additionally, for this treatment group, there was a sig-
nificant negative correlation between the length of the exposure
(number of days) and the average percentage of fertilization
(R2 D 0.80; P 0.001), indicating that the effects of BPA on
fertilization became progressively more pronounced over the
exposure period. BPA spawned a significantly greater number of eggs per female
when compared to all other treatment groups (P 0.01); this
increased egg production intensified throughout the exposure
period (Fig. 1A). During the pre-exposure, fertilization success
remained consistently high with no significant differences
between groups and an overall mean fertilization rate of 85.6%. During the 15 d exposure, fertilization success in colonies
exposed to 1 mg/L BPA significantly declined (P D 0.001;
Fig. 1B). Effects of bisphenol A on reproduction During the 10 d pre-exposure period there were no differences
in cumulative egg production between treatment groups
(P D 0.098). During the exposure, groups treated with 1 mg/L 528
L. V. LAING ET AL. 528 Figure 1. A) Cumulative number of eggs per female per day in breeding groups
exposed to 0.01, 0.1, and 1 mg/L BPA. Data is presented for a 10 d pre-exposure
followed by a 15 d chemical exposure periods (n D 3 replicate groups per treat-
ment). Statistical comparisons were conducted in R (version 3.0.2), and the lme4
package was used to fit mixed effects linear models, followed by repeated meas-
ures ANOVA and Chi-squared Wald test to determine the effects of the exposure
to BPA compared to the solvent control. B) Mean fertilization success (%) during
the 15 d chemical exposure period (n D 3 replicate groups per treatment). Statisti-
2 Effects of bisphenol A on gene transcription Additionally, for this treatment group, there was a sig-
nificant negative correlation between the length of the exposure
(number of days) and the average percentage of fertilization
(R2 D 0.80; P 0.001), indicating that the effects of BPA on
fertilization became progressively more pronounced over the
exposure period. As in the liver, dnmt1 was significantly downregulated in ova-
ries following exposure to all three BPA concentrations tested (P
D 0.032, 0.032, 0.032, respectively). Although no significant
group-wise changes in dnmt1 transcription were observed in the
testis (Fig. 2; Supporting Information Fig. S4), the expression of
dnmt1 in the testis was associated with BPA exposure concentra-
tion (R2 D 0.110; P D 0.046; Supporting Information Table S4). In addition, changes in mbd2 transcription were observed in the
testis, with a significant increase in transcription measured in
males exposed to 0.01 mg/L BPA (P D 0.020), but reduced expres-
sion in males exposed to 1 mg/L BPA (P D 0.030; Fig. 2; Support-
ing Information Fig. S4). EPIGENETICS
529 Figure 2. Transcript profiles for target genes in the livers of females (A) and males (B), and in the ovary (C) and testis (D) following exposure to 0.01, 0.1, and 1 mg/L BPA
for 15 d. Data were collected for 6-8 fish per treatment, and data points classified as outliers (using the Chauvenet’s criterion) and for which the expression was below
the detection limit of the assay were excluded from analysis. Where amplification was detected in more than 70% of individuals, data are represented as fold-change rela-
tive to the expression in the solvent control group. Where amplification was detected in less than 70% of individuals, data are presented as the proportion of individuals
for which the target genes were amplified. Asterisks represent significant differences between treatment groups compared to the solvent control group (P < 0.05,
P < 0.01, P < 0.001). Figure 2. Transcript profiles for target genes in the livers of females (A) and males (B), and in the ovary (C) and testis (D) following exposure to 0.01, 0.1, and 1 mg/L BPA
for 15 d. Data were collected for 6-8 fish per treatment, and data points classified as outliers (using the Chauvenet’s criterion) and for which the expression was below
the detection limit of the assay were excluded from analysis. Effects of bisphenol A on gene transcription Where amplification was detected in more than 70% of individuals, data are represented as fold-change rela-
tive to the expression in the solvent control group. Where amplification was detected in less than 70% of individuals, data are presented as the proportion of individuals
for which the target genes were amplified. Asterisks represent significant differences between treatment groups compared to the solvent control group (P < 0.05,
P < 0.01, P < 0.001). Effects of bisphenol A on global DNA methylation Analysis of global DNA methylation in the gonads revealed sig-
nificant decreases in the proportion of global methylation fol-
lowing exposure to 1 mg/L BPA in both males (by 3.2%;
P D 0.029; Fig. 4A) and females (by 4.9%; P D 0.041; Fig. 4B). Targeted DNA methylation profiling in the promoter region of
amh revealed that exposure to 1 mg/L BPA caused a small but
significant increase in methylation compared to the solvent
control for the first of the three CpG sites assessed in the testes Figure 3. Principal components (PC) score plots showing the relative similarity of gonadal transcription profiles for zebrafish exposed to solvent, 0.01, 0.1, and 1 mg/L BPA
for 15 d. A) Ovary. B) Testis. Points represent PC scores for individual fish along PCs 1 and 2. Circles represent a general characterization of the PC space occupied by each
treatment group and were calculated using the prcomp package in R (version 3.0.2). Figure 3. Principal components (PC) score plots showing the relative similarity of gonadal transcription profiles for zebrafish exposed to solvent, 0.01, 0.1, and 1 mg/L BPA
for 15 d. A) Ovary. B) Testis. Points represent PC scores for individual fish along PCs 1 and 2. Circles represent a general characterization of the PC space occupied by each
treatment group and were calculated using the prcomp package in R (version 3.0.2). 530
L. V. LAING ET AL. 30
L. V. LAING ET AL. Figure 4. Global DNA methylation profiles in the gonads of adult zebrafish following exposure to 0.01, 0.1, and 1 mg/L BPA. Graphs present the percentage of global DNA
methylation in ovaries (A) and testis (B). Data are presented as boxplots (n D 6-8 for each group). Asterisks indicate significant differences compared to the solvent control
(P < 0.05, P < 0.01, P < 0.001). Figure 4. Global DNA methylation profiles in the gonads of adult zebrafish following exposure to 0.01, 0.1, and 1 mg/L BPA. Graphs present the percentage of global DNA
methylation in ovaries (A) and testis (B). Data are presented as boxplots (n D 6-8 for each group). Asterisks indicate significant differences compared to the solvent control
(P < 0.05, P < 0.01, P < 0.001). testes (males). Although group-wise comparisons of this region
revealed no significant differences in the female ovaries (Figs. Effects of bisphenol A on global DNA methylation 6
and 7), dnmt1 promoter methylation was significantly corre-
lated with BPA exposure at various sites (positions 4, 5, 6 and
8; Supporting Information Table S4). (P D 0.032; Fig. 5, see Supporting Information Fig. S6 for the
position of this CpG site), with DNA methylation at this site
being significantly correlated with BPA exposure concentration
(R2 D 0.1625; P D 0.013). No differences in DNA methylation
were seen for this region in ovaries from exposed female fish
(Fig. 5). BPA was also not associated with altered DNA methyl-
ation at two CpG sites in the 50 flanking region of the esr1 gene
in either the liver or gonads (Supporting Information Fig. S7). The analysis of 11 CpG sites across the promoter of dnmt1
identified significant increases in DNA methylation for a num-
ber of sites in the liver (in both males and females) and the Discussion Exposure to BPA resulted in a consistent downregulation of
dnmt1 transcription in the ovary and in the liver of both males
and
females
following
exposure
to
BPA,
including
at Figure 5. Gene specific DNA methylation profiles for three CpG sites in the promoter region of anti-M€ullerian hormone (amh) in the ovaries (A) and testes (B) of adult
zebrafish following exposure to 0.01, 0.1, and 1 mg/L BPA. C) Example pyrogram of three CpG sites in the 50 flanking regions of the amh gene. Data are presented as box-
plots (n D 6-8 for each group). Asterisks indicate significant differences compared to the solvent control (P < 0.05, P < 0.01, P < 0.001). Figure 5. Gene specific DNA methylation profiles for three CpG sites in the promoter region of anti-M€ullerian hormone (amh) in the ovaries (A) and testes (B) of adult
zebrafish following exposure to 0.01, 0.1, and 1 mg/L BPA. C) Example pyrogram of three CpG sites in the 50 flanking regions of the amh gene. Data are presented as box-
plots (n D 6-8 for each group). Asterisks indicate significant differences compared to the solvent control (P < 0.05, P < 0.01, P < 0.001). EPIGENETICS
531 EPIGENETICS
531 fic DNA methylation profiles for 11 CpG sites in the promoter region of DNA (cytosine-5)-methyltransferase 1 (dnmt1) in the ovaries (A) and testis (B)
owing exposure to 0.01, 0.1, and 1 mg/L BPA. C) Example pyrogram of 11 CpG sites in the 50 flanking regions of the dnmt1 gene. Data are presented
or each group). Asterisks indicate significant differences compared to the solvent control (P < 0.05, P < 0.01, P < 0.001). Figure 6. Gene-specific DNA methylation profiles for 11 CpG sites in the promoter region of DNA (cytosine-5)-methyltransferase 1 (dnmt1) in the ovaries (A) and testis (B)
of adult zebrafish following exposure to 0.01, 0.1, and 1 mg/L BPA. C) Example pyrogram of 11 CpG sites in the 50 flanking regions of the dnmt1 gene. Data are presented
as boxplots (n D 6-8 for each group). Asterisks indicate significant differences compared to the solvent control (P < 0.05, P < 0.01, P < 0.001). pathways, as previously reported for a range of organisms.39–41
We have investigated the effects of BPA on the expression of
transcripts involved in reproductive function and known to be
directly or indirectly regulated by estrogens. environmentally relevant concentrations in females. Discussion In associa-
tion with this, we found a reduction in global DNA methylation,
probably due to the decrease in dnmt1 expression. At the highest
concentration tested, BPA caused reduced fertilization, poten-
tially via estrogenic mechanisms. Together, our data provide evi-
dence of the molecular mechanisms of action of BPA and the
potential for it to cause adverse health impacts in vertebrates. We found no evidence for significant alterations in the tran-
scription of esr1 or DNA methylation across the esr1 promoter
in the gonads and livers of both sexes, but a significant associa-
tion between BPA concentration and decreased transcription
was found for the livers of females, and a trend for reduced
expression was also observed in the ovaries and testis, similar
to that described previously.31 Disruption of ESR1 has been
associated with alterations of spermatogenesis and subse-
quently infertility in mice,42,43 therefore suggesting that the
apparent decrease in esr1 transcript in the testis may contribute
toward the observed decline in fertilization success at this
concentration. Reproductive effects of BPA on adult zebrafish In parallel, BPA induced a significant increase
in the transcription of the egg yolk protein, vtg1, and an
increase in HSI in males, likely as a result of increased vitello-
genin production in hepatocytes, indicating an association
between the induction of esr2b in males and the induction of
vtg1, as previously reported for fathead minnows.44 Together,
these findings suggest that the effects of BPA on reproduction
involve disruption of estrogen receptor signaling principally via
esr1 and esr2b in the liver, and esr2a in the gonads. transcription was reported in ovaries of Gobiocypris rarus
exposed to 0.05 mg/L BPA for 35, d and was associated with
disruption of oogenesis and the occurrence of atretic follicles.31
These findings concur with previous studies reporting that
esr2a is more sensitive compared to esr1, to the natural estro-
gen, 17b-estradiol (E2).41 In contrast, BPA caused increased
transcription of esr2b in the livers of males and females but not
in the gonads, and, importantly, for females this effect was
observed at the environmentally relevant concentration of
0.01 mg/L BPA. In parallel, BPA induced a significant increase
in the transcription of the egg yolk protein, vtg1, and an
increase in HSI in males, likely as a result of increased vitello-
genin production in hepatocytes, indicating an association
between the induction of esr2b in males and the induction of
vtg1, as previously reported for fathead minnows.44 Together,
these findings suggest that the effects of BPA on reproduction
involve disruption of estrogen receptor signaling principally via
esr1 and esr2b in the liver, and esr2a in the gonads. In the testis, a decrease in amh transcription was associated
with increased BPA exposure concentrations. Similarly, in
mammals, downregulation of AMH has been reported follow-
ing exposure to BPA.46,47 Exposure to 1 mg/L BPA also caused
significant DNA hypermethylation in the amh promoter in the
testis (CpG 1), demonstrating that exposure to BPA caused epi-
genetic alterations at this specific gene locus. There was also a
significant correlation between the level of methylation in CpG
1 and amh transcription, and with BPA exposure concentra-
tion. This suggests that epigenetic mechanisms may be playing
a role in the observed decline in amh transcript in testis tissue,
which in turn could have consequences for the functioning of
the testis, resulting in de-masculinization. Fertilization success decreased over time with the mean fer-
tilization rate dropping from 89% on day 1 to 69% by day 15. Reproductive effects of BPA on adult zebrafish We provide evidence that BPA exposure results in an
impairment of reproductive function in breeding zebrafish. These effects included an increase in the number of eggs
spawned and a decrease in fertilization success in groups
exposed to 1 mg/L BPA. A number of mechanisms may con-
tribute to the observed effect of BPA on reproduction, includ-
ing stimulation of estrogen responsive processes via the
interaction of BPA or its metabolites with estrogen signaling BPA was found to downregulate esr2a in both ovaries and
testes, but not in the liver. Similarly, a decrease in esr2a 532
L. V. LAING ET AL. 532
L. V. LAING ET AL. Figure 7. Gene specific DNA methylation profiles for 11 CpG sites in the promoter region of DNA (cytosine-5)-methyltransferase 1 (dnmt1) in the livers of female (A) and
male (B) adult zebrafish following exposure to 0.01, 0.1, and 1 mg/L BPA. Data are presented as boxplots (n D 6-8 for each group). Asterisks indicate significant differences
compared to the solvent control (P < 0.05, P < 0.01, P < 0.001). Figure 7. Gene specific DNA methylation profiles for 11 CpG sites in the promoter region of DNA (cytosine-5)-methyltransferase 1 (dnmt1) in the livers of female (A) and
male (B) adult zebrafish following exposure to 0.01, 0.1, and 1 mg/L BPA. Data are presented as boxplots (n D 6-8 for each group). Asterisks indicate significant differences
compared to the solvent control (P < 0.05, P < 0.01, P < 0.001). following a long term exposure to BPA,19 and studies using the
aromatase knockout (ArKO) mouse found ArKO males to
have reduced fertility,45 demonstrating the critical role of aro-
matase in gametogenesis in males. transcription was reported in ovaries of Gobiocypris rarus
exposed to 0.05 mg/L BPA for 35, d and was associated with
disruption of oogenesis and the occurrence of atretic follicles.31
These findings concur with previous studies reporting that
esr2a is more sensitive compared to esr1, to the natural estro-
gen, 17b-estradiol (E2).41 In contrast, BPA caused increased
transcription of esr2b in the livers of males and females but not
in the gonads, and, importantly, for females this effect was
observed at the environmentally relevant concentration of
0.01 mg/L BPA. Reproductive effects of BPA on adult zebrafish These findings are consistent with those of Haubruge et al.,
who reported declines in sperm count of 40-75% in guppies
exposed to 0.274 or 0.549 mg/L BPA.23 BPA exposure has been
linked to male sexual dysfunction in humans, and urinary con-
centrations of BPA have been associated with declines in sperm
concentration, motility, and morphology in men.48 The mecha-
nism by which disruption of normal spermatogenesis takes
place is hypothesized to be via disruption of the Sertoli cells,
which are directly sensitive to xenobiotic chemicals, and whose
functions are essential during spermatogenesis.23 Our data are
in agreement with these findings and further document the In addition to the disruption in estrogen receptor signaling,
changes in sex steroid biosynthesis may have contributed to the
observed disruption of reproduction in colonies exposed to
1 mg/L BPA. We found a significant decrease in cyp19a1a tran-
script in the testis of males exposed to 1 mg/L BPA, and a sig-
nificant association between transcription and BPA exposure
concentration. In ovaries, a decreasing trend was also observed. These findings suggest potential feedback mechanisms were
activated to counteract the estrogen/androgen ratio imbalance
caused by BPA, through reducing the irreversible conversion of
testosterone into estrogens. Similar findings have recently been
reported for the Chinese rare minnow (Gobiocypris rarus) EPIGENETICS
5 EPIGENETICS 533 importance of Sertoli cells as targets for BPA toxicity, by dem-
onstrating its effects on amh and cyp19a1a, both expressed in
these cells in the testis. measured a significant decrease in global DNA methylation in the
testis of fish exposed to 1 mg/L BPA, suggesting that the BPA-
induced reduction in global methylation is likely to be functionally
linked to the decrease in dnmt1 transcription. These data align
with the reported hypomethylation of sperm associated with the
presence of BPA in urine, in a study of male factory workers in
China.37 There is evidence to suggest that DNA demethylation and
methylation establishment events during early development are
guided by the paternal DNA methylation program instructed by
the sperm chromosomes.55,56 Therefore, it is plausible that changes
to the global DNA methylation pattern in testes such as those
reported for fish exposed to 1 mg/L BPA may have the potential to
impact on the epigenetic reprogramming of embryos, with poten-
tial consequences for their subsequent development. Effects of BPA on epigenetic regulation There is now strong evidence demonstrating that BPA has the
potential to induce changes in DNA methylation at both gene-
specific and genome-wide levels in exposed organisms32,33;
however, this has rarely been studied in fish. It is important to note that global DNA methylation in this
study, measured using the LUMA assay, provides only an esti-
mate of the total DNA methylation across all areas of the
genome and all cell types in a given tissue. Decreased dnmt1
transcription may be causing demethylation of specific areas of
the genome or within specific cell types, but this may not be
detectable by a global measurement of DNA methylation,
including all cell types simultaneously. This may explain why
dnmt1 transcription appears to be more sensitive to BPA expo-
sure compared to global methylation measurements. In our study, we found a significant decrease in the expression of
the DNA methylation maintenance enzyme, dnmt1, for all three
BPA concentrations tested in ovaries of females—including at
environmentally relevant concentrations—and the DNA methyla-
tion pattern in the promoter region of the dnmt1 gene was found
to be significantly associated with BPA exposure concentrations for
four CpG sites. The expression of dnmt1 is known to be associated
with changes in global DNA methylation, and inactivation of
dnmt1 has been shown to cause global demethylation of the
genome.52 In this regard, it was interesting that global DNA meth-
ylation levels were significantly decreased in ovarian tissue of fish
exposed to 1 mg/L BPA, potentially as a consequence of the sup-
pression in dnmt1 transcription. In contrast, previous studies in
Gobiocypris rarus, have reported global DNA hypermethylation in
ovaries exposed to 0.015 mg/L BPA for 35, d19 suggesting these epi-
genetic effects may be concentration- and time-dependent, and
potentially vary across vertebrate species. Importantly, dnmt1 is
reported to be an important maternal transcript involved in the
regulation of DNA methylation during the first stages of embryo
development, particularly prior to the zygote genome activa-
tion.53,54 Therefore, the significant decrease in the expression of
dnmt1 observed in ovaries of females exposed to all three concen-
trations of BPA could have potential consequences for the appro-
priate development of offspring, in addition to influencing the level
of DNA methylation in the ovary of exposed females. The transcript profile for mbd2 was significantly altered fol-
lowing exposure to BPA in both male testis and female livers. Reproductive effects of BPA on adult zebrafish Changes in fertilization success may have occurred not only
due to effects of BPA on spermatogenesis but also due to BPA-
induced alterations in egg quality. Females exposed to 1 mg/L
BPA produced an increased number of eggs, but these eggs may
have lacked the quality required for fertilization success and
embryo survival. Many factors contribute to egg quality, of
which the hormonal environment during oogenesis is a critical
one.49 The observed changes in the expression of estrogen recep-
tors and the trends observed for cyp19a1a in females indicate a
disruption of the estrogen/androgen balance within ovaries and
consequent alterations in sex steroid signaling pathways, puta-
tively leading to alterations in oogenesis and oocyte quality. This
hypothesis is supported by previous studies in which BPA was
shown to affect oogenesis.50 In addition, a study in pregnant
mice exposed to BPA found gross abnormalities in the meiotic
prophase of oogenesis, including synaptic defects, which were
suggested to occur via Esr2 (ERb) signaling.51 Interestingly, in
the present study, changes were also observed in the expression
of an ERb subtype (esr2a) in the gonads of both sexes, suggesting
similar mechanisms could be occurring. In the liver, we observed a significant decrease in dnmt1
transcription in males and females, including at environmen-
tally relevant concentrations, demonstrating the very significant
impact of BPA on the expression of this key DNA methylation
maintenance enzyme. In addition, we report significant hyper-
methylation of the promoter region of the dnmt1 gene in both
male and female livers. Based on the positive association
between the expression of this gene and global DNA methyla-
tion, it is plausible that the suppression of dnmt1 may impact
on global methylation as seen in other tissues. However, this
could not be measured in the liver due to technical limitations
related to the amount of DNA obtained from this tissue. The
fact that changes in the transcript and methylation profile for
dnmt1 occur at environmentally relevant concentrations high-
lights the potential for BPA to cause epigenetic effects in
exposed organisms within current exposure scenarios. Exposures of breeding zebrafish to bisphenol A The selected 15 groups that showed consistent breeding and
behavioral patterns during the initial acclimation period were
subjected to a 10 d pre-exposure period, followed by a 15 d expo-
sure period. Reproductive data for the 10 d pre-exposure period
were collected to ensure that all breeding groups were reproduc-
ing consistently and there were no differences between reproduc-
tive measurements for any of the breeding groups prior to the
chemical exposure period. Three independent replicate breeding
groups were assigned at random to each treatment. A flow-
through system was used to dose the tanks for 15 d with three
concentrations of BPA (0.01, 0.1, and 1 mg/L) using ethanol
(0.0005%) as a solvent. An absolute control receiving water alone
and a solvent control receiving the same concentration of ethanol
as the chemical exposures were also included. Effects of BPA on epigenetic regulation mbd2 belongs to a family of nuclear proteins capable of binding
specifically to methylated DNA, and may also function to
repress transcription from methylated gene promoters.57 We
found also a significant decrease in mecp2 transcription in male
livers, a gene involved in transcriptional repression by associat-
ing with methylated CpG dinucleotides where it silences tran-
scription by recruiting histone deacetylases, resulting in
chromatin remodeling.58 In addition, in male livers a significant
decrease in hdac3 transcription was also observed. These find-
ings suggest that BPA is not only interacting with the processes
linked to DNA methylation, but also has the potential to dis-
rupt processes linked to chromatin structure and potentially
impact on gene function via these mechanisms. For males, dnmt1 transcription was also negatively associated
with BPA exposure concentrations and a significant hypermethyla-
tion of two CpG sites in the promoter region of the dnmt1 gene in
fish exposed to 0.1 mg/L BPA was observed. In addition, we Despite the advances in our understanding of the epigenetic
and transcriptional consequences of BPA in a model vertebrate,
there are some limitations to the methodologies used: the locus-
specific DNA methylation measurements conducted were 534 L. V. LAING ET AL. 534 based on the sodium bisulphite treatment of genomic DNA
and, therefore, cannot distinguish between DNA modifications
such as 5-hydroxymethylcytosine (5hmC), 5-formylcytosine
(5fC), and methylcytosine (5mC), which have unknown func-
tional significance.59 In addition, we explored the methylation
status of specific CpG positions, within the regulatory regions
of select target genes, hypothesized to be targets of BPA toxic-
ity. This hypothesis-driven approach was successful in identify-
ing some important mechanisms of BPA toxicity but may have
missed other interesting effects outside these targeted regions,
as suggested by the effects of BPA on global methylation levels. In addition, the global and locus-specific methylation measure-
ments reported in this study are single measurements of DNA
methylation across multiple cellular populations and cell types
within each tissue. Both the gonad and liver are comprised of a
mixture of cell types, whose genomic methylation and tran-
scriptional activity is unique to the function of each cell type. In the testis for example, a large percentage of the cellular com-
position is made up of sperm cells containing very little cyto-
plasm and limited transcriptional activity, and the genomic
DNA of sperm cells is also known to be hypermethylated. Fish husbandry Wild type WIK strain adult zebrafish (originating from a stock
population at the University of Exeter) were maintained
according to the conditions reported in Paull et al.60 Prior to
the start of the experiment, fish were randomly allocated into
18 breeding groups of 4 males and 4 females, kept in individual
15-L flow-through tanks and were allowed to breed naturally
during an acclimation period of 7 d. After this period, colonies
that failed to spawn consistently were removed prior to the start
of the experiment. Mains tap water was filtered by reverse
osmosis [Environmental Water Systems (UK) Ltd.] and recon-
stituted with Analar-grade mineral salts to standardized syn-
thetic freshwater (final concentrations to give a conductivity of
300 mS: 122 mg/L CaCl22H2O, 9.4 mg/L NaHCO3, 50 mg/L
MgSO47H2O, 2.5 mg/L KCl, 50 mg/L Tropic Marin Sea Salt),
aerated, and heated to 28C in a reservoir, before it was sup-
plied to each aquarium using a flow-through system. Tanks
were aerated and supplied with a flow rate of 48 L/day of
water.60 Tank water was maintained at 28 § 0.5C and pH 7-
7.5 and fish were maintained under a 12 h light:dark cycle,
including dawn and dusk transition periods of 30 min. Fish
were fed live Artemia nauplii once daily (ZM Premium Grade
Artemia; ZM Ltd.) and TetraMin tropical flake food (Tetra;
Melle, Germany) twice daily, to satiation. Effects of BPA on epigenetic regulation In
contrast, the ovary contains oocytes characterized by very large
cytoplasm where transcripts are stored to support the initial
stages of embryogenesis before embryonic genome activation. Therefore, the datasets collected for these tissues are strongly
dependent on the cellular composition of the tissue. In future
studies, a genome-wide approach to measure methylation and
also histone modifications, as well as analysis of single cells or
pure populations of cells, may help to further characterize the
effects of BPA on epigenetic signaling pathways. Fish husbandry Conclusions Overall, we have found evidence that BPA caused significant
disruption to reproduction in breeding zebrafish exposed to
1 mg/L BPA, likely via estrogenic mechanisms. The potential
for BPA to cause disruption of reproduction shown here raises
concerns for its toxicity when organisms are exposed to BPA in
environments affected by other stressors, including other envi-
ronmental endocrine disruptors with similar mechanistic path-
ways that may act additively to cause reproductive disruption. Importantly, BPA also caused significant alterations in the tran-
scription of a number of genes involved in epigenetic regulation
in both liver and gonad tissue, most notably on dnmt1, which
occurred in conjunction with decreases in global DNA methyl-
ation. Of note, some changes were observed after exposure to
environmentally relevant concentrations of BPA (0.01 mg/L),
corresponding to current exposure scenarios for both humans
and wildlife. These findings provide evidence of the adverse
effects of BPA in a model vertebrate and advocate for BPA’s
replacement within consumer products and its reduction in the
environment. On day one of the exposure period, tanks were spiked with
the appropriate amount of BPA to achieve the required expo-
sure concentrations. Flow rates were monitored daily to ensure
the chemical concentrations remained consistent and dosing
stocks were replaced every day. Water samples from each tank
were collected on days 5, 10, and 15 of the exposure, and were
stored at ¡20C until chemical analysis. The effects of BPA on reproduction were determined by
measuring the egg production and fertilization success of indi-
vidual groups. Eggs were collected each morning approximately
one hour post-fertilization (hpf), washed and transferred to
petri dishes for analysis. The numbers of fertilized and unfertil-
ized eggs were determined by visual inspection for each treat-
ment using a dissection microscope (Motic DM143, Hong
Kong). Water chemistry For analysis of the concentrations of BPA in the exposure
water, methanol, acetonitrile and water, both HPLC and LC-
MS grade, HiPerSolv CHROMANORM®, were purchased from
VWR Int. One mL of each water sample was added to a glass
vial and mixed with 1 mL of HPLC-grade acetonitrile. Before
LC-MS/MS analysis, aliquots were vortexed and diluted in a
mixture of acetonitrile and water (1:3 v/v). Analyses were per-
formed using a Surveyor MS Pump Plus HPLC pump with an
HTC PAL autosampler coupled to a TSQ Vantage triple quad-
rupole mass spectrometer equipped with heated electrospray Transcript profiling Transcript profiling of genes encoding epigenetic regulatory
proteins and genes involved in reproductive function was con-
ducted using real-time quantitative PCR (RT-QPCR) as previ-
ously described.61 Beacon Designer 3.0 software (Premier
Biosoft International, Paulo Alto, CA) was used for designing
primers for each target gene using zebrafish NCBI RefSeq
sequences, and primers were purchased from MWG-Biotech
(Ebersburg, Germany). Assays for each transcript were opti-
mized and standard curves were generated as previously
described.61 Primer specificity was confirmed by observation of
a single amplification product of the expected melting tempera-
ture throughout the range of detection. The linear correlation
(R2) between the mean Ct and the logarithm of the cDNA dilu-
tion was > 0.99 in each case, and efficiencies were between
1.86-2.24. The primer sequences, annealing temperatures, PCR
product sizes and PCR efficiencies for each primer pair are
shown in Supporting Information Table S2. Template preparation and pyrosequencing was carried out
as described by Tost and Gut (2007) 66 on bisulfite-treated
DNA from 8 individual fish (gonads and livers) per treatment
group. Briefly, genomic DNA (500ng) was treated with sodium
bisulfite using the EZ-96 DNA Methylation-Gold Kit (Zymo
Research, CA, USA) according to the manufacturers’ standard
protocol. Water negative controls were run in duplicate to ver-
ify the absence of DNA contamination. Bisulfite-PCR amplifi-
cation was performed in duplicate using the primers and assay
conditions provided in Supporting Information Table S3. Unmodified DNA samples were included during primer opti-
mization to confirm primer specificity for bisulfite-modified
DNA. Chemicals All chemicals were obtained from Sigma-Aldrich, UK, unless
stated otherwise. g
On day 15 of the exposure period, all fish were sacrificed
humanely using a lethal dose of benzocaine followed by EPIGENETICS EPIGENETICS 535 et al. 2015) 63 using the Biomart portal.64 Zebrafish esr1 (ENS-
DARG00000004111)
has
3
known
transcripts
[esr1-001
(3449 bp), esr1-201 (3502 bp) and esr1-202 (212 bp)] and 2
transcription start sites (TSSs). The dnmt1 gene (ENS-
DARG00000030756) also has 2 TSSs and 3 transcripts [dnmt1-
001
(4896
bp),
dnmt1-201
(4893
bp)
and
dnmt1-202
(5031 bp)]. amh (ENSDARG00000014357) has one transcript
(amh-001, 3243 bp) and one TSS (Supporting Information
Fig. S6). Target sites within the promoter sequences were cho-
sen based on their proximity to the TSSs and estrogen-respon-
sive elements (EREs), identified using JASPAR,65 and the
matrix models ESR1 (MA0112) and ESR2 (MA0258). PCR and
bisulfite pyrosequencing assays were designed using the Pyro-
Mark Assay design software (Qiagen, Hilden, Germany). Pyro-
sequencing primers and their corresponding target sequences
are shown in Supporting Information Table S3. destruction of the brain, in accordance with UK Home Office
regulations. The wet weight and fork length were recorded, and
the condition factor for each fish was calculated (k) D [weight
(g) £ 100]/[fork length (cm)]3. The gonads and livers were dis-
sected and weighed, and the gonadosomatic index (GSI) D
gonad weight (mg)/[total weight (mg)- gonad weight (mg)] £
100 and hepatosomatic index (HSI) D liver weight (mg)/[total
weight (mg)- liver weight (mg)] £ 100 were calculated. Gonads
and livers were collected, snap frozen in liquid nitrogen and
stored at ¡80C until analysis for transcript profiling and DNA
methylation. Luminometric-based assay (LUMA) for global DNA
methylation pp
g
RNA and DNA were extracted together from the livers and
gonads of 8 male and 8 female fish from each treatment group
using the AllPrep DNA/RNA Micro Qiagen Kit (Qiagen, Hil-
den, Germany) according to the manufacturer’s instructions,
which allows for extraction of both RNA and DNA from the
same tissue sample. NanoDrop ND-1000 Spectrophotometer
(NanoDrop Technologies, Wilmington, USA) was used to
assess RNA and DNA purity and concentration. RNA was
treated with DNase I (Qiagen) to remove any potential DNA
contamination. cDNA was synthesized from 2 mg of total RNA
using random hexamers (MWG-Biotech, Ebersberg, Germany)
and M-MLV reverse transcriptase (Promega, Madison, USA),
according to manufacturer’s instructions. cDNA was then
diluted 1:2 and RT-QPCR was performed in duplicate using an
iCycler iQ Real-time Detection System (Bio-Rad Laboratories,
Hercules, CA) and SYBR Green chemistry, as previously
described.61 On each plate, a template-minus negative control
was run in duplicate to verify the absence of cDNA contamina-
tion. Efficiency-corrected relative expression levels were deter-
mined after normalization to a control gene, ribosomal protein
l8 (rpl8), which has been shown to have stable expression in the
livers and gonads following exposures to estrogens in another
cyprinid fish species.44,62 The LUMA assay was performed as described by Karimi et al. (2006) using DNA extracted from gonad samples from 8 indi-
vidual fish per treatment.67 Sufficient quantities of DNA were
not available to perform the LUMA assay in liver samples;
therefore, analysis of global DNA methylation were conducted
only for gonad samples. Each DNA sample (250 ng) was
digested in duplicate with HpaII and MspI, and data were nor-
malized to the EcoRI peak to account for any technical differ-
ences between samples.68 Global DNA methylation values were
calculated according to the formula [HpaII(G)/EcoRI(T)]/
[MspI(G)/EcoRI(T)], where G and T refer to the peak heights
for HpaII or MspI (methylation) and EcoRI (input DNA),
respectively. Bisulfite pyrosequencing DNA sequence data for the promoter regions of esr1, amh, and
dnmt1 were obtained from Ensembl (release 83; Cunningham 536
L. L. V. LAING ET AL. 536 (HESI II) source (ThermoFisher Scientific, Hemel Hempstead,
UK). Chromatographic separation was achieved using a
reversed-phase, 3 mm particle size, C18 Hypersil GOLD col-
umn 50 mm £ 2.1 mm i.d. (Thermo Scientific, San Jose CA,
USA). Analytes were separated using a linear gradient of water
and methanol. The initial conditions for the gradient consisted
of 10% methanol, which was increased to 100% in 4.5 min and
maintained for 1 min before returning to the initial 10% metha-
nol. The flow rate was 500 mL/min. The temperature of the
autosampler was set at 8C, and the column was kept at a room
temperature. The HESI probe was operating in the negative
mode and an ion-spray voltage of ¡4.0 kV was applied. The
heated capillary temperature was set at 275C and the vaporizer
temperature was 60C. Nitrogen was employed as sheath and
auxiliary gas at a pressure of 30 and 5 arbitrary units, respec-
tively. The argon CID gas was used at a pressure of 1.5 mTorr
and the optimum collision energy (CE) for each transition was
selected. Quantification of BPA was performed using two char-
acteristic multiple reaction monitoring (MRM) transitions of
precursor ion 227.1! 212.1 (CE: 20 V) and 227.1 ! 133.1
(CE: 28 V). For transcript profiles, data points classified as outliers
(using Chauvenet’s criterion) and data points for which the
expression was below the assay detection limit were excluded
from analysis. Where amplification was detected in more than
70% of individuals, data were represented as fold-change rela-
tive to the expression in the water control group and groups
were then compared using one-way ANOVA and Kruskal-
Wallis tests with post-hoc tests as described previously. Where
amplification was detected in less than 70% of individuals, data
were represented as the proportion of individuals for which the
target genes were detected, and analysis was conducted using a
binomial generalized linear model. In the gonadal data sets,
PCA was also performed using the prcomp function to identify
the main trends in gene expression. In order to determine if there were associations between the
methylation levels for specific loci in the promoter regions of
genes of interest and their transcription, correlation analysis was
conducted. Bisulfite pyrosequencing Where data was normally distributed Pearson corre-
lation was used, and where data did not meet the assumptions
of parametric testing, Spearman correlation analysis was per-
formed. Correlation analyses were also conducted to determine
the relationship between global methylation and dnmt1 tran-
scription, as above. The relationship between BPA concentration
and transcript expression or methylation was also determined
using regression analysis, calculated using linear modeling. Funding We thank the Aquatic Resources Center technical team for support with
zebrafish husbandry. This work was funded by a PhD studentship from
the Fisheries Society of the British Isles (http://www.fsbi.org.uk/) and the
University of Exeter (http://www.exeter.ac.uk/) to LVL and EMS. TMUW
was funded by a Natural Environment Research Council CASE PhD stu-
dentship (grant no. NE/I528326/1) and the Salmon & Trout Association
(http://www.salmon-trout.org/). No potential conflicts of interest were disclosed. No potential conflicts of interest were disclosed. Statistical analysis Statistical analyses were carried out using R (version 3.0.2).69
Prior to analysis, data were tested for equal variance and for
normality using the Shapiro–Wilk test. Proportional data and
variables with non-Gaussian distributions or non-homoge-
neous variances were subjected to variance-stabilizing arcsine
transformations or log transformations. Non-parametric statis-
tics were used when transformations did not result in distribu-
tions meeting the assumptions for parametric tests. All graphs
were plotted using untransformed data for ease of interpreta-
tion. For the mean fertilization rates, comparisons between
treatments were performed using Kruskal-Wallis tests followed
by the Wilcoxon signed rank test. The Regression coefficient
(R2) was calculated using linear modeling for fertilization rates. Linear mixed effects models were generated using the lme4
package 70 in order to explore the effect of BPA concentration
and length of exposure on egg numbers. Non-significant terms
were removed from models; models were compared based on
likelihood ratio testing to give the appropriate minimum ade-
quate model. Model results were inspected to ensure residuals
were normally distributed. All graphs were plotted using untransformed data for ease of
interpretation, and were created using the R packages ggplot2,71
gplots,72 beeswarm,73 and ggbiplot.74 References Reduced sperm counts in guppies (
Poecilia reticulata) following exposure to low levels of tributyltin and
bisphenol A. Proc R Soc Lind B 2000; 267:2333-7; PMID:11413652;
http://dx.doi.org/10.1098/rspb.2000.1288 7. Calafat AM, Kuklenyik Z, Reidy JA, Caudill SP, Ekong J, Needham LL. Urinary concentrations of bisphenol A and 4-nonylphenol in a human
reference population. Environ Health Persp 2004; PMID:15811827;
113:391-5; http://dx.doi.org/10.1289/ehp.7534 p
g
p
24. Kwak H, Bae M, Lee M, Lee Y, Lee B, Kang K, Chae CH, Sung HJ,
Shin JS, Kim JH, et al. Effects of nonylphenol, bisphenol A, and their
mixture on the viviparous swordtail fish ( Xiphophorus helleri). Envi-
ron Toxicol Chem 2001; 20:787-95; PMID:11345455; http://dx.doi. org/10.1002/etc.5620200414 p
g
p
8. Zhang Z, Alomirah H, Cho H, Li Y, Liao C, Minh TB, Mohd MA,
Nakata H, Ren N, Kannan K. Urinary bisphenol A concentrations
and their implications for human exposure in several asian countries. Environ Sci Technol 2011; 45(16):7044-50; PMID:21732633; http://
dx.doi.org/10.1021/es200976k g
25. Lang IA, Galloway TS, Scarlett A, Henley WE, Depledge M, Wallace RB,
Melzer D. Association of urinary bisphenol A concentration with medical
disorders and laboratory abnormalities in adults. JAMA 2008; 300:1303-
10;PMID:18799442;http://dx.doi.org/10.1001/jama.300.11.1303 9. Takeuchi T, Tsutsumi O. Serum bisphenol A concentrations showed
gender differences, possibly linked to androgen levels. Biochem Bio-
phys Res Commun 2002; 291:76-8; PMID:11829464; http://dx.doi. org/10.1006/bbrc.2002.6407 10;PMID:18799442;http://dx.doi.org/10.1001/jama.300.11.1303 26. Braun JM, Kalkbrenner AE, Calafat AM, Yolton K, Ye X, Dietrich KN,
Lanphear BP. Impact of early-life bisphenol A exposure on behavior
and executive function in children. Pediatrics 2011; 128:873-82;
PMID:22025598; http://dx.doi.org/10.1542/peds.2011-1335 10. Ikezuki Y, Tsutsumi O, Takai Y, Kamei Y, Taketani Y. Determination
of bisphenol A concentrations in human biological fluids reveals sig-
nificant early prenatal exposure. Hum Reprod 2002; 17:2839-41;
PMID:12407035; http://dx.doi.org/10.1093/humrep/17.11.2839 27. Ho SM, Tang WY, Belmonte de Frausto J, Prins GS. Developmental
exposure to estradiol and bisphenol A increases susceptibility to pros-
tate carcinogenesis and epigenetically regulates phosphodiesterase
type 4 variant 4. Cancer Res 2006; 66:5624-32; PMID:16740699;
http://dx.doi.org/10.1158/0008-5472.CAN-06-0516 11. Kang JH, Kondo F, Katayama Y. Human exposure to bisphenol A. Toxicology
2006;
226:79-89;
PMID:16860916;
http://dx.doi.org/
10.1016/j.tox.2006.06.009 12. Staples CA, Woodburn K, Caspers N, Hall AT, Klecka GM. A weight
of evidence approach to the aquatic hazard assessment of bisphenol
A. Hum Ecol risk Assess 2002; 8:1083-105; PMID:21130487; http://
dx.doi.org/10.1080/1080-700291905837 28. Sugiura-Ogasawara M, Ozaki Y, Sonta S, Makino T, Suzumori K. Exposure to bisphenol A is associated with recurrent miscarriage. Hum Reprod 2005; 20:2325-9; PMID:15947000; http://dx.doi.org/
10.1093/humrep/deh888 13. References 1. Ramakrishnan S, Wayne NL. Impact of bisphenol-A on early embry-
onic development and reproductive maturation. Reprod Toxicol 2008;
25:177-83;
PMID:18191535;
http://dx.doi.org/10.1016/j. reprotox.2007.11.002 In order to determine the effects of BPA on the reproductive
and molecular endpoints measured, statistical comparisons were
performed between the solvent control and the groups exposed
to BPA, and comparisons between the water control and the sol-
vent control were also conducted to confirm that no significant
differences occurred as a result of the presence of the solvent. Comparisons between treatments were performed using one-way
analysis of variance (ANOVA) and Kruskal-Wallis tests. Where
ANOVA analysis found a P 0.05, post-hoc testing was carried
out using the pairwise multiple comparisons of means method
with false discovery rate P value adjustment. Where the Kruskal-
Wallis test was used, post-hoc testing was carried out using the
Wilcoxon signed rank test accounting for repeated measures
within the data sets. P values of 0.05 were considered to be
significant. All data are presented as mean § SEM. 2. Vandenberg LN, Hauser R, Marcus M, Olea N, Welshons WV. Human exposure to bisphenol A (BPA). Reprod Toxicol 2007;
24:139-77;
PMID:17825522;
http://dx.doi.org/10.1016/j. reprotox.2007.07.010 2. Vandenberg LN, Hauser R, Marcus M, Olea N, Welshons WV. Human exposure to bisphenol A (BPA). Reprod Toxicol 2007;
24:139-77;
PMID:17825522;
http://dx.doi.org/10.1016/j. reprotox.2007.07.010 3. Mileva G, Baker SL, Konkle ATM, Bielajew C. Bisphenol-A: epigenetic
reprogramming and effects on reproduction and behavior. Int J Envi-
ron Res Public Health 2014; 11:7537-61; PMID:25054232; http://dx. doi.org/10.3390/ijerph110707537 g
j p
4. Liao C, Kannan K. Concentrations and profiles of bisphenol A and
other bisphenol analogues in foodstuffs from the United States and
their implications for human exposure. J Agric Food Chem 2013;
61:4655-62; PMID:23614805; http://dx.doi.org/10.1021/jf400445n g
j
5. von Goetz N, Wormuth M, Scheringer M, Hungerb€uhler K. Bisphenol
a: how the most relevant exposure sources contribute to total EPIGENETICS
53 537 Aquat Toxicol 2015; 169:204-14; PMID:26562050; http://dx.doi.org/
10.1016/j.aquatox.2015.10.020 consumer exposure. Risk Anal 2010; 30:473-87; PMID:20136739;
http://dx.doi.org/10.1111/j.1539-6924.2009.01345.x 22. Bhandari RK, Vom Saal FS, Tillitt DE. Transgenerational effects from
early developmental exposures to bisphenol A or 17a-ethinylestradiol
in medaka, Oryzias latipes. Sci Rep 2015; 5:9303; PMID:25790734;
http://dx.doi.org/10.1038/srep09303 6. Rudel RA, Camann DE, Spengler JD, Korn LR, Brody JG. Phthalates,
alkylphenols, pesticides, polybrominated diphenyl ethers, and other
endocrine-disrupting compounds in indoor air and dust. Environ Sci
Technol 2003; 37:4543-53; PMID:14594359; http://dx.doi.org/10.1021/
es0264596 23. Haubruge E, Petit F, Gage MJG. References Belfroid A, van Velzen M, van der Horst B, Vethaak D. Occurrence of
bisphenol A in surface water and uptake in fish: evaluation of field
measurements. Chemosphere
2002;
49:97-103;
PMID:12243336;
http://dx.doi.org/10.1016/S0045-6535(02)00157-1 29. Bonefeld-Jørgensen EC, Long M, Hofmeister M V, Vinggaard AM. Endocrine-disrupting potential of bisphenol A, bisphenol A dimetha-
crylate, 4-n-nonylphenol, and 4-n-octylphenol in vitro: new data and
a
brief
review. Environ
Health
Persp
2007;
115:69-76;
PMID:18174953; http://dx.doi.org/10.1289/ehp.9368 14. Li Y, Luh CJ, Burns KA, Arao Y, Jiang Z, Teng CT, Tice RR, Korach
KS. Endocrine-Disrupting Chemicals (EDCs): in vitro mechanism of
estrogenic activation and differential effects on ER target genes. Envi-
ron Health Persp 2013; 121:459-66; PMID: 23384675; http://dx.doi. org/10.1289/ehp.1205951 30. Honma S, Suzuki A, Buchanan DL, Katsu Y, Watanabe H, Iguchi T. Low dose effect of in utero exposure to bisphenol A and diethylstilbes-
trol on female mouse reproduction. Reprod Toxicol 2002; 16:117-22;
PMID:11955942; http://dx.doi.org/10.1016/S0890-6238(02)00006-0 g
p
15. Jobling S, Nolan M, Tyler CR, Brighty G, Sumpter JP. Widespread
sexual disruption in wild fish. Environ Sci Technol 1998; 32:2498-506;
http://dx.doi.org/10.1021/es9710870 31. Zhang Y, Gao J, Xu P, Yuan C, Qin F, Liu S, Zheng Y, Yang Y, Wang
Z. Low-dose bisphenol A disrupts gonad development and steroido-
genic genes expression in adult female rare minnow Gobiocypris
rarus. Chemosphere 2014; 112:435-42; PMID:25048937; http://dx.doi. org/10.1016/j.chemosphere.2014.04.089 g
16. Waring RH, Harris RM. Endocrine disrupters: a human risk? Mol Cell
Endocrinol 2005; 244:2-9; PMID:16271281; http://dx.doi.org/10.1016/
j.mce.2005.02.007 32. Kundakovic M, Champagne FA. Epigenetic perspective on the devel-
opmental effects of bisphenol A. Brain Behav Immun 2011; 25:1084-
93; PMID:21333735; http://dx.doi.org/10.1016/j.bbi.2011.02.005 17. vom Saal FS, Akingbemi BT, Belcher SM, Birnbaum LS, Crain DA,
Eriksen M, Farabollini F, Guillette LJ, Jr, Hauser R, Heindel JJ, et al. Chapel Hill bisphenol A expert panel consensus statement: integration
of mechanisms, effects in animals and potential to impact human
health at current levels of exposure. Reprod Toxicol 2007; 24:131-8;
PMID:17768031; http://dx.doi.org/10.1016/j.reprotox.2007.07.005 33. Kim JH, Sartor MA, Rozek LS, Faulk C, Anderson OS, Jones TR, Nahar
MS, Dolinoy DC. Perinatal bisphenol A exposure promotes dose-
dependent alterations of the mouse methylome. BMC Genomics 2014;
15:30; PMID:24433282; http://dx.doi.org/10.1186/1471-2164-15-30 18. Villeneuve DL, Garcia-Reyero N, Escalon BL, Jensen KM, Cavallin
JE, Makynen EA, Durhan EJ, Kahl MD, Thomas LM, Perkins EJ,
et al. Ecotoxicogenomics to support ecological risk assessment: a
case study with bisphenol A in fish. Environ Sci Technol 2012;
46:51-9; PMID:21786754; http://dx.doi.org/10.1021/es201150a 34. Singh S, Li SS. References Epigenetic effects of environmental chemicals bisphe-
nol
A
and
phthalates. Int
J
Mol
Sci
2012;
13:10143-53;
PMID:22949852; http://dx.doi.org/10.3390/ijms130810143 35. Dolinoy DC, Huang D, Jirtle RL. Maternal nutrient supplemen-
tation counteracts bisphenol A-induced DNA hypomethylation
in early development. Proc Natl Acad Sci U. S. A 2007;
104:13056-61;
PMID:17670942;
http://dx.doi.org/10.1073/
pnas.0703739104 19. Liu Y, Yuan C, Chen S, Zheng Y, Zhang Y, Gao J, Wang Z. Global and
cyp19a1a gene specific DNA methylation in gonads of adult rare min-
now Gobiocypris rarus under bisphenol A exposure. Aquat Toxicol
2014;
156:10-6;
PMID:25125231;
http://dx.doi.org/10.1016/j. aquatox.2014.07.017 36. Morgan HD, Sutherland HG, Martin DI, Whitelaw E. Epigenetic
inheritance at the agouti locus in the mouse. Nat Genet 1999; 23:314-
8; PMID:10545949; http://dx.doi.org/10.1038/15490 20. Sohoni P, Tyler CR, Hurd K, Caunter J, Hetheridge M, Williams T,
Woods C, Evans M, Toy R, Gargas M, et al. Reproductive effects of
long-term exposure to bisphenol A in the fathead minnow ( Pime-
phales
promelas). Environ
Sci
Technol
2001;
35:2917-52;
PMID:11478243; http://dx.doi.org/10.1021/es000198n 37. Miao M, Zhou X, Li Y, Zhang O, Zhou Z, Li T, Yuan W, Li R, Li DK. LINE-1 hypomethylation in spermatozoa is associated with Bisphenol
A exposure. Andrology 2014; 2:138-44; PMID:24293158; http://dx. doi.org/10.1111/j.2047-2927.2013.00166.x p
g
21. Chen J, Xiao Y, Gai Z, Li R, Zhu Z, Bai C, Tanguay RL, Xu X, Huang
C, Dong Q. Reproductive toxicity of low level bisphenol A exposures
in a two-generation zebrafish assay: evidence of male-specific effects. g
j
38. Crain DA, Eriksen M, Iguchi T, Jobling S, Laufer H, LeBlanc GA,
Guillette LJ, Jr. An ecological assessment of bisphenol-A: evidence 538
L. V. LAING ET AL. L. V. LAING ET AL. 538 from
comparative
biology. Reprod
Toxicol
2007;
24:225-39;
PMID:17604601; http://dx.doi.org/10.1016/j.reprotox.2007.05.008 zebrafish early embryos. Cell 2013; 153:773-84; PMID:23663777;
http://dx.doi.org/10.1016/j.cell.2013.04.041 56. 56. Potok ME, Nix DA, Parnell TJ, Cairns BR. Reprogramming the mater-
nal zebrafish genome after fertilization to match the paternal methyla-
tion pattern. Cell 2013; 153:759-72; PMID:23663776; http://dx.doi. org/10.1016/j.cell.2013.04.030 39. Peretz J, Gupta RK, Singh J, Hernandez-Ochoa I, Flaws JA. Bisphenol A
impairs follicle growth, inhibits steroidogenesis, and downregulates rate-
limiting enzymes in the estradiol biosynthesis pathway. Toxicol Sci 2011;
119:209-17; PMID:20956811; http://dx.doi.org/10.1093/toxsci/kfq319 40. Peretz J, Craig ZR, Flaws JA. Bisphenol A inhibits follicle growth and
induces atresia in cultured mouse antral follicles independently of the
genomic
estrogenic
pathway. Biol
Reprod
2012;
87:1-11;
PMID:22743301; http://dx.doi.org/10.1095/biolreprod.112.101899 57. Turek-Plewa J, Jagodzinski P. References The role of mammalian DNA methyl-
transferases in the regulation of gene expression. Cell Mol Biol Lett
2005; 10:631-47; PMID:16341272 58. Fuks F, Hurd PJ, Wolf D, Nan X, Bird AP, Kouzarides T. The methyl-
CpG-binding protein MeCP2 links DNA methylation to histone
methylation. J Biol Chem 2003; 278:4035-40; PMID:12427740; http://
dx.doi.org/10.1074/jbc.M210256200 p
g
p
41. Tohyama S, Miyagawa S, Lange A, Ogino Y, Mizutani T, Tatarazako N,
Katsu Y, Ihara M, Tanaka H, Ishibashi H, et al. Understanding the
molecular basis for differences in responses of fish estrogen receptor sub-
types to environmental estrogens. Environ Sci Technol 2015; 49:7439-47;
PMID:26032098; http://dx.doi.org/10.1021/acs.est.5b00704 g
j
59. Yu M, Hon GC, Szulwach KE, Song CX, Zhang L, Kim A, Li X, Dai Q,
Shen Y, Park B, et al. Base-resolution analysis of 5-hydroxymethylcy-
tosine
in
the
mammalian
genome. Cell
2012;
149:1368-80;
PMID:22608086; http://dx.doi.org/10.1016/j.cell.2012.04.027 p
g
42. Eddy EM, Washburn TF, Bunch DO, Gouding EH, Gladen BC, Luban
DB, Korach KS. Targeted disruption of the estrogen receptor gene in
male mice causes alteration of spermatogenesis and infertility. Endo-
crinology
1996;
137:4796-805;
PMID:8895349;
http://dx.doi.org/
10.1210/endo.137.11.8895349#sthash.SbK32OFU.dpuf p
g
j
60. Paull GC, Van Look KJW, Santos EM, Filby AL, Gray DM, Nash JP, Tyler
CR. Variability in measures of reproductive success in laboratory-kept col-
onies of zebrafish and implications for studies addressing population-level
effects of environmental chemicals. Aquat Toxicol 2008; 87:115-26;
PMID:18308405; http://dx.doi.org/10.1016/j.aquatox.2008.01.008 p
43. Cooke HJ, Saunders PTK. Mouse models of male infertility. Nat Rev
Genet 2002; 3:790-801; PMID:12360237; http://dx.doi.org/10.1038/
nrg911 61. Uren-Webster TM, Laing L, Florence H, Santos EM. Effects of glypho-
sate and its formulation, Roundup®, on reproduction in zebrafish (
Danio rerio). Environ Sci Technol 2014; 48:1271-9; PMID:24364672;
http://dx.doi.org/10.1021/es404258h 44. Filby AL, Tyler CR. Molecular characterization of estrogen receptors
1, 2a, and 2b and their tissue and ontogenic expression profiles in fat-
head minnow ( Pimephales promelas). Biol Reprod 2005; 73:648-62;
PMID:15930325; http://dx.doi.org/10.1095/biolreprod.105.039701 62. Filby AL, Tyler CR. Appropriate “housekeeping” genes for use in
expression profiling the effects of environmental estrogens in fish. BMC Mol Biol 2007; 8:10; PMID:17288598; http://dx.doi.org/10.1186/
1471-2199-8-10 45. Robertson KM, Simpson ER, Lacham-Kaplan O, Jones MEE. Charac-
terization of the fertility of male aromatase knockout mice. J Androl
2001; 22:825-30; PMID:11545296; http://dx.doi.org/10.1002/j.1939-
4640.2001.tb02587.x 63. Cunningham F, Amode MR, Barrell D, Beal K, Billis K, Brent S, Car-
valho-Silva D, Clapham P, Coates G, Fitzgerald S, et al. Ensembl 2015. Nucleic Acids Res 2015; 43:D662-9; PMID:25352552; http://dx.doi. References org/10.1093/nar/gku1010 46. Rhee JS, Kim BM, Lee CJ, Yoon YD, Lee YM, Lee JS. Bisphenol A
modulates expression of sex differentiation genes in the self-fertilizing
fish, Kryptolebias marmoratus. Aquat Toxicol 2011; 104:218-29;
PMID:21632026; http://dx.doi.org/10.1016/j.aquatox.2011.04.020 64. Kinsella RJ, K€ah€ari A, Haider S, Zamora J, Proctor G, Spudich G,
Almeida-King J, Staines D, Derwent P, Kerhornou A, et al. Ensembl
BioMarts: a hub for data retrieval across taxonomic space. Database
(Oxford)
2011;
2011:bar030;
PMID:21785142;
http://dx.doi.org/
10.1093/database/bar030 47. Li Y, Zhang W, Liu J, Wang W, Li H, Zhu J, Weng S, Xiao S, Wu T. Pre-
pubertal bisphenol A exposure interferes with ovarian follicle develop-
ment and its relevant gene expression. Reprod Toxicol 2014; 44:33-40;
PMID:24051130; http://dx.doi.org/10.1016/j.reprotox.2013.09.002 65. Sandelin A, Alkema W, Engstrom P, Wasserman WW, Lenhard B. JASPAR: an open-access database for eukaryotic transcription factor
binding profiles. Nucleic Acids Res 2004; 32:D91-4; PMID:14681366;
http://dx.doi.org/10.1093/nar/gkh012 48. Meeker JD, Ehrlich S, Toth TL, Wright DL, Calafat AM, Trisini AT,
Ye X, Hauser R. Semen quality and sperm DNA damage in relation to
urinary bisphenol A among men from an infertility clinic. Reprod
Toxicol 2010; 30:532-9; PMID:20656017; http://dx.doi.org/10.1016/j. reprotox.2010.07.005 p
g
g
66. Tost J, Gut IG. DNA methylation analysis by pyrosequencing. Nat
Protoc 2007; 2:2265-75; PMID:17853883; http://dx.doi.org/10.1038/
nprot.2007.314 49. Brooks S, Tyler CR, Sumpter JP. Quality in fish: what makes a good
egg? Rev Fish Biol Fisher 1997; 7:387-416; PMID:23083410; http://dx. doi.org/10.1023/A:1018400130692 67. Karimi M, Johansson S, Ekstr€om TJ. Using LUMA: A luminometric-
based assay for global DNA-methylation. Epigenetics 2006; 1:45-48;
PMID:17998810; http://dx.doi.org/10.1111/1755-0998.12244 50. Hunt PA, Lawson C, Gieske M, Murdoch B, Smith H, Marre A, Has-
sold T, VandeVoort CA. Bisphenol A alters early oogenesis and follicle
formation in the fetal ovary of the rhesus monkey. Proc Natl Acad Sci
U. S. A 2012; 109:17525-30; PMID:23012422; http://dx.doi.org/
10.1073/pnas.1207854109 68. Head JA, Mittal K, Basu N. Application of the LUminometric Methyl-
ation Assay to ecological species: Tissue quality requirements and a
survey of DNA methylation levels in animals. Mol Ecol Resour 2014;
14:943-952; PMID:24576185 51. Susiarjo M, Hassold TJ, Freeman E, Hunt PA. Bisphenol A exposure
in utero disrupts early oogenesis in the mouse. PLoS Genet 2007; 3:e5;
PMID:17222059; http://dx.doi.org/10.1371/journal.pgen.0030005 69. R Core Team. R: A language and environment for statistical comput-
ing. R Found Stat Comput Vienna, Austria. 2012; -. Available: http://
www.r-project.org/ 52. Bestor TH. The DNA methyltransferases of mammals. Hum Mol Gen
2000; 9:2395-402; PMID:11005794; http://dx.doi.org/10.1093/hmg/
9.16.2395 70. References Bates D, Maechler M, Bolker B, Walker S. lme4: Linear mixed-effects
models using Eigen and S4. R Package version 11-7. 2014. Available:
http://CRAN.R-project.org/packageDlme4> 53. Andersen IS, Lindeman LC, Reiner AH, ; strup O, Aanes H, Alestr€om
P, Collas P. Epigenetic marking of the zebrafish developmental pro-
gram. Curr Top Dev Biol 2013; 104:85-112; PMID:23587239; http://
dx.doi.org/10.1016/B978-0-12-416027-9.00,003-6 71. Wickham H. ggplot2: elegant graphics for data analysis. Springer New
York. 2009 72. Warnes GR, Bolker B, Bonebakker L, Gentleman R, Liaw WHA, Lumley T,
et al. gplots: Various R Programming Tools for Plotting Data. R Package
version 2170 2015. Available: http://cran.r-project.org/packageDgplots 54. Aanes H, Winata CL, Lin CH, Chen JP, Srinivasan KG, Lee SGP, Lim
AY, Hajan HS, Collas P, Bourque G, et al. Zebrafish mRNA sequenc-
ing deciphers novelties in transcriptome dynamics during maternal to
zygotic transition. Genome Res 2011; 21:1328-38; PMID:21555364;
http://dx.doi.org/10.1101/gr.116012.110 73. Eklund A. Beeswarm: The Bee Swarm Plot, an Alternative to Strip-
chart. R Package version 020 2015. Available: http://cran.r-project. org/packageDbeeswarm p
g
g
55. Jiang L, Zhang J, Wang JJ, Wang L, Zhang L, Li G, Yang X, Ma X, Sun
X, Cai J, et al. Sperm, but not oocyte, DNA methylome is inherited by g p
g
74. Vincent QV. ggbiplot: A ggplot2 based biplot. R Packag version 055. 2011. Available: http://github.com/vqv/ggbiplot
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Nonlinear behaviour of liquefied natural gas tanks with different seismic isolation systems
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Građevinar
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DOI: https://doi.org/10.14256/JCE.3767.2023
Primljen / Received:
7.4.2023.
Ispravljen / Corrected: 15.6.2023.
Prihvaćen / Accepted:
4.8.2023.
Dostupno online / Available online: 10.11.2023.
Građevinar 10/2023
Nonlinear behaviour of liquefied natural gas
tanks with different seismic isolation systems
Authors:
Research Paper
İbrahim Hüner, Bülent Akbaş, Abdullah Cem Koç
Nonlinear behaviour of liquefied natural gas tanks with different seismic
isolation systems
İbrahim Hüner, PhD. CE
Pamukkale University, Denizli, Turkey
Department of Civil Engineering
fsm-1453@hotmail.com
Corresponding author
This study determines the effects of different types of base isolator systems on the
seismic performance of liquefied natural gas (LNG) storage tanks. Nonlinear time-history
analyses of the non-isolated and three different isolated models were performed for the
average acceleration of seven ground motions scaled to achieve a specified safe shutdown
earthquake. The ANSYS Workbench program was used in the modelling studies of the
LNG liquid, inner steel tank, outer shell, ring beam, roof and concrete foundation and side
wall insulation. The LS-DYNA program was used for the nonlinear analyses of the LNG
liquid, inner steel tank and concrete foundation. The results of the total base shear force,
sloshing height, steel tank stresses and lateral deflection were compared. The results
indicated that there was no difference between the convective and impulsive modes for
the LNG tanks with isolators. It was concluded that the wave motion of the liquid was
different from the oscillation of the structure and the earthquake isolation times did not
affect the sloshing motion. In the non-isolated system, the stress reached 400 MPa,
whereas it was 350 MPa on average in the LNG tanks with isolators.
Prof. Bülent Akbaş, PhD. CE
Key words:
Gebze Institute of Technology, Gebze, Kocaeli,
liquefied natural gas tanks, HDRB, LRB, FPS, LS-DYNA, ANSYS workbench, nonlinear analyses
Turkey
Department of Civil Engineering
akbasb@gyte.edu.tr
Prethodno priopćenje
İbrahim Hüner, Bülent Akbaş, Abdullah Cem Koç
Nelinearno ponašanje spremnika za ukapljeni prirodni plin s različitim
sustavima potresne izolacije
Prof. Abdullah Cem Koç, PhD. CE
Pamukkale University, Denizli, Turkey
Department of Civil Engineering
a_c_koc@pau.edu.tr
Ovaj rad pobliže određuje učinke različitih tipova izolacijskih sustava na ponašanje
spremnika za ukapljeni prirodni plin (engl. liquefied natural gas - LNG) tijekom potresa.
Provedene su nelinearne analize primjenom vremenskog zapisa neizoliranih i triju različitih
izoliranih modela za prosječno ubrzanje sedam vrsta gibanja tala stupnjevanih kako bi se
postigla određena moguća sigurna obustava rada postrojenja tijekom potresa. Program
ANSYS Workbench primijenjen je za modeliranje tekućine ukapljenog plina, unutarnjeg
čeličnog spremnika, vanjske stijenke, serklaže, krova, betonskih temelja i izolacije stražnje
stijenke. Program LS-DYNA primijenjen je za nelinearne analize tekućine LNG-a, unutarnjeg
čeličnog spremnika i betonskih temelja. Uspoređeni su rezultati ukupne poprečne potresne
sile u podnožju, visine zapljuskivanja, naprezanja čeličnog spremnika i bočnog pomaka.
Rezultati su pokazali da nema razlike između konvektivnih i impulsnih modova za izolirane
spremnike LNG-a. Zaključeno je da se valno gibanje tekućine razlikuje od osciliranja
konstrukcije, a razdoblja protupotresnih izolacija nisu utjecala na zapljuskivanje. U
neizoliranom su sustavu vrijednosti naprezanja dosegle 400 MPa, dok su te vrijednosti
u izoliranim spremnicima za LNG prosječno iznosile 350 MPa.
Ključne riječi:
spremnik za ukapljeni prirodni plin, gumeni ležajevi s velikim prigušenjem (HDRB), gumeni ležajevi s
olovnom jezgrom (LRB), klizni ležajevi s njihalom (FPS), nelinearne analize
GRAĐEVINAR 75 (2023) 10, 979-995
979
Građevinar 10/2023
1. Introduction
Natural gas is a hydrocarbon-based gas that consists largely
of methane and has a very low density (0.66 kg/m3). The
transportation of natural gas, which plays an important role
in meeting the energy demands of the world, from source to
end user, is a critical process. Road transport has become an
important alternative to natural gas supply owing to problems
such as natural disasters and security issues in natural gas
transmission lines located at sea and on land. Liquefied
natural gas (LNG) (430–480 kg/m3) is used in land and sea
transportation and it is obtained by the condensation of
natural gas at −168 °C temperature and normal atmospheric
pressure conditions. It should be noted that there is a
difference of approximately 600 times between the densities
of liquid natural gas and gaseous gas. Different types of
storage and transport tanks are used to provide reasonably
fast and predictable transportation of LNG. These tanks
are generally divided into different classes, such as heavytonnage pickup truck, ship, terminal and storage tanks [1]. The
tank type covered in this study is the fixed LNG storage tank
with very large storage volume. These tanks, which are called
fully contained storage tanks, consist of an inner tank made
of cryogenic steel (9 % nickel) and an outer cylindrical shell
wall made of typical post-tensioned reinforced concrete along
the vertical and radial directions. The base slab and spherical
roof dome are made of reinforced concrete. Adequate thermal
insulation is provided between the tanks [2]. In the more
common cases, where the sole plate is in direct contact with
the ground, freezing is prevented using electric heating plates
[1].
In API 620 [3], API625 [4] and NFPA 59A [5], the design
requirements for LNG tanks are stringent because they
store a high-energy chemical substance. LNG storage tanks
are designed according to three levels of seismic action.
The operational basis earthquake (OBE) ground motion is
represented by an acceleration response spectrum with a
10 % probability of exceedance over a 50 years period (mean
return interval of 475 years). The tank system should be
designed to continue operating during and after the OBE.
After a safe shutdown earthquake (SSE), no deformation
should occur in the tank support systems, insulation layers,
or isolators. The accepted maximum considered earthquake
(MCER), determined based on site-specific research, has a 2
% probability of exceedance within a 50 years period (mean
recurrence interval of 2475 years). The aftershock level
earthquake (ALE) ground motion is defined as half the SSE.
An LNG tank system subjected to an ALE should maintain
the primary container volume at its maximum operating level
with no loss from the secondary container [3-5].
The behaviour of structures exposed to fluid pressure caused
by earthquakes was first investigated by Westergaard in
1933 [6]. In the studies carried out by Jacobsen [7, 8], the
rigid tank containing the liquid and the support legs carrying
980
İbrahim Hüner, Bülent Akbaş, Abdullah Cem Koç
the horizontally accelerated liquid tank were analysed. In
studies conducted by Housner [9, 10], the hydrodynamic
pressure created by the liquid in the tank was separated into
its components and the effects of the impulsive pressure
caused by the liquid part accelerating with the tank and
the convective pressure caused by the agitated liquid part
were investigated. The convective component was then
modelled using a single degree-of-freedom (DOF) oscillator
[11, 12]. Haroun and Housner [13] developed a 3-DOF
model for ground-supported deformable tanks, including
flexible behaviour. Veletsos and Tang [14] also conducted
similar studies. Malhatro et al. [15] considered the pulsed
and convective modes and modified the properties of the
mechanical analogue to include higher modes in the resulting
base shear and base overturning moments.
The seismic risks of LNG storage tanks are higher than those
of traditional buildings because they can lead to secondary
disasters such as explosions and environmental pollution,
which would result in significant property damage or loss of
life. For example, the destruction of an LNG tank during the
1964 Niigata earthquake in Japan caused fires and explosions,
resulting in serious societal losses and pollution [16]. Since
the 1990s, several studies have been conducted on the
dynamic analysis of LNG storage tanks [17, 18]. Basic isolation
techniques, such as lead-core rubber bearings (LRBs), highdamping rubber bearings (HDRBs), steel hysteretic shock
absorbers and friction pendulum bearings are used in the
seismic design of LNG tanks and in many other applications
in modern structural design [19-22]. Three different LNG
tank structural configurations, that is, with a fixed bottom,
seismically isolated with rubber bearings only and seismically
isolated with rubber bearings and steel hysteretic dampers,
were tested in [19]. The test results confirmed that the
use of steel hysteretic dampers drastically decreased large
displacements. Christovasilis and Whittaker [23] investigated
the seismic response of a conventional and isolated 150,000
m3 capacity vertical cylindrical LNG tank by applying finite
element analysis to mechanical models. The base shear and
overturning moment in the seismically isolated LNG tank
were 10 %–15 % of the values computed for the conventional
tank and the wave heights were unaffected by the use of a
seismic isolation system. Gregoriou et al. [20, 21] analysed
the seismic response of three typical LNG tanks isolated
using high-damping rubber bearings and lead-core rubber
bearings. As a result of these processes, problems related to
the base shear force and deflection of the inner steel were
reduced by approximately 70 %–60 %. However, an increase
in the agitation height was observed compared to the nonisolated tank, especially in the case of high-damping rubber
support insulation. Marti et al. [1] studied a typical modern
LNG tank with a capacity of 160,000 m3 and they reported
that seismically isolated tanks can be used when the design
peak ground accelerations are in the range of approximately
0.30–0.90 g. Ruifu et al. [22] analysed the seismic response of
GRAĐEVINAR 75 (2023) 10, 979-995
Nonlinear behaviour of liquefied natural gas tanks with different seismic isolation systems
a vertical, cylindrical, extra-large and insulated LNG tank with
a multiple friction pendulum system (MFPS). They presented
statistically classified data, including pile shear, wave height,
impulsive acceleration, convective acceleration and outer
tank acceleration and showed that the isolation system
could adapt perfectly to different liquid levels and was very
effective in controlling the seismic response of extra-large
LNG tanks. Datoli et al. [24] examined an LNG tank with a
capacity of approximately 172,000 m3 during an earthquake
of 7.1 Richter magnitude. They constructed a finite element
model (FEM) model consisting of a flat anchored base and a
cylindrical metal wall in contact with the LNG and reported
that the fluid motion and fluid–structure interactions were
responsible for a failure type known as the elephant’s foot.
Zhao et al. [25] investigated the effects of liquid filling rate
and earthquake motion periods on the efficiency of a lead–
rubber bearing system using the ABAQUS program. Zhao et
al. [26] investigated the liquid filling ratio and earthquake
motion periods to determine the efficiency of a lead–rubber
bearing system using the general finite element code
ABAQUS. They analysed a 160,000 m3 LNG prestressed
storage tank for 12 earthquake waves under four site classes
using stress distributions on the outer and inner tanks, as
well as tip displacement and base shear. The maximum
stress of the inner tank was greater than 500 MPa at the 100
% liquid level in all four site classes, creating significant safety
hazards. Design optimisation and establishment of an early
warning system are imperative for controlling high liquid
levels. Barone and Sartori [27] investigated two elevated LNG
tanks with a full working capacity of 10,130 m3 and 91 pieces
of friction pendulum systems (FPSs) isolated at the Corsini
port of Ravenna, Italy. In their experiments, they observed
that a high lateral flexibility and a high friction coefficient
effectively separated the motion of the ground from the
structure and dissipated some of the seismic energy. Kilic
et al. [28] studied the performance of one broad tank, one
medium tank and one slender tank with two- and threedimensional elastomeric bearings, which were isolated along
only the horizontal direction and in both directions (horizontal
and vertical), respectively, by performing nonlinear dynamic
time history analyses. It was observed that 3D isolators
provided more efficient results than 2D isolators. Chen et
al. [29] performed shake table tests and numerical models
of an LRB isolated LNG tank. Although the base shear force,
overturning moment and acceleration spectra of the tank
Građevinar 10/2023
were significantly reduced, the displacement of the tank
posed a danger to the piping system connected to the tank.
Sharari et al. [30] examined a 160,000 m3 full containment
LNG tank considering the soil structure and fluid–structure
interactions while assessing the impacts of the depth of soil
liquefaction on the performance of different components of
the system. According to the nonlinear time history results,
the seismic forces on the inner steel and outer reinforced
concrete tank walls decreased as the liquefaction depth
increased. However, increases in the lateral displacements,
shear forces and bending moments of the pile head were
observed. The positive effects of the isolators on the LNG
tank in earthquakes of 0.4 g and above were compared. To
calculate the sloshing height in the wide and high LNG tanks
with a long period of 9.80 s, earthquakes with a minimum of
27 s and above were selected. The effects of the isolators
on the sloshing height of the LNG liquid were investigated.
The analysis results of medium and strong earthquakes in
anchored and isolated LNG tanks were evaluated.
This paper presents a case study analysing the behaviour of
an LNG tank without seismic isolation and with three different
types of base seismic isolation.
2. Description and modelling of the liquefied
natural gas (LNG) tank
For this study, an LNG tank with a volume of 232.000 m3
constructed for high-seismic hazard areas was selected.
The effects of the insulation type on the spherical bottom
slip, LNG sloshing wave height, lateral displacements on the
steel tank wall, stresses in the tank and earthquake isolation
force–displacement diagrams were investigated. In this LNG
tank, the height-to-radius ratio is 1. The LNG tank consists
of an inner layer, insulation layer and outer layer. The height
of the inner and outer tanks is 45 m and their diameters
are 84 and 88 m, respectively. The maximum height of the
liquid is 42 m. The outer shell has a constant thickness (0.8
m along its height), whereas the thickness of the inner shell
increases from top to bottom, from 12 to 32 mm. The space
(1.20 m) between the two shells was filled with perlite to
provide thermal insulation. While the outer shell was placed
directly on a circular foundation slab (1.80 m), a layer of
foam glass (0.70 m) was placed between the inner shell
base and foundation slab for thermal insulation. Analyses
were performed considering the geometric properties listed
Table 1. Material properties of the LNG tank
Description
Young modulus E (MPa) [MPa]
Poisson ratio ν
Mass density γ [kg/m3]
Inner steel
210000
0.30
7850
Concrete
37000
0.20
2500
LNG
2000
-
480
Perlite isolation
7500
-
240
Foam glass isolation
1200
-
150
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Table 2. Geometric dimensions of the LNG tank
Description
Dimension
Height of inner steel tank
45.00 m
Diameter of inner steel tank
84.00 m
Thickness of inner steel tank wall
32 mm-12 mm
Inner steel tank bottom thickness
5 mm
Height of LNG
42.00 m
Height of outer concrete tank
61.00 m
Diameter of outer concrete tank
88.00 m
Wall thickness of outer concrete tank
0.80 m
Sphere thickness of outer concrete tank
0.40 m
Outer concrete wall with between inner steel tank perlite insulation thickness
1.20 m
Thickness of bottom base insulation (foam glass)
0.70 m
Thickness of foundation
1.80 m
in Table 1 and the material properties listed in Table 2. The
steel tank forming the inner layer is composed of a nickelcontaining steel alloy (9 % Ni). Because of this steel alloy,
the inner layer tank exhibits excellent low-temperature
resistance, good weldability and low susceptibility to cold
cracks [3, 26]. As shown in Fig. 1, the inner tank consists of
six different layers with a height of 8 m.
DOF rectangular shell elements. The fibre glass and pearlite
isolation layers were modelled using eight-nodded 12-DOF
solid elements. The fluid content was modelled using eightnodded 12-DOF fluids. The fluid–structure interaction was
approximated by determining the appropriate coupling
equations at the nodes of the fluid–structure interface. The
outer concrete wall, inner steel tank, concrete dome and
ring beam, foundation slab, bottom foam glass and side
perlite insulation were modelled using quadrilateral shell
elements. In the analyses, the fluid side was considered
an acoustic element and was combined with the structural
side with the combining option to obtain results consistent
with the theoretical calculations.
Table 3. Thickness of each layer in the inner tank
Layer
Level [m]
Height [m]
Thickness [mm]
1
+2.50 to +10.50
8.00
32
2
+10.50 to +18.50
8.00
28
3
+18.50 to +25.50
8.00
24
Figure 1. Schematic view of the basic design section of the liquefied
4
+25.50 to +33.50
8.00
20
natural gas (LNG) storage tank with a volume of 232,000 m3
5
+33.50 to +41.50
8.00
16
For example, the R1/32 Layer refers to the first layer with a
thickness of 32 mm at the bottom. The thicknesses of these
layers gradually increase from top to bottom, as presented
in Table 3. When the inner tank is filled with 100 % LNG,
the maximum stress in it exceeds 500 MPa in the four site
classes. Considering that the yield strength of the alloy steel
used is between 500 and 600 MPa, this stress value poses
a risk to structural safety [26, 31]. The selected LNG tanks
were modelled using the ANSYS Workbench finite element
program [32]. The outer and inner shells as well as the dome
and foundation plate were modelled with four-point, 24-
6
+41.50 to +47.50
5.00
12
982
The seismic analysis FEM developed in ANSYS Workbench
was converted to LS-DYNA and seismic transient nonlinear
analyses were carried out in LS-DYNA. The shell and solid
elements had similar DOFs and types. The MAT_24 piecewise
multilinear material model was used for the metal parts and
the MAT_01 elastic model was adopted for the concrete and
isolation parts. For the fluid side, MAT_NULL with Lagrangian
options was used with automatic node-to-surface coupling
of the sloshing effects have been presented.
GRAĐEVINAR 75 (2023) 10, 979-995
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Figure 2. Finite element network structure for the LNG tank system
3. Modelling of the isolation systems
The base isolation systems used in this study included a series
of similar rubber bearings homogeneously distributed under
the foundation slab. The total number of required supports
was calculated considering that a single element supports a
maximum of 10 to 12 m2 of foundation slab area [20, 21]. As this
study aimed to examine the behavioural differences of isolator
types under the same system, combined isolator placement was
not performed. Therefore, the estimated number of bearings for
a tank with a volume of 232,000 m3 was 597. The frequency for
extra-large LNG tanks is generally between 2 and 10 Hz, which
is the specific resonance range for earthquake-induced ground
movements [18].
used to model the seismic isolation system [32]. Using the
SpaceClaim program, the bearings were converted to beam/
link elements to maintain proper locations and the connection
points were linked to the concrete base to create bonding. In
the ANSYS Workbench program, it was assumed that the link
elements were rigidly connected to the concrete and a fixed
boundary condition was applied on the other side. For the
seismic simulations, these link elements were converted to
bushing elements using Beam ELFORM 6. The beam elements
in LS-DYNA were MAT_197_SEISMIC_ISOLATOR, whose
characteristics are described below..
Figure 3. Details A and B
An FEM of the previously described LNG tank was developed to
introduce seismic isolators at the base of the structure during
the modal analyses. The bearings are assumed to have infinite
vertical stiffness. Based on this assumption, the displacement
constraint in the direction of the earthquake motion at the base
of the tank walls was relaxed and nonlinear horizontal springs
were placed to connect the base of the walls to the ground.
In the ANSYS Workbench program, link elements (LINK8) that
act as truss elements and combine nonlinear materials were
GRAĐEVINAR 75 (2023) 10, 979-995
Figure 4. Earthquake isolation arrangement plan for high-damping
rubber bearing (HDRB), lead-core rubber bearing (LRB) and
friction pendulum system (FPS) isolators
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Table 4. Engineering characteristics and bilinear spring parameters of HDRB SI-N 900/204 and LRB SI-N 900/225-185
Description
HDRB SI-N 900/204
LRB SI-N 900/225-185
Vertical stiffness Kv [kN/mm]
2317
2198
Effective horizontal stiffness Ke [kN/mm]
2.50
2.47
Elastic horizontal stiffness K1 [kN/mm]
19.67
17.33
Yield displacement d1 [mm]
12
18
Plastic horizontal stiffness K2 [kN/mm]
1.97
1.77
Maximum seismic displacement d2 [mm]
400
400
Maximum vertical load at load combinations including
the seismic action [kN]
7980
6630
Elastomer stiffness [mm]
900
900
Elastomer thickness te [mm] of total design
204
225
Dynamic shear modulus Gdin na γ =1 [MPa]
0.8
0.6
Table 5. Engineering characteristics and bilinear spring parameters of FPS FIP-D M 1600/800 (3700)
Description
Vertical stiffness Kv [kN/mm]
36715
Restoring stiffness Kr [kN/mm]
0.6376
Effective horizontal stiffness Ke [kN/mm]
1.145
Friction force developed by the isolator F0 [kN]
203.5
Maximum horizontal force Fmax [kN]
458
Minimum friction coefficient µ [%]
4.872
Effective viscous damping ratio ξe
0.2826
Equivalent radius of curvature R [mm]
3700
Maximum seismic displacement d [mm]
400
Isolator diameter excluding anchoring elements D [mm]
940
Isolator height excluding dowels H [mm]
199
3.1. High damping rubber bearings (HDRB)
HDRBs are one of three main subtypes of steel-reinforced
elastomeric bearings [33]. They consist of thin rubber layers
reinforced with steel plates. According to a preliminary design
[34], the number of high damping rubber bearings (SI-N
900/204) that should be used for an LNG tank with a volume of
232,000 m3 is 597. The properties used in this study are listed
in Table 4.
3.2. Lead core rubber bearings (LRB)
Lead-core rubber bearings are composed of steel plate
layers, rubber layers and a lead core. Similar to the steel
shims in natural rubber bearings, the steel layers provide
vertical stiffness, the rubber layers provide high lateral
flexibility and the lead core provides these devices with extra
stiffness and damping properties. According to a preliminary
984
FIP-D M 1600/800 (3700)
design [34], the number of lead core rubber bearings (SI-N
900/225-185) that should be used for an LNG tank with a
volume of 232,000 m3 is 597. The properties used in this
study are listed in Table 4.
3.3. Friction pendulum system (FPS)
Friction pendulum bearings are curved surface sliding
bearings consisting of an articulated slider and a cover
plate. The slider used in the system is covered with a selflubricating composite liner. During an earthquake, the
articulated slider on the bearing side along the concave
surface enables the structure to move with gentle
pendulum movements. According to a preliminary design
[34], the number of the friction pendulum bearings [FIP-D
M 1600/800 (3700)] that should be used for an LNG tank
with a volume of 232,000 m3 is 597. The properties used in
this study are listed in Table 5.
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Table 6. Earthquake specifications used for the time history analyses
No
Earthquake ground motions (PEER
register number)
Station
Abbrev.
Year
Mw
Time
[s]
SSE scale
factor
PGA
(SSE)
1
Imperial Valley / SAD (RSN6)
El Centro Array
IV2
1940
6.95
53.72
4.85
1.022
2
Kern County / SAD (RSN15)
Taft Lincoln School
KC
1952
7.36
54.35
5.75
1.037
3
Borrego Mtn / SAD (RSN36)
El Centro Array
BM
1968
6.80
79.99
7.20
0.414
4
Imperial Valley-I / SAD (RSN162)
Calexico Fire
Station
IV06-1
1979
6.53
37.86
6.30
1.282
5
Imperial Valley-II / SAD (RSN169)
Delta
IV06-2
1979
6.53
70.00
3.95
0.931
6
Victoria / Meksiko (RSN266)
Chihuahua
VM
1980
6.33
27.00
4.50
0.679
7
Irpinia / Italija (RSN286)
Bisaccia
IITA
1980
6.90
38.26
5.05
0.482
4. Analysis results
4.1. Earthquake ground motions
Analyses of the LNG tank system and the filling systems added
to this system were conducted for three types of seismic ground
motions: OBEs, SSEs and ALEs. According to the definitions in
API 625 [4] and NFPA 59A [5] the ground motion of the OBE is
represented by an acceleration response spectrum with a 10 %
probability of deflection over a 50-year period (an average return
interval of 475 years). An SSE is defined as the ground motion of
an accepted maximum earthquake (MCER) over a 50-year period
(mean recurrence interval of 2475 years) based on site-specific
research. The ALE ground motion is defined as half of the SSE.
The magnitudes used for the analyses of past earthquakes are
given in Table 6, including the year, time, scale factor of the
SSE, peak ground acceleration of the SSE and ground motion.
A linear scaling approach was used to match the acceleration
time series during the period of interest. The specified period
range was selected as 0.1 and 12 s. This range represents a
very broad spectrum band that matches the target acceleration
spectrum, owing to the long agitation period (Tc1 = ~9.7 s) of
the stored LNG. Seven pairs of earthquake ground motions,
called initial or core, were used to match the SSE spectrum. The
calculations indicated that the impulsive mode of the median
spectral coordinates matched well with the design spectrum
for the specified periods, whereas the convective mode was
significantly lower than the design spectrum for these periods.
The adopted soil class was D and the ground motion and design
level acceleration parameter values were determined as Ss
= 1.926, S1 = 0.660, SDS = 1.926 (SSE) and SD1 = 1.123 (SSE).
Seven pairs of earthquake ground motions were used as seeds
to match the SSE spectra.
4.2. Modal analysis results
4.2.1. Inner steel tank and LNG fluid
The results of the inner steel tank and LNG fluid modal analyses
for the non-isolated (anchored bottom) tank and systems with
HDRB, LRB and FPS isolators are listed in Table 7. Figure 6
shows the first, second, third and fourth convective modes of
the FEM. The convective mode shapes represent the agitation
of the liquid in the tank without the tank wall. The impulsive
mode shapes represent the combined
motion of the tank and liquid. Figure 7
shows the first, second, third and fourth
impulsive modes of the FEM.
The results of static and dynamic
analyses of the LNG tank are presented
in this section. The first horizontal
convective and impulsive mode periods
were 10 and 0.2302 s, respectively. The
high-period (low-frequency) convective
mode caused the agitation of the
liquid and damaged the tank roof. The
difference between the impulsive and
convective mode frequencies indicated
that the interaction effects between
Figure 5. Acceleration site-specific response spectra for the design earthquake increased with
the safe shutdown earthquake (SSE) scale factor
these modes were not significant.
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Figure 6. Convective mode types of the finite element model (FEM) for the non-isolated LNG tank: a) First convective mode T1 = 9.8232 s;
b) Second convective mode T2 = 7.4460 s; c) Third convective mode T3 = 6.6269 s; d) Fourth convective mode T4 = 6.3291 s
Figure 7. Impulsive mode types of the FEM for the non-isolated LNG tank: a) First impulsive mode T1 = 0.2302 s; b) Second impulsive mode
T2 = 0.2301 s; c) Third impulsive mode T3 = 0.2178 s; d) Fourth impulsive mode T4 = 0.2158 s
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Table 7. Natural periods of the inner model
Steel tank and
LNG fluid
LNG Fluid
Part
Mode
number
Non-isolated
HDRB isolator
LRB isolator
FPS isolator
Frequency
[Hz]
Period
[s]
Frequency
[Hz]
Period
[s]
Frequency
[Hz]
Period
[s]
Frequency
[Hz]
Period
[s]
Mod 1
0.1018
9.8232
0.1011
9.8912
0.1011
9.8912
0.1006
9.9404
Mod 2
0.1343
7.4460
0.1343
7.4460
0.1343
7.4460
0.1343
7.4460
Mod 3
0.1509
6.6269
0.1509
6.6269
0.1509
6.6269
0.1509
6.6269
Mod 4
0.1580
6.3291
0.1580
6.3291
0.1580
6.3291
0.1580
6.3291
Mod 5
0.1780
5.6180
0.1780
5.6180
0.1780
5.6180
0.1780
5.6180
Mod 6
0.1784
5.6054
0.1782
5.6117
0.1782
5.6117
0.1781
5.6148
Mod 1
4.3436
0.2302
3.9718
0.2518
3.9706
0.2519
3.4718
0.2880
Mod 2
4.3445
0.2301
4.0986
0.2440
4.0975
0.2441
3.8823
0.2576
Mod 3
4.5913
0.2178
4.1754
0.2395
4.1741
0.2396
3.9885
0.2507
Mod 4
4.633
0.2158
4.7762
0.2094
4.7417
0.2109
4.099
0.2440
Mod 5
5.016
0.1994
5.0602
0.1976
5.0593
0.1977
4.8246
0.2073
Mod 6
5.301
0.1886
5.7193
0.1748
5.7174
0.1749
5.010
0.1996
4.2.2. Outer concrete
Modal analyses were also performed for the external
prestressed concrete walls, ring beams, domes and mat
foundations. The results of the outer concrete modal analyses
for the non-isolated tank and systems with HDRB, LRB and
FPS isolators are listed in Table 8. Figure 8 shows the first,
second and third mode shapes of the FEM. In the first mode,
Figure 8. Outer concrete mode shapes of the FEM for the non-isolated tank: a) First mode T1 = 0.2302 s; b) Second mode T2 = 0.2178 s;
c) Third mode T3 = 0.1994 s
GRAĐEVINAR 75 (2023) 10, 979-995
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Table 8. Natural periods of the outer concrete model
Non-isolated
HDRB isolator
LRB isolator
FPS isolator
Part
Mode
No
Outer
concrete
tank
Mod 1
4.3445
0.2302
3.9718
0.2518
3.9716
0.2518
3.4718
0.2880
Mod 2
4.5913
0.2178
4.7762
0.2094
4.7174
0.2120
3.8823
0.2576
Mod 3
5.0163
0.1994
5.7193
0.1748
5.7174
0.1749
4.8246
0.2073
Frequency
[Hz]
Period
[s]
Frequency
[Hz]
the walls were deformed in the radial direction; however, the
roof diaphragm did not move laterally. In the second mode, the
walls were displaced laterally, whereas the ring beam, dome
and foundation were rigid. In the third mode, the prestressed
concrete walls moved along their axes in the positive and
negative directions.
4.3. Times history analysis results
The ground motion acceleration was defined using the
LOAD_BODY_X option in the LS-DYNA program. The ground
was fixed to provide the structure with inertia. Additionally,
LOAD_BODY_Z was defined to conserve gravity. Because
a large structure with a fluid domain was analysed, an
inclined interface time was used to neglect the initial
gravitational effect and avoid unnecessary strain on the
structure. The LS-DYNA software was used to simulate
the fluid–structure interaction in a tank filled with fluid.
This software offers significant advantages, particularly
Period
[s]
Frequency
[Hz]
Period
[s]
Frequency
[Hz]
Period
[s]
for solving dynamic contact problems. In addition, the LSDYNA program provides materials and standardised contact
formulations that can be used to model fluids and represent
the interaction between the tank shell and fluid during
seismic excitation.
4.3.1. Inner steel tank stresses
The inner tank maximum von Mises stress values (MPa)
and percentages (%) of reduction with respect to the nonisolated values occurring during a seven-scale earthquake
in the non-isolated and HDRB-, LRB- and FPS-isolated LNG
tanks fully filled with LNG are given in Table 9. The maximum
stress distributions of the non-isolated and HDRB-, LRBand FPS-isolated LNG tanks for the 1940 Imperial Valley
Earthquake El Centro Array Base Station data are shown
in Fig. 9. Compared with conventional tanks, HDRB, LRB
and FPS seismic isolators provide an average reduction of
15 % to 5 % in repulsive modes. Considering the fixed base
Figure 9. Inner steel tank wall tension stresses for non-isolated and HDRB-, LRB- and FPS-isolated tanks: a) Non-isolated; b) HDRB isolator;
c) LRB isolator; d) FPS isolator
988
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Table 9. Inner tank maximum von Mises stress values (MPa) and reduction rates (%) with respect to the non-isolated values
Earthquake ground motions
Base type
Non-isolated
HDRB isolator
LRB isolator
FPS isolator
IV2-1940
406
369
9%
358
12 %
347
15 %
KC-1952
378
354
6%
354
6%
349
8%
BM-1968
380
348
8%
348
8%
346
9%
IV06-I-1979
392
357
9%
346
12 %
350
11 %
IV06-II-1979
405
348
14 %
355
12 %
345
15 %
VM-1980
382
352
8%
352
8%
345
10 %
IITA-1980
375
351
6%
356
5%
328
12 %
conditions, the reduction in the maximum stresses in the
inner shell of approximately 15 % compared with the nonisolated situation reveals the importance of isolator use.
Dynamic loads generate much greater stress than static
loads, which is critical for LNG tanks. Considering that the
yield strength of 9 % Ni steel is between 515 MPa and 585
MPa, the maximum stress in the inner tank of the anchored
LNG tank exceeding 400 MPa poses a risk to the safety of
the structure [26, 31].
4.3.2. LNG fluid maximum sloshing wave height
The LNG fluid maximum sloshing wave height values (mm)
and percentages (%) of reduction with respect to the nonisolated values occurring during a seven-scale earthquake
in the non-isolated and HDRB-, LRB- and FPS-isolated LNG
tanks fully filled with LNG are given in Table 10. The maximum
sloshing wave heights of the non-isolated and HDRB-, LRBand FPS-isolated LNG tanks for the 1979 Imperial Valley
earthquake Calexico Fire Station data are shown in Fig. 10.
The FPS and LRB earthquake isolators exhibited the best
performance at the height of the agitated liquid in the LNG
tank, which was examined for seven different earthquakes
and four different systems. This result can be explained by
a friction force of approximately 200 kN when the FPS-type
earthquake isolator was used. This frictional force absorbs
the horizontal forces that occur during an
earthquake. In an LRB-type isolator, the lead core in the
centre absorbs horizontal forces. Higher sloshing heights
than those in the LRB, FPS and fixed-bottom LNG tanks were
observed when the HDRB-type isolator was used. The LNG
tank oscillated because of the horizontal force originating
Figure 10. LNG fluid maximum sloshing wave height for non-isolated and HDRB-isolated, LRB-isolated and FPS-isolated tanks: a) Non-isolated;
b) HDRB isolator; c) LRB isolator; d) FPS isolator
GRAĐEVINAR 75 (2023) 10, 979-995
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Table 10. LNG fluid maximum sloshing wave height values (mm)
Structural bearing type
IV2-1940
KC-1952
BM-1968
IV06-I-1979
IV06-II-1979
VM-1980
IITA-1980
Fixed
2463
2119
3317
3421
2006
2567
3355
HDRB
2577
1920
3638
3433
1959
2836
3488
LRB
2379
1934
3560
3428
1880
2470
3438
FPS
2090
1932
3550
3348
1899
2760
3276
from the rubber system. As a result, the LNG liquid height
in the convective mode increased during an earthquake. The
sloshing height in conventional tanks was higher than that
in LNG tanks with isolators in the Kern County and Imperial
Valley-II earthquakes and the earthquake oscillations in the
convective mode continued with high accelerations.
4.3.3. The maximum lateral displacement of inner steel
tank wall
The maximum lateral displacement values (mm) and reduction
rates (%) of the inner steel tank with respect to the non-isolated
values occurring during a seven-scale earthquake in the non-
Figure 11. Maximum lateral displacement values (mm) at the steel wall for non-isolated and HDRB-, LRB- and FPS-isolated tanks: a) Nonisolated; b) HDRB isolator; c) LRB isolator; d) FPS isolator
Table 11. Maximum lateral displacement values (mm) at the steel tank wall and reduction rates (%) with respect to the non-isolated values
Base type
Earthquake ground
motions
Non-isolated
IV2-1940
898
496
45 %
424
53 %
604
33 %
KC-1952
578
361
38 %
369
36 %
329
43 %
990
HDRB isolator
LRB isolator
FPS isolator
BM-1968
369
322
13 %
278
25 %
345
7%
IV06-I-1979
706
553
22 %
430
39 %
299
58 %
IV06-II-1979
1120
287
74 %
287
74 %
401
64 %
VM-1980
449
444
1%
312
30 %
440
2%
IITA-1980
171
263
-53 %
253
-48 %
297
-74 %
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isolated and HDRB-, LRB- and FPSisolated LNG tanks fully filled with LNG
are given in Table 11. The maximum
lateral displacements of the inner steel
tank wall values of the non-isolated
and HDRB-, LRB- and FPS-isolated
LNG tanks for the 1979 Imperial Valley
Earthquake Delta Station data are
shown in Fig. 11. It was observed that
the horizontal displacement movements
of the inner tanks in the isolated LNG
tank were less than those of the fixed
system during high-acceleration and
Figure 12. LNG tank base shear force non-isolated and HDRB-, LRB- and FPS-isolated tanks
long oscillating earthquakes. However,
the elephant foot deformation type, which is observed in lowintensity earthquakes, is less common in LNG tanks fixed to the
principle, the base shear forces that occur in these systems
foundation with anchor bolts than in structures with isolators.
during an earthquake are less than those of the LRB and HDRB
When using an LRB-type isolator in severe earthquakes, less
isolator systems. Consequently, LNG tanks with frictionlateral deformation occurs in the inner tank compared with that
based pendulum-type isolators performed much better than
generated with other isolator systems. This is because of the
other rubber-based isolator systems when compared to the
ability of the lead core in the centre of the LRB-type isolators to
base shear forces occurring during earthquakes.
inhibit lateral displacement.
The comparative analysis results of the maximum inner steel
tank von Mises stresses, LNG fluid sloshing wave heights, inner
steel tank lateral displacements and base shear forces are
4.3.4. Maximum base shear force of inner steel tank
shown in Fig. 13.
The maximum base shear forces of the inner steel tank (kN)
and reduction rates (%) with respect to the non-isolated
4.3.5. Force–displacement diagrams for LNG isolation
types
values occurring during a seven-scale earthquake for nonisolated and HDRB-, LRB- and FPS-isolated LNG tanks fully
filled with LNG are given in Table 12. The maximum base shear
The force (kN) and displacement (mm) values during a
force values of the non-isolated and HDRB-, LRB- and FPSseven-scale earthquake for the HDRB-, LRB- and FPSisolated LNG tanks for the 1979 Imperial Valley earthquake
isolated LNG tanks fully filled with LNG are listed in Table
Delta Station data are shown in Fig. 12. The maximum base
13. The force–displacement graphics of the LNG tank base
shear forces of the inner steel tank for the HDRB and LRB
isolators of the HDRB-, LRB- and FPS-isolated LNG tanks
isolator systems were between 50 % and 10 % lower than
for the 1940 Imperial Valley earthquake El Centro Array Base
those of the non-isolated LNG tanks. It has been observed
Station data are shown in Fig. 14. While the LRB earthquake
that the base shear forces formed in LNG tanks with an FPS
isolator shows less displacement than the other isolators
are 67 %–49 % lower than those in systems without isolators.
under the effect of a large shear force coming from the LNG
Because systems with FPS operate according to the friction
tank in high-acceleration and long-oscillating earthquakes,
Table 12. Maximum base shear forces (kN) and reduction rates (%) with respect to the non-isolated tanks for HDRB-, LRB- and FPS-isolated tanks
Base type
Earthquake ground
motions
Non-isolated
IV2-1940
6.78 × 105
5.26 × 105
22 %
4.48 × 105
34 %
3.10 × 105
54 %
KC-1952
5
5.92 × 10
3.98 × 10
33 %
3.94 × 10
5
34 %
2.08 × 10
5
65 %
BM-1968
4.87 × 10
5
3.70 × 10
24 %
3.38 × 10
5
31 %
2.18 × 10
5
55 %
IV06-I-1979
6.08 × 10
5
5.47 × 10
10 %
4.33 × 10
5
29 %
2.01 × 10
5
67 %
IV06-II-1979
6.44 × 10
5
3.19 × 10
50 %
3.27 × 10
5
49 %
2.36 × 10
5
63 %
VM-1980
6.29 × 10
5
4.72 × 10
25 %
3.56 × 10
5
43 %
2.53 × 10
5
60 %
IITA-1980
3.89 × 10
3.21 × 10
17 %
3.09 × 10
5
21 %
1.97 × 10
5
49 %
5
GRAĐEVINAR 75 (2023) 10, 979-995
HDRB isolator
5
5
5
5
5
5
LRB isolator
FPS isolator
991
Građevinar 10/2023
İbrahim Hüner, Bülent Akbaş, Abdullah Cem Koç
Figure 13. Nonlinear analysis results: a) Von Mises stress (MPa); b) sloshing wave height (mm); c) lateral displacement (mm); d) base shear force
(kN)
Figure 14. LNG tank base isolators force–displacement graphics: ) HDRB isolator; b) LRB isolator; c) FPS isolator
Table 13. LNG tank base isolators maximum force–displacement values
Earthquake ground
motions
992
Isolator type
HDRB
LRB
FPS
Force [kN]
Displacement [mm]
Force [kN]
Displacement [mm]
Force [kN]
Displacement [mm]
IV2-1940
880
340
746
286
506
499
KC-1952
656
225
658
232
345
229
BM-1968
615
205
564
173
363
256
IV06-I-1979
904
360
710
274
336
210
IV06-II-1979
534
164
544
164
395
302
VM-1980
791
296
595
194
422
346
IITA-1980
536
165
518
145
328
195
GRAĐEVINAR 75 (2023) 10, 979-995
Nonlinear behaviour of liquefied natural gas tanks with different seismic isolation systems
the FPS isolators perform better in moderate-intensity and
low-oscillation earthquakes. This is because the horizontal
displacement force–displacement angle ratio of the LNG
tank in earthquakes when FPS-type isolators are used and
the friction-damped force is exceeded is higher than that
with LRB- and HDRB-type isolator systems. Compared to
the HDRB isolator system, the lead in the LRB core limited
the horizontal displacement movement of the LNG tank. The
horizontal displacement of LNG tanks during an earthquake
is critical because of the mechanical and carrier pipe systems;
in this respect, the LRB isolator type is more advantageous
than the FPS and HDRB.
5. Conclusions
In this study, an LNG tank with a volume of 232,000 m3
placed on four different base types was analysed under seven
different earthquake effects. The height-to-radius ratio was
selected as 1 for maximum LNG fluid. The base shear force,
sloshing height, inner steel tank lateral deflection, von Mises
stresses in the inner steel tank and force–displacement of the
isolators were investigated.
-- When comparing the LNG tanks with the HDRB, LRB and
FPS isolators with that fixed to the foundation with anchor
bolts, no difference was observed between the convective
and impulsive modes. It was concluded that the wave
motion of the liquid was different from the oscillation of the
structure and the earthquake isolation times did not affect
the sloshing motion.
-- An average stress of 270 MPa occurred in the inner steel
tank owing to the static loading. The stresses varied
depending on the intensity of the earthquake and whether
the system was isolated or fixed. While a stress value of
400 MPa occurred in the fixed system, these values were
350 MPa on average for the LNG tanks with the HDRB,
LRB and FPS-type earthquake isolator systems. The yield
strength of the steel tank is between 515 and 585 MPa.
According to the API 620 standard, 80 % of this value (412–
468 MPa) should not be exceeded for the SSE. Otherwise,
there is a significant risk to LNG tanks. Therefore, base
isolator systems should be used in structures under risk.
-- The sloshing time varies between 9.82–9.94 s for tanks
without isolator and with HDRB-, LRB- and FPS-type
isolators. For the isolated and anchored systems, the wave
height varies depending on the agitation time of the LNG
liquid. The sloshing wave height of the liquid is proportional
to the harmonic movements of the earthquake acceleration
of the structure rather than the numerical magnitude of the
earthquake accelerations. For example, the sloshing wave
height for acceleration values varying between +0.3 g and
−0.3 g of the BM-1968 earthquake within a period of 10 s is
greater than that for the IV06-II-1979 earthquake.
-- Except for earthquakes such as IV06-I-1979 with very high
GRAĐEVINAR 75 (2023) 10, 979-995
Građevinar 10/2023
acceleration values, the sloshing wave height observed
as a result of the regular harmonic motion acceleration
values formed in anchored LNG tanks was less than that
in structures with isolators. This result can be attributed
to the damping function oscillating between high and
low acceleration values. However, the acceleration values
decrease owing to the damping provided by the lead core
in the LRB centre and the frictional force generated in the
FPS, resulting in a lower sloshing height in tanks using
these types of isolators than that in tanks with the HDRB
isolator.
-- In moderate and weak earthquakes, such as the Irpina
earthquake, the pressure of the impulsive mass in the
inner steel tank is blocked by the anchors and the lateral
displacement movement of the structure is not allowed.
This is why there is buckling with less movement than that
in structures with isolators. However, in earthquakes of
+0.4 g and above, the lateral displacement of the inner steel
tank wall in the anchored LNG tank is greater than that in
tanks with LRB, HDRB and FPS isolators. Therefore, elastoplastic buckling of the inner steel tank, which we call the
elephant foot strain, was observed in the anchored tanks.
-- With an increase in average ground acceleration, the total
bottom shear force acting on the LNG tank increased.
Moreover, with an increase in the maximum shear force
on the base, the percentage of shear force reduction in
systems with isolators also increased. The bottom shear
forces were found to be similar in the LNG tanks with
HDRB and LRB isolators. However, better performance was
obtained in LNG tanks with an FPS system compared with
that of the other two isolator types. This can be attributed
to the fact that each FPS isolator generated a frictional
force that absorbed a base shear force of 200 kN.
-- LNG transported by pipes from a ship docking at a port
is discharged from the roof of the LNG tank to the inner
steel tank. During this time, it is desirable that the
horizontal displacement of the LNG tank be limited during
an earthquake to avoid damage to the mechanical and
piping systems. When the force–displacement graphs
of the HDRB, LRB and FPS isolator-supported LNG tanks
are examined, it can be observed that, compared with the
other earthquake isolators, the LRB isolator has the least
horizontal displacement. When the acceleration–time
graphs of seven earthquakes with 0.4 g and above ground
movements are examined, the FPS showed more lateral
displacement than the HDRB and LRB LNG tanks because
of the amount of horizontal force acting on each isolator.
This can be attributed to the earthquake forces exceeding
the friction-damped force and reaching a higher horizontal
displacement motion. This situation is undesirable for LNG
tanks. Meanwhile, the LRB system provides a 10 %–15 %
advantage over the HDRB isolator owing to the horizontal
damping of the middle lead core.
993
Građevinar 10/2023
-- It was observed that earthquake isolators used in LNG
storage tanks perform well even during very strong
earthquakes, which may occur once every 2475 years.
The use of isolator systems is recommended for the
earthquake safety of LNG storage tanks, as they may
cause major environmental disasters and destruction.
Systems with LRB isolators outperformed those with
FPS and HDRB isolators and fixed support systems in all
criteria, except for the base shear force.
İbrahim Hüner, Bülent Akbaş, Abdullah Cem Koç
Acknowledgments
This study was funded by the Pamukkale University
Scientific Research Project Coordination Unit (grant number
2019FEBE055). The authors would like to thank to Associate
Prof. Temel Varol from the Metallurgical and Materials
Engineering Department, Karadeniz Technical University, Ph.D.
student Murat Çelik from the Civil Engineering Department,
Istanbul Technical University and M.Sc. Mechanical Engineer
Hakan Balaban for their valuable contributions.
REFERENCES
[1]
Marti, J., Crespo, M., Martinez, F.: Seismic isolation of liquefied
natural gas tanks: A comparative assessment, The Journal of the
Anti-Seismic Systems International Society, 1 (2010) 1, pp. 125140, doi:10.2140/siaps.2010.1.125.
[13] Haroun, M.A., Housner, G.W.: Dynamic interaction of liquid storage
tanks and foundation soil, in Dynamic Response of Structures:
Experimentation: Observation, Prediction and Control, American
Society of Civil Engineers, ASCE, New York, USA, 1981.
[2]
Summers, P.B., Castellano, M.G., Bergamo, G., Gatti, F., Marti,
J., Poggianti, A.: Seismic risk reduction at petrochemical and
LNG acilities: Main results from in-depth project, 14th World
Conference on Earthquake Engineering, Beijing, China, October,
2006.
[14] Veletsos, A.S., Tang, Y.: Soil-structure interaction effects for
laterally excited liquid-storage tanks., Earthquake Engineering
and Structural. Dynamics, 19 (1990) 4, pp. 473-496, doi:10.1002/
eqe.4290190402.
[3]
API Standard 620, Design and construction of large, welded, lowpressure storage tanks, American Petroleum Institute Publishing
Services, 12th Edition, Addendum 1, API, Washington D.C., USA,
2014.
[4]
API Standard 625, Tank systems for refrigerated liquefied gas
storage, American Gas Association Publishing Services, 1st Edition,
Addendum 2, AGA, Washington D.C., USA, 2014.
[5]
NFPA 59A, Standard for the production, storage and handling of
liquefied natural gas LNG, National Fire Protection Association
Publishing Services, NFPA , Washington D.C., USA, 2016.
[17] Bomhard, H., Stempniewski, L.: LNG tanks for seismically highly
affected sites, Intl. Post SMiRT Conference Seminar on Isolation,
Energy Dissipation and Control of Vibrations of Structures, IAEA,
Capri, Italy, 1993.
[6]
Westergaard, H.M.: Water pressures on dams during earthquakes,
Transactions of the American Society of Civil Engineers, 98 (1933)
2, Washington D.C., USA.
[18] Tajirian, F.F.: Base isolation design for civil components and civil
structures, in Proceedings of the Structural Engineers World
Conference, San Francisco, California, USA, July, 1998.
[7]
Jacobsen, L.S.: Impulsive hydrodynamics of fluid inside a cylindrical
tank and of fluid surrounding a cylindrical pier, Bulletin of the
Seismological Society of America, 39 (1949) 3, pp. 189-204,
doi:10.1785/BSSA0390030189.
[8]
Jacobsen, L.S., Ayre, R.S.: Hydrodynamic experiments with rigid
cylindrical tanks subjected to transient motions, Bulletin of the
Seismological Society of America, 41 (1951) 4, pp. 313-346,
doi:10.1785/BSSA0410040313.
[19] Castellano, M.G., Infanti, S., Dumoulin, C., Ducoup, L., Martelli, A.,
Dusi, A.: Shaking table tests on a liquefied natural gas storage
tank mock-up seismically protected with elastomeric isolators
and steel hysteretic torsional dampers, 12th World Conference on
Earthquake Engineering, Auckland, New Zealand, February, 1999.
[9]
Housner, G.W.: Dynamic pressures on accelerated fluid containers,
Bulletin of the Seismological Society of America, 47 (1957) 1, pp.
15-35, doi:10.1785/BSSA0470010015.
[10] Housner, G.W.: The dynamic behavior of water tanks, Bulletin of
the Seismological Society of America, 1963, 53 (1963) 2, pp. 381387, doi:10.1785/BSSA0530020381.
[11] Veletsos, A.: Seismic effects in flexible liquid storage tanks,
in Proceedings of the 5th World Conference on Earthquake
Engineering, Rome, Italy, June, 1974.
[12] Veletsos, A., Yang J.: Dynamics of fixed-base liquid storage
tanks, in Proceedings of the US–Japan Seminar for Earthquake
Engineering Research with Emphasis on Lifeline Systems, Tokyo,
Japan, November, 1976.
994
[15] Malhotra, P.K., Wenk, T., Wieland, M.: Simple procedures
for seismic analyses of liquid storage tanks, Structural
Engineering International, 10 (2000) 3, pp. 197-201,
doi:10.2749/101686600780481509.
[16] Zhao, Y., Li, H.N., Fu, X., Zhang, S., Mercan, O.: Seismic analyses of
a large LNG tank considering the effect of liquid volume, Hindawi,
(2020), Article ID 8889055, pp. 1-18, doi:10.1155/2020/8889055.
[20] Gregoriou, V.P., Tsinopoulos, S.V., Karabalis, D.L.: Base isolated LNG
tanks: Seismic analyses and comparison studies, First European
Conference on Earthquake Engineering and Seismology, Geneva,
Switzerland, September, 2006.
[21] Gregoriou, V.P., Tsinopoulos, S.V., Karabalis, D.L.: Dynamic
analyses of liquefied natural gas tanks seismicly protected with
energy dissipating base isolation systems, 3rd ECCOMAS Thematic
Conference on Computational Methods in Structural Dynamics
and Earthquake Engineering, Corfu, Greece, May, 2011.
[22] Ruifu, Z., Dagen W., Xiaosong, R.: Seismic analyses of a LNG
storage tank isolated by a multiple friction pendulum system,
Earthquake Engineering and Engineering Vibration, 10 (2011) 2,
pp. 253-262, doi:10.1007/s11803-011-0063-3.
[23] Christovasilis, I.P., Whittaker, A.S.: Seismic analyses of conventional
and isolated LNG tanks using mechanical analogs, Earthquake
Spectra, 2008, 24 (2008) 3, pp. 599-616, doi:10.1193/1.2945293.
GRAĐEVINAR 75 (2023) 10, 979-995
Nelinearno ponašanje spremnika za ukapljeni prirodni plin s različitim sustavima potresne izolacije
[24] Datoli, R., Danilo, B., Perillo, M.: Sloshing response of a LNG
storage tank subjected to seismic loading, 6th European LS-DYNA
Users’ Conference, Gothenburg, Sweden, May, 2007.
[25] Zhou, Y., Li, X., Chen, Z.: Seismic responses analyses of baseisolated LNG storage tank, in Proceeding of GeoShanghai
2018 International Conference: Advances in Soil Dynamics and
Foundation Engineering, Singapore, May, 2018.
[26] Zhou, Y., Li, H., Zhang S., Mercan, O., Caiyan, Z.: Seismic analyses
of a large LNG tank considering different site conditions, Applied
Sciences, 10 (2020) 22, pp. 8121, doi:10.3390/app10228121.
[27] Barone, S., Sartori, M.: Seismic isolation of LNG storage tanks
in Italy with curved surface sliders, 17th World Conference on
Earthquake Engineering, Sendai, Japan, September, 2020.
[28] Kılıç, S., Akbaş, B., Shen, J., Paolacci, F.: Seismic behavior of
liquid storage tanks with 2D and 3D base isolation systems,
Structural Engineering and Mechanics, 83 (2022) 5, pp. 627-644,
doi:10.12989/sem.2022.83.5.627.
Građevinar 10/2023
[30] Sharari, N., Behzad, F., Hokmabadi A., Xu, R.: Seismic resilience
of extralarge LNG tank built on liquefable soil deposit capturing
soilpilestructure interaction, Bulletin of Earthquake Engineering,
20 (2022) 4, pp. 3385-3441, doi:10.1007/s10518-022-01384-1.
[31] Standardization Administration Committee of the People’s
Republic of China: 9 % Nickel Steel Plates for Pressure Vessels
with Specified Low Temperature Properties, SAC, Beijing, China,
2010.
[32] ANSYS Workbench 20, LS-DYNA 2018, Southpointe, 275
Technology Drive, PA 15317, ANSYS Inc., Canonsburg, USA, 2022.
[33] Vaiana, N., Sessa, S., Marmo, F., Rosati, L.: An accurate and
computationally efficient uniaxial phenomenological model
for steel and fiber reinforced elastomeric bearings, Composite
Structures, 211 (2019) 11, pp. 196-212, https://doi.org/10.1016/j.
compstruct.2018.12.017.
[34] FIP MEC Srl., Via Scapacchio, 41/35030 Selvazzano Dentro, Italy,
2022.
[29] Chen, Z., Xu, Z., Teng, L., Fu, J., Xu, T., Zhao, Z.: Experimental and
numerical investigation for seismic performance of a large-scale
LNG storage tank structure model, Applied Sciences, 12 (2022)
17, pp. 8390, doi:10.3390/app12178390.
GRAĐEVINAR 75 (2023) 10, 979-995
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Computing all hybridization networks for multiple binary phylogenetic input trees
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METHODOLOGY ARTICLE Open Access © 2015 Albrecht. 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: benjamin.albrecht@bio.ifi.lmu.de
Institut für Informatik, Ludwig-Maximilians-Universität, Amalienstr. 17, 80333
München, Germany Abstract Background: The computation of phylogenetic trees on the same set of species that are based on different
orthologous genes can lead to incongruent trees. One possible explanation for this behavior are interspecific
hybridization events recombining genes of different species. An important approach to analyze such events is the
computation of hybridization networks. Results: This work presents the first algorithm computing the hybridization number as well as a set of representative
hybridization networks for multiple binary phylogenetic input trees on the same set of taxa. To improve its practical
runtime, we show how this algorithm can be parallelized. Moreover, we demonstrate the efficiency of the software
Hybroscale, containing an implementation of our algorithm, by comparing it to PIRNv2.0, which is so far the best
available software computing the exact hybridization number for multiple binary phylogenetic trees on the same set
of taxa. The algorithm is part of the software Hybroscale, which was developed specifically for the investigation of
hybridization networks including their computation and visualization. Hybroscale is freely available1 and runs on all
three major operating systems. Conclusion: Our simulation study indicates that our approach is on average 100 times faster than PIRNv2.0. Moreover, we show how Hybroscale improves the interpretation of the reported hybridization networks by adding
certain features to its graphical representation. Keywords: Hybridization networks, Maximum acyclic agreement forests, Phylogenetics Albrecht BMC Bioinformatics (2015) 16:236
DOI 10.1186/s12859-015-0660-7
METHODOLOGY ARTICLE
Open Access
Computing all hybridization networks for
multiple binary phylogenetic input trees
Benjamin Albrecht
Abstract
Background: The computation of phylogenetic trees on the same set of species that are based on different
orthologous genes can lead to incongruent trees. One possible explanation for this behavior are interspecific
hybridization events recombining genes of different species. An important approach to analyze such events is the
computation of hybridization networks.
Results: This work presents the first algorithm computing the hybridization number as well as a set of representative
hybridization networks for multiple binary phylogenetic input trees on the same set of taxa. To improve its practical
runtime, we show how this algorithm can be parallelized. Moreover, we demonstrate the efficiency of the software
Hybroscale, containing an implementation of our algorithm, by comparing it to PIRNv2.0, which is so far the best
available software computing the exact hybridization number for multiple binary phylogenetic trees on the same set
of taxa. The algorithm is part of the software Hybroscale, which was developed specifically for the investigation of
hybridization networks including their computation and visualization. Hybroscale is freely available1 and runs on all
three major operating systems.
Conclusion: Our simulation study indicates that our approach is on average 100 times faster than PIRNv2.0.
Moreover, we show how Hybroscale improves the interpretation of the reported hybridization networks by adding
certain features to its graphical representation.
Keywords: Hybridization networks, Maximum acyclic agreement forests, Phylogenetics Albrecht BMC Bioinformatics (2015) 16:236
DOI 10.1186/s12859-015-0660-7
METHODOLOGY ARTICLE
Open Access
Computing all hybridization networks for
multiple binary phylogenetic input trees
Benjamin Albrecht
Abstract
Background: The computation of phylogenetic trees on the same set of species that are based on different
orthologous genes can lead to incongruent trees. One possible explanation for this behavior are interspecific
hybridization events recombining genes of different species. An important approach to analyze such events is the
computation of hybridization networks. Results: This work presents the first algorithm computing the hybridization number as well as a set of representative
hybridization networks for multiple binary phylogenetic input trees on the same set of taxa. To improve its practical
runtime, we show how this algorithm can be parallelized. Moreover, we demonstrate the efficiency of the software
Hybroscale, containing an implementation of our algorithm, by comparing it to PIRNv2.0, which is so far the best
available software computing the exact hybridization number for multiple binary phylogenetic trees on the same set
of taxa. The algorithm is part of the software Hybroscale, which was developed specifically for the investigation of
hybridization networks including their computation and visualization. Hybroscale is freely available1 and runs on all
three major operating systems. Conclusion: Our simulation study indicates that our approach is on average 100 times faster than PIRNv2.0. Moreover, we show how Hybroscale improves the interpretation of the reported hybridization networks by adding
certain features to its graphical representation. Keywords: Hybridization networks, Maximum acyclic agreement forests, Phylogenetics Albrecht BMC Bioinformatics (2015) 16:236
DOI 10.1186/s12859-015-0660-7 Computing all hybridization networks for
multiple binary phylogenetic input trees Benjamin Albrecht Background its parents, which ensures a reduction of the gene flow
between its parental genotypes. Due to these circum-
stances, homoploid hybrid speciation is considered as a
rare phenomenon. Note that, after such a new hybrid
species has successfully established itself, there can still
occur gene flow back from the hybrid species to their
parent species, which is denoted as introgression [1]. Recombinational or homoploid hybrid speciation [1] is a
mechanism influencing the evolution of species by merg-
ing a sizable percentage of the genomes of two different
species. It has been discovered especially in plants [2, 3],
but also in certain animals [4]. If two individuals each
belonging to different species hybridize, a new species,
containing genes from both parental individuals, can arise
under the following certain circumstances. First of all,
the resulting hybrid has to produce viable gametes, which
is often a problem due to the two genetically different
parental sets of chromosomes preventing a correct mei-
otic pairing. Second, if these two sets are similar enough
and, thus, the hybrid is able to produce any progeny, its
early recombinants have to find and successfully colonize
its own unexploited niche that is different from either of Now, regarding a well-established homoploid hybrid
species resulting from such a evolutionary process as
described above, we can reconstruct its evolutionary his-
tory by taking two different scenarios each corresponding
to one of its parental species into account. This is typ-
ically done by, first, computing two rooted phylogenetic
trees each based on those genes corresponding to one
of both parental gene sets and, second, by reconciling
these two topologically different trees into one rooted
phylogenetic network, whose reticulate nodes (nodes of
in-degree ≥2) represent certain putative hybridization
events. Because of those major hurdles a hybrid species
has to face, hybridization events rarely happen and, thus, Albrecht BMC Bioinformatics (2015) 16:236 Page 2 of 15 from a biological point of view, only those networks con-
taining a minimum number of reticulate nodes are of high
interest. regarding the interpretation of hybridization networks, we
decided to discuss those rather complex theoretical issues
in a forthcoming paper [13]. Due to hybridization, the genome of hybrid species,
however, can obviously contain more than just two genes
having different evolutionary histories. Background Thus, given a set
T of rooted binary gene trees sharing the same set of
taxa, the general problem is to compute a rooted phylo-
genetic network displaying T by a minimum hybridiza-
tion number as defined later by Eq. 2. Unfortunately,
this is a well-known NP-hard problem, which is how-
ever fixed-parameter tractable, even for the simplest case
when only just two binary input trees are given [5]. In the general case, however, if the input consists of
more than two rooted binary trees, the problem still
remains fixed-parameter tractable as recently shown by
van Iersel and Linz [6]. More precisely, this means that
the problem is exponential in some parameter related
to the problem itself, namely the hybridization num-
ber, and only polynomial in the size of the input trees. Note that this is an important feature, which facili-
tates the development of exact algorithms as it is used
by our algorithm for some subproblems to maximize
efficiency. Given a hybridization network displaying several input
trees, it is often visually challenging for a user to figure
out the embedding of those trees. Thus, we have devel-
oped the software Hybroscale providing a function for
highlighting each input tree by coloring its corresponding
edges within a resulting network, which makes it easier
for a biologist to analyze hybridization events. Moreover,
Hybroscale sorts the set of computed networks by support
values indicating how often a certain hybridization event
occurs in the set of representative networks. To demonstrate the efficiency of our implementation,
we computed the hybridization number for a specific syn-
thetic dataset and compared the respective runtime with
the best currently available software PIRNv2.0 [7, 8]. Note
that there are two main differences between our approach
and the one corresponding to PIRN. On the one hand, our
software provides the better practical runtime for com-
puting hybridization numbers because of parallelization,
certain reduction steps, and other algorithmic issues as
discussed in the upcoming part of this paper. On the other
hand, our approach additionally enables the computation
of all representative networks allowing the assignment of
meaningful support values to each internal node repre-
senting a putative hybridization event which helps biolo-
gists to figure out hybridization events that might played
an important role. Background Note that the networks reported by
PIRN2.0 are either also calculated by our approach or are
not considered as being relevant because there exist other
networks representing these networks as described in the
upcoming part of this paper. y
In this work, we tackle this NP-hard problem by pre-
senting the first algorithm that is able to compute the
exact hybridization number as well as a certain set con-
taining all representative networks for, not just only two,
but an arbitrary number of rooted binary phylogenetic
X -trees all sharing the same set of taxa. Note that, until
now, the software PIRNv2.0 [7, 8] is the most efficient
software that guarantees the computation of the exact
hybridization number for multiple input trees. In most
cases, however, PIRN runs only reasonable efficient if the
number of hybridization events is relatively small and,
moreover, PIRN does usually output only a small sub-
set of those networks that are computed by our method
which plays an important role for the interpretation of
the networks as shown later. The algorithm, presented
in this work, is based on previous work of Albrecht
et al. [9] describing an algorithm for just two input trees,
which itself is based on several works including Baroni
et al. [10], Bordewich and Semple [11], and Whidden et al. [12]. Moreover, this previous approach could only com-
pute a subset of all representative networks and, thus, the
motivation for this work was to extend this former algo-
rithm such that now all of those networks for an arbitrary
number of input trees can be computed. Methods In this section, we first introduce the notation and termi-
nology that is used throughout the paper and then present
the algorithm ALLHNETWORKS. Albrecht BMC Bioinformatics (2015) 16:236 Additionally, by T|X ′ we define the subgraph that
is obtained from T(X ′) by suppressing all nodes of both
in- and out-degree 1. Moreover, given a tree T, through-
out this paper we use T to denote the tree that is obtained
from T by suppressing each node of both in- and out-
degree 1. Now, given a rooted phylogenetic X -tree T and a taxa
set X ′
⊆
X , we define T(X ′) as the minimal con-
nected subgraph of T whose leaf set contains each taxon
in X ′. Additionally, by T|X ′ we define the subgraph that
is obtained from T(X ′) by suppressing all nodes of both
in- and out-degree 1. Moreover, given a tree T, through-
out this paper we use T to denote the tree that is obtained
from T by suppressing each node of both in- and out-
degree 1. Hybridization networks. A hybridization network N is
a rooted phylogenetic network, which is a rooted acyclic
digraph not containing nodes of both in- and out-degree 1
and whose leaves are all labeled one-to-one by a taxon
of the taxa set X (cf. Fig.1(a)). Each node v of in-degree
greater than 1 is called a hybridization node and each
edge directed into v is called a reticulation edge or, in the
context of hybridization, a hybridization edge. We say a
hybridization network N on X displays a rooted phyloge-
netic X ′-tree T′, with X ′ ⊆X , if we can delete a set of
hybridization edges E′ followed by suppressing each node
of both in- and out-degree 1 such that the resulting rooted
phylogenetic X -tree T contains T′ as restricted subtree on
X ′. In such a case, we say that E′ refers to T′ (cf. Fig.1(b)). Since a network can display a tree in potentially several
ways, E′ is not necessarily unique. To quantify the num-
ber of reticulation events of a network N, the reticulation
number r(N) is defined by Agreement forests. Let T1 and T2 be two rooted binary
phylogenetic X -trees. For technical purpose, we regard
the root of both trees T1 and T2 as being a node that has
been attached to the original roots and to a taxon ρ ̸∈X . Now, an agreement forest for T1 and T2 is a set of compo-
nents F = {Fρ, F1, . . . Albrecht BMC Bioinformatics (2015) 16:236 , Fk} on X ∪{ρ} with the following
properties. (1) Each component Fi with taxa set Xi refers to the
restricted subtree T1|Xi and T2|Xi, respectively. i
i
(2) There is exactly one component, denoted as Fρ,
containing ρ. (3) Let Xρ, X1, . . . , Xk be the taxa sets corresponding to
Fρ, F1, . . . , Fk. Then, all trees in
{T1(Xi)|i ∈{ρ, 1, . . . , k}} and
{T2(Xi)|i ∈{ρ, 1, . . . , k −1}} are node disjoint
subtrees of T1 and T2, respectively. r(N) =
v∈V:δ−(v)>0
(δ−(v) −1),
(1) (1) (1) A maximum agreement forest is an agreement forest
of minimum size, which implies there does not exist a
smaller set of components fulfilling each property listed
above. Moreover, we call an agreement forest F for two
rooted binary phylogenetic X -trees T1 and T2 acyclic, if
its underlying ancestor-descendant graph AG(T1, T2, F)
does not contain any directed cycles (cf. Fig. 2). More
specifically, this graph AG(T1, T2, F) contains one node
corresponding to exactly one component of F. Moreover, where δ−(v) denotes the in-degree of node v. Moreover,
for a set T of rooted phylogenetic X -trees, we define the
hybridization number h(T ) as where δ−(v) denotes the in-degree of node v. Moreover,
for a set T of rooted phylogenetic X -trees, we define the
hybridization number h(T ) as h(T ) = min{r(N) : N displays each T ∈T }. (2) h(T ) = min{r(N) : N displays each T ∈T }. (2) h(T ) = min{r(N) : N displays each T ∈T }. (2) (2) Fig. 1 a A hybridization network N with taxa set X = {a, b, c, d, e} whose reticulation edges are consecutively numbered. b A phylogenetic X-tree
T that is displayed by N. Based on N, both edge sets {3, 6, 1} and {3, 6, 2} refer to T. c The restricted network N|E′,X ′ with E′ = {3, 6, 1} and
X ′ = {b, c, d, e} still containing nodes of both in- and out-degree 1 Fig. 1 a A hybridization network N with taxa set X = {a, b, c, d, e} whose reticulation edges are consecutively numbered. b A phylogenetic X-tree
T that is displayed by N. Based on N, both edge sets {3, 6, 1} and {3, 6, 2} refer to T. Albrecht BMC Bioinformatics (2015) 16:236 Albrecht BMC Bioinformatics (2015) 16:236 Page 3 of 15 Page 3 of 15 Page 3 of 15 Now, given a hybridization network N on X and an edge
set E′ referring to an embedded rooted phylogenetic X ′-
tree T′ in N, the reduced network N|E′,X ′, with X ′ ⊆X , is
computed as follows. First, E′ is deleted and, second, each
node of out-degree 0 that is unlabeled or not labeled by a
taxon in X ′ is removed repeatedly. The resulting directed
graph corresponds to T′|X ′ but still contains nodes of both
in- and out-degree 1, and, thus, each node in N|E′,X ′ can
be mapped back to exactly one specific node of the unre-
stricted network N (cf. Fig.1(c)). Moreover, the network
N(v) denotes a network rooted at v that is computed by,
first, removing each node that cannot be reached from
v and, second, by suppressing each node of both in- and
out-degree 1. is contained in the subtree rooted at v. Given a set F of
trees, the label set L(F) is simply the union of each label
set L(Fi) of a tree Fi ∈F. is contained in the subtree rooted at v. Given a set F of
trees, the label set L(F) is simply the union of each label
set L(Fi) of a tree Fi ∈F. Now, given a rooted phylogenetic X -tree T and a taxa
set X ′
⊆
X , we define T(X ′) as the minimal con-
nected subgraph of T whose leaf set contains each taxon
in X ′. Additionally, by T|X ′ we define the subgraph that
is obtained from T(X ′) by suppressing all nodes of both
in- and out-degree 1. Moreover, given a tree T, through-
out this paper we use T to denote the tree that is obtained
from T by suppressing each node of both in- and out-
degree 1. is contained in the subtree rooted at v. Given a set F of
trees, the label set L(F) is simply the union of each label
set L(Fi) of a tree Fi ∈F. Now, given a rooted phylogenetic X -tree T and a taxa
set X ′
⊆
X , we define T(X ′) as the minimal con-
nected subgraph of T whose leaf set contains each taxon
in X ′. Preliminaries
h The upcoming definitions used for describing and dis-
cussing our algorithm follow the work of Huson et al. [14]. We assume that the user is familiar with general
graph-theoretic concepts. Phylogenetic trees. A rooted phylogenetic X -tree T is a
directed tree, whose edges are directed from the root to
the leaves and whose nodes, except the root, have a degree
not equal to 2. If T is a binary tree its root has in-degree
0 and out-degree 2, each inner node an in-degree of 1 and
an out-degree of 2, and each leaf an in-degree of 1 and
an out-degree of 0. Moreover, each leaf is labeled one-to-
one by a taxon of the taxa set X , which usually consists of
certain species or genes and is also denoted by L(T). For
a node v of T, the label set L(v) contains each taxon that As we state that our algorithm guarantees the compu-
tation of the exact hybridization number, we are aware of
the fact that this algorithm raises some questions regard-
ing its correctness. However, since in this paper we want
to focus on the efficiency of the presented algorithm as
well as on the advantages of our software Hybroscale Albrecht BMC Bioinformatics (2015) 16:236 c The restricted network N|E′,X ′ with E′ = {3, 6, 1} and
X ′ = {b, c, d, e} still containing nodes of both in- and out-degree 1 Albrecht BMC Bioinformatics (2015) 16:236 Page 4 of 15 Fig. 2 An agreement forest F of two phylogenetic X-trees T1 and T2 together with the corresponding ancestor-descendant graph AG(T1, T2, F). Note that, as AG(T1, T2, F) does not contain any directed cycles, F is acyclic Fig. 2 An agreement forest F of two phylogenetic X-trees T1 and T2 together with the corresponding ancestor-descendant graph AG(T1, T2, F). Note that, as AG(T1, T2, F) does not contain any directed cycles, F is acyclic two nodes Fi and Fj with i ̸= j are connected via a directed
edge (Fi, Fj) if either two nodes Fi and Fj with i ̸= j are connected via a directed
edge (Fi, Fj) if either as already described in Baroni et al. [10], as follows. First,
select the node vρ of in-degree 0, which corresponds to
Fρ, and remove vρ by deleting this node together with all
its incident edges. Next, again choose a node v1 with in-
degree 0 and remove this node. By continuing this way
until finally all nodes have been removed, one receives the
ordering = (vρ, v1, . . . , vk). In the following, we call the
ordering of components corresponding to , denoted by
(Fρ, F1, . . . , Fk), an acyclic ordering of F. As during each
of those steps there can occur multiple nodes of in-degree
0, especially if F contains components only consisting of
isolated nodes, such an acyclic ordering is in general not
unique. (i) the root of T1(Xi) is an ancestor of the root of T1(Xj)
or
(ii) the root of T2(Xi) is an ancestor of the root of T2(Xj), (ii) the root of T2(Xi) is an ancestor of the root of T2(Xj), where Xi, Xj ⊆X denotes the taxa set of Fi and Fj,
respectively. Again, we call an acyclic agreement forest
of minimum size a maximum acyclic agreement forest. Note that for a maximum acyclic agreement forest for two
rooted binary phylogenetic X -trees T1 and T2 containing
k components there exists a hybridization network whose
reticulation number is k −1 [15]. This means, in particu-
lar, if a maximum acyclic agreement forest for T1 and T2
contains only one component, both trees are equal. where Xi, Xj ⊆X denotes the taxa set of Fi and Fj,
respectively. Again, we call an acyclic agreement forest
of minimum size a maximum acyclic agreement forest. Note that for a maximum acyclic agreement forest for two
rooted binary phylogenetic X -trees T1 and T2 containing
k components there exists a hybridization network whose
reticulation number is k −1 [15]. This means, in particu-
lar, if a maximum acyclic agreement forest for T1 and T2
contains only one component, both trees are equal. Representative networks. As mentioned above, our
algorithm ensures the computation of all representative
networks, which are those hybridization networks with
minimum hybridization number (cf. Eq. two nodes Fi and Fj with i ̸= j are connected via a directed
edge (Fi, Fj) if either (2)) fulfilling
an additional property that is based on the following If F is acyclic and, thus, AG(T1, T2, F) does not contain
any directed cycles, one can compute an acyclic ordering, If F is acyclic and, thus, AG(T1, T2, F) does not contain
any directed cycles, one can compute an acyclic ordering, Albrecht BMC Bioinformatics (2015) 16:236 Page 5 of 15 the hybridization number or all representative networks
showing the embedding of those input trees. Similar to
the approach described in the work of Albrecht et al. [9],
ALLHNETWORKS can be separated into three phases. The
reduction phase (consisting of a subtree reduction follow-
ing the work of Bordewich and Semple [16] and a cluster
reduction following the work of Baroni et al. [10] and Linz
[17]), the exhaustive search phase, and the output phase
(combining the result of all clusters and undoing each sub-
tree reduction). Whereas the reduction and the output
phase can be conducted in polynomial time, the second
phase solves an NP-hard problem and, thus, its runtime is
exponential [5]. However, as recently shown by van Iersel
and Linz [6], certain parts of the problem still remain
fixed-parameter tractable, which, as already noted in the
introduction, is an important feature that is exploited by
our algorithm to maximize its efficiency. observation. Given a hybridization network containing a
node v with in-degree of at least 3, one can generate fur-
ther networks by simply dragging some of its hybridiza-
tion edges upwards resulting in a stack of hybridization
nodes. More precisely, such a stack is a path (v1, . . . , vn)
in which each hybridization node vi is connected through
a hybridization edge to vi+1 (cf. Fig. 3). From a biologi-
cal point of view, such a stack implies that a hybridiza-
tion event belonging to a hybridization node vi happened
before those corresponding to the in-edges of a hybridiza-
tion node vj with i < j. However, as for each of those
networks there exists a network where each stack is fully
compressed, we only consider those compressed networks
as being relevant. Consequently, the set of representative networks con-
sists only of those networks with minimum hybridization
number not containing any stacks of hybridization nodes
leaving the interpretation of the ordering of the hybridiza-
tion events open. two nodes Fi and Fj with i ̸= j are connected via a directed
edge (Fi, Fj) if either Moreover, just for simplicity, we claim
that each of those networks has to be binary not contain-
ing any nodes of out-degree greater than 2. By introducing
multifurcating nodes, which are nodes having an out-
degree of at least 3, the set of representative networks
typically shrinks because due to those nodes a network
can display several binary networks. At this point, we have to give a remark regarding the cor-
rectness of the cluster reduction. The well-known work of
Baroni et al. [10] contains a proof showing that the exact
hybridization number of two binary phylogenetic X -trees
can also be computed by adding up the exact hybridiza-
tion numbers of its minimum common clusters. A more
general proof, showing that this concept also holds for
multiple binary phylogenetic X -trees, can be found in our
forthcoming paper [13]. Lastly, given two representative networks N1 and N2, we
say that N1 differs from N2 if either their graph topologies
(disregarding edge labels) are not isomorphic or their edge
sets indicating the embedding of each input tree differ. In the upcoming part, we will briefly discuss the exhaus-
tive search phase and its parallelization. A description
of the other two phases is omitted but can be looked
up in the work of Albrecht et al. [9]. The exhaustive
search phase runs for an increasing parameter k bounding
the reticulation number of each computed network. If a
hybridization network with reticulation number less than
or equal to k does not exist, the search is continued with
k + 1 until a hybridization network displaying all input
trees can be computed. The algorithm ALLHNETWORKS For each edge set Ei referring
to the embedded tree Ti|L(F′) in N, the set VA
s of source
nodes of Type A contains all nodes v with N|Ei,L(F′)(v) iso-
morphic to Ti|L(F′)(vsib), where vsib denotes the sibling
of the node v′ with L(v′) = L(Fi) in Ti|L(F′)∪L(Fi). Note
that, due to the restriction of the network to L(F′), this
set usually consists of more than one node. Source Nodes of Type B. The set VB
s of source nodes of
Type B is computed such that it contains each node v of a
subtree, whose root is a sibling of a node in VA
s and which
does not contain any taxa of L(F′). Moreover, its leaf set
L(v) has to consist only of several subsets representing the
total taxa set L(F) of a component F in F, which means
that v must not be part of a subtree corresponding to a
component that is added afterward. The input tree Ti is added to a so far computed net-
work N by adding hybridization edges connecting certain
parts of N. Given an edge set E′ referring to a phyloge-
netic X -tree T′ that is displayed by N, such parts can
be derived from the components of a maximum acyclic
agreement forest for T′ and Ti. Again, in order to guar-
antee the computation of all representative networks, the
insertion of Ti has to be performed for all maximum
acyclic agreement forests referring to Ti and each phylo-
genetic X -tree T′ that is embedded in N, and, additionally,
for all edge sets E′ referring to T′. Note that, given two
rooted binary phylogenetic X -trees, the computation of
all maximum acyclic agreement forests follows the algo-
rithm ALLMAAFS [18]. For a better understanding the definitions of source and
target nodes are illustrated in Fig. 4. Now, given a valid pair (s, t) of source and target nodes,
a new hybridization edge is inserted as follows. The in-
edge of the source node s is split in a way that there is a
new node s′ that is connected to s and to the parent of
s. If the parent of t is of in-degree one the in-edge of t
is split in the same way. Otherwise, its parent node acts
as t′ which allows the computation of networks contain-
ing nodes of in-degree greater than 2. The algorithm ALLHNETWORKS In this section, we give a high level description of our algo-
rithm ALLHNETWORKS. More information, involving a
more detailed description of the upcoming steps as well as
some theoretical issues, will be discussed in a forthcoming
paper [13]. The input of the algorithm is a set T
of rooted
binary phylogenetic X -trees and its output is either just Exhaustive search phase. Given a set T consisting of
n rooted binary phylogenetic X -trees and a parameter Fig. 3 An illustration of a stack of hybridization nodes. The hybridization node with in-degree 4 of the left tree T1 can be resolved, amongst others,
into two different stacks of hybridization nodes (v1, v2, v3) as demonstrated by T2 and T3, respectively. Note that by resolving a hybridization node
into a stack of hybridization nodes the set of trees that are displayed in the original network remains unchanged Fig. 3 An illustration of a stack of hybridization nodes. The hybridization node with in-degree 4 of the left tree T1 can be resolved, amongst others,
into two different stacks of hybridization nodes (v1, v2, v3) as demonstrated by T2 and T3, respectively. Note that by resolving a hybridization node
into a stack of hybridization nodes the set of trees that are displayed in the original network remains unchanged Albrecht BMC Bioinformatics (2015) 16:236 Albrecht BMC Bioinformatics (2015) 16:236 Albrecht BMC Bioinformatics (2015) 16:236 Page 6 of 15 Page 6 of 15 k ∈N, in a first step we choose an ordering of T , which is
for convenience (T1, T2, . . . , Tn) in the following. Second,
each tree of this ordering is added sequentially to a set N
of networks in all possible ways. At the beginning, N only
consists of the first tree of the ordering, which is T1 in this
case. By adding an upcoming input tree Ti (i > 1), the size
of N grows rapidly, because in general there exist multi-
ple ways of how this can be achieved (cf. Fig. 6). Since we
do not delete any edges from a so far computed network
N, we can disregard those networks whose reticulation
number exceeds k. Note that, in order to guarantee the
computation of all representative networks, this step must
be performed for each possible ordering of T . Source Nodes of Type A. The algorithm ALLHNETWORKS Note that this is an
optional step that is necessary to ensure that each com-
puted hybridization network does not contain any stacks
of hybridization nodes such that it applies to the definition
of a representative network. Finally, the two nodes s′ and
t′ are connected through a path P consisting of two edges. This is done because, on the one hand, we only allow
nodes of in-degree one as source nodes, but, on the other
hand, in order to compute all representative networks,
we have to enable that a target node can additionally be
attached to hybridization edges. Due to this fact, however,
before reporting a network embedding all input trees, one
still has to suppress all nodes of both in- and out-degree
1. By referring to the terminology used above, in Fig. 5 we A maximum acyclic agreement forest F of Ti and T′
is added to N by, first, computing an acyclic ordering
(Fρ, F1, . . . , Fk) of F which can be done with the help of
the directed graph AG(T′, Ti, F) as previously described. Next, each component Fi, beginning with F1, is added to
N by inserting a new hybridization edge connecting a cer-
tain source and target node such that, after all components
of F have been inserted, N displays the considered input
tree Ti. In order to guarantee the computation of the exact
hybridization number, all acyclic orderings and all valid
combinations of source and target nodes, as described
below, have to be taken into account. More precisely, in
order to avoid directed cycles, we consider a pair (s, t) of
source and target nodes as being valid if the source node
s cannot be reached from t. Note that the way of how
we add a tree to a network is similar to the algorithm
HYBRIDPHYLOGENY [10]. Fig. 4 An illustration of the definitions of target (left) and source
nodes (right) for a component Fi (p, q > i) in which red nodes
correspond to target nodes, blue nodes to source nodes of Type A,
and green nodes to source nodes of Type B. The algorithm ALLHNETWORKS Moreover, dashed edges
and dotted edges are those edges that are disregarded when
considering the restricted network in terms of the chosen embedded
tree and the taxa set of the so far added components, respectively The set of target and source nodes corresponding to
a component Fi in F is defined as follows. Let F′ =
{Fρ, F1, . . . , Fi−1} ⊂F = {Fρ, F1, . . . , Fk} be the set of
components that has been added so far. Note that, since N
is initialized with Fρ, at the beginning L(F′) equals L(Fρ)
and the first component that is added is F1. Fig. 4 An illustration of the definitions of target (left) and source
nodes (right) for a component Fi (p, q > i) in which red nodes
correspond to target nodes, blue nodes to source nodes of Type A,
and green nodes to source nodes of Type B. Moreover, dashed edges
and dotted edges are those edges that are disregarded when
considering the restricted network in terms of the chosen embedded
tree and the taxa set of the so far added components, respectively Target Nodes. The set Vt of target nodes contains all
nodes v with N|E′,L(F′)∪L(Fi)(v) isomorphic to Ti|L(Fi). Due to the restriction of the network to L(F′), this set
usually contains more than one node. Target Nodes. The set Vt of target nodes contains all
nodes v with N|E′,L(F′)∪L(Fi)(v) isomorphic to Ti|L(Fi). Due to the restriction of the network to L(F′), this set
usually contains more than one node. Albrecht BMC Bioinformatics (2015) 16:236 Page 7 of 15 give a short example of how a certain input tree is added
to a network. representative networks, one still has to compute for each
network all possible combinations of edge sets each refer-
ring to the embedding of one input tree. For clarity, the algorithm ALLHNETWORKS has been
described so far with respect to one single embedding
of each input tree. Given n input trees, to generate all Lastly, we give some high level ideas why the algo-
rithm
ALLHNETWORKS is correct, i.e., calculates all p
p
g
Fig. 5 An illustration of how an input tree Ti is inserted into a network Ni−1 with the help of an embedded tree T′. a The network Ni−1 together with
an embedded tree T′. The algorithm ALLHNETWORKS b The input tree Ti, which will be embedded into Ni−1 by inserting the maximum acyclic agreement F forest of Ti and T′
consisting of three components Fρ, F1, and F2. c, d All important elements that have to be considered during the insertion of both components F1
and F2, respectively. Blue dots correspond to source nodes and red nodes to target nodes. Note that, regarding N(1)
i
, there is only one valid pair of
source and target nodes. Dashed edges are those edges that are disregarded when considering the restricted network in terms of T′ and the taxa set
of the so far added components of F. e The resulting network Ni, which is obtained from N(3)
i
by suppressing each node of both in- and out-degree 1 Fig. 5 An illustration of how an input tree Ti is inserted into a network Ni−1 with the help of an embedded tree T′. a The network Ni−1 together with
an embedded tree T′. b The input tree Ti, which will be embedded into Ni−1 by inserting the maximum acyclic agreement F forest of Ti and T′
consisting of three components Fρ, F1, and F2. c, d All important elements that have to be considered during the insertion of both components F1
and F2, respectively. Blue dots correspond to source nodes and red nodes to target nodes. Note that, regarding N(1)
i
, there is only one valid pair of
source and target nodes. Dashed edges are those edges that are disregarded when considering the restricted network in terms of T′ and the taxa set
of the so far added components of F. e The resulting network Ni, which is obtained from N(3)
i
by suppressing each node of both in- and out-degree 1 Albrecht BMC Bioinformatics (2015) 16:236 Page 8 of 15 hybridization number k in parallel. As mentioned above,
the insertion of an input tree Ti to a so far computed
network results in several new networks, which are then
processed by inserting the next input tree Ti+1 of the
chosen ordering (cf. Fig. 6). Since the processing of net-
works runs independently from each other, these steps can
be parallelized in a simple manner. Based on the num-
ber of hybridization edges of a so far computed network,
each of those steps is more or less likely to result in a
representative network. The algorithm ALLHNETWORKS Thus, we set up a priority queue
to process the most promising networks first, which, on
the one hand, depends on the number of so far inserted
input trees and, on the other hand, on its reticulation
number. representative networks for a set of rooted binary phy-
logenetic X -trees. We refer readers who are interested
in a detailed proof to our forthcoming paper [13]. Let
N′ be a representative network displaying a subset T ′
of all input trees T and let N be a hybridization net-
work (not containing any stacks of hybridization nodes)
that is based on N′ and displays a further input tree
Ti ̸∈T . Then, one can obtain N from N′ by insert-
ing a set E′ of reticulation edges whose source and target
nodes can be derived from an acyclic agreement for-
est F for Ti and a certain tree displayed by N′. This
is due to the fact that N′ must contain such an agree-
ment forest F so that each of its components, except Fρ,
is rooted at a target node of an edge in E′ whose inci-
dent nodes are contained in Vt and VA
s ∪VB
s , respectively. Moreover, one can show that by constructing networks
for all possible orderings of the input trees, it suffices
to take only maximum acyclic agreement forests into
account. Such a priority queue, however, does only speed up the
computation of the hybridization number, since in this
case all computational paths can be aborted immediately
as far as the first minimum hybridization network could
be computed successfully. For the computation of all rep-
resentative networks, however, each computational path
has to be processed anyway until either it can be early
aborted (which is the case if the reticulation number of
the corresponding network exceeds k) or it leads to a
representative network. Parallelization In order to improve the practical runtime of the algorithm
ALLHNETWORKS, our implementation is able to run the
exhaustive search looking for hybridization networks with Fig. 6 An illustration of how the insertion of the input trees is conducted by the algorithm ALLHNETWORKS in respect of the parameter k bounding
the maximal reticulation number of resulting networks. Beginning with the first input tree T1, repeatedly, first, an embedded tree T′ of a so far
computed network N is extracted, and, second, the current input tree Ti is inserted into N by sequentially adding the components of a maximum
acyclic agreement forest for T′ and Ti. As soon as the reticulation number of a so far computed network exceeds k one can be sure that this network
cannot lead to a network with reticulation number smaller or equal to k and, thus, the corresponding computational path can be aborted Fig. 6 An illustration of how the insertion of the input trees is conducted by the algorithm ALLHNETWORKS in respect of the parameter k bounding
the maximal reticulation number of resulting networks. Beginning with the first input tree T1, repeatedly, first, an embedded tree T′ of a so far
computed network N is extracted, and, second, the current input tree Ti is inserted into N by sequentially adding the components of a maximum
acyclic agreement forest for T′ and Ti. As soon as the reticulation number of a so far computed network exceeds k one can be sure that this network
cannot lead to a network with reticulation number smaller or equal to k and, thus, the corresponding computational path can be aborted Albrecht BMC Bioinformatics (2015) 16:236 Albrecht BMC Bioinformatics (2015) 16:236 Albrecht BMC Bioinformatics (2015) 16:236 Page 9 of 15 Moreover, as the algorithm computes networks for all
different orderings of input trees and all different acyclic
orderings of maximum acyclic agreement forests, a rep-
resentative network can be computed multiple times. As
a consequence, to ensure that the output only consists
of unique networks, one has to filter the set of networks
obtained from the exhaustive search step. For this pur-
pose, we first group this set after the sum of support values
computed for each network (as defined later) and then
check each of those subgroups for isomorphic networks
in parallel. Due to the typically large number of computed
networks (cf. Additional features Given just the extended newick format [19] of a hybridiza-
tion network, its topology is in general hard to interpret. Although there exist software packages, which are able to
display rooted phylogenetic networks, e.g., the software
Dendroscope [14], most of them are not able to visualize
the embedding of all input trees, which is a preferable fea-
ture for studying hybridization events. In order to close
this gap, we have developed the software Hybroscale,
which is specifically designed for studying hybridization
networks. Besides the computation of a graphical lay-
out of rooted trees and rooted networks, which is opti-
mized by minimizing the number of crossings between all
hybridization edges, Hybroscale can additionally highlight
each hybridization edge that is necessary for displaying all
embedded input trees by assigning a specific color to each
tree (cf. Fig 7). Thus, Hybroscale is a software that, on the
one hand, enables an easy handling of our algorithm and,
on the other hand, ensures the readability of the computed
networks. Our synthetic dataset is freely available2 and consists
of several tree sets each containing multiple rooted phy-
logenetic X -trees. Each X -tree is generated by ranging
over all different combinations of four parameters, namely
the number of input trees n, the number of leaves ℓ, an
upper bound for the hybridization number k, and the clus-
ter degree c as defined below. Each of the n input trees
is obtained from a bicombining network N, which means
that N only contains hybridization nodes of in-degree 2. This network N is computed in respect to these four dif-
ferent parameters as follows. In a first step, a random
binary tree T with ℓleaves is computed which is done in
the following way. First, at the beginning, two nodes u and
v of a specific set V, which is initialized by ℓnodes of both
in- and out-degree 0, are randomly selected. Those two
selected nodes u and v are then connected to a new node
w and, finally, V is updated by replacing u and v by its par-
ent node w. This process is repeated until V consists only
of one node corresponding to the root of T. In a second
step, k hybridization edges are created in T with respect
to parameter c such that the resulting network N contains
exactly k hybridization nodes of in-degree 2. Simulation study
T
h
h To show the efficiency of our implementation, we
have integrated our algorithm into the Java software
Hybroscale and conducted a simulation study compar-
ing its runtime to PIRNv2.0 [7, 8], which is so far the
best available software for computing exact hybridization
numbers for multiple rooted binary phylogenetic X -trees. 2 Results and discussion In this section, we first report a simulation study indicat-
ing that our approach is much faster than other existing
methods and then illustrate how Hybroscale can be used
for studying hybridization networks by applying the soft-
ware to a well known grass (Poaceae) dataset. Parallelization Tables 1, 2), the restriction of the filtering
step to small subgroups usually provokes a large speedup. Note that, as already mentioned above, we consider two
networks as being different if either their graph topologies
(disregarding edge labels) are not isomorphic or their sets
of edges that are necessary for displaying each input tree
differ. is {austrodant, karoochloa}. Second, we determine the
fraction of networks containing (v). This step ensures
that the user can instantly look at those networks con-
taining the most promising hybridization events, which is
an important feature, because usually a large number of
networks is reported (cf. Tables 1, 2). Additional features Furthermore, Hybroscale assigns each hybridization
node a support value indicating the fraction of networks
containing this node and, additionally, sorts the reported
networks by the sum of those values in decreasing order. More specifically, the computation of support values is
done as follows. Given a network N, each edge set Ei refer-
ring to one of the input trees Ti, and a certain hybridiza-
tion node v, we, first, compute the following ordering of
taxa sets (v) = (L(N(v)|E1,X ), . . . , L(N(v)|En,X )). More
precisely, each element L(N(v)|Ei,X ) consists of those taxa
adhering to each leaf that can be reached from v by
directed paths only crossing those hybridization edges in
Ei indicating the embedding of Ti. For example, regarding
Fig. 7, the set referring to the hybridization edges indicat-
ing the embedding of Tree 2 and the node labeled by 22 % In this context, the cluster degree is an ad hoc con-
cept influencing the computational complexity of a tree
set similar to the concept of the tangling degree intro-
duced in the work of Scornavacca et al. [18]. When adding
a hybridization edge e with target node v2 and source
node v1, we say that e respects cluster degree c, if v1 can-
not be reached from v2 and there is a path of length less
than or equal to c leading from v2 to a certain node p
such that v1 can be reached from p. Consequently, net-
works providing a small cluster degree in general contain
more minimum common clusters than networks of large Albrecht BMC Bioinformatics (2015) 16:236 Page 10 of 15 Fig. 7 Our software Hybroscale showing a hybridization network displaying the embedding of four input trees by the colors blue, red, green, and
orange Fig. 7 Our software Hybroscale showing a hybridization network displaying the embedding of four input trees by the colors blue, red, green, and
orange Fig. 7 Our software Hybroscale showing a hybridization network displaying the embedding of four input trees by the colors blue, red, green, and
orange cluster degrees and, thus, typically can be processed quite
fast when applying a cluster reduction beforehand. For a
better understanding, in Fig. 8 an example of this concept
is depicted. Additional features 4
6
8
10
12
14
16
0
200
400
600
800
1000
1200
Upper Bound of the Hybridization Number
Mean REAL Runtime (s)
89 %
26 %
17 %
100 %
100 %
97 %
PIRN
Hybroscale
n=3
Fig. 9 The figure shows the mean average runtime corresponding to
Hybroscale and PIRNv2.0 grouped by parameter k denoting the
hybridization number of the network that was used to obtain the tree
set T from. Thus, this parameter k acts as an upper bound of the
hybridization number of T . Each percentage indicates the
proportion of tree sets that could be computed within the time limit
of 20 minutes To compare the efficiency, both programs have been run
on a grid computer providing 16 cores and 40 GB RAM
for our synthetic dataset containing tree sets with param-
eters n ∈{3, 4, 5}, ℓ∈{10, 25, 50}, k ∈{5, 10, 15}, and c ∈
{1, 3, 5}. More precisely, we have generated for all 81 com-
binations of the four parameters 30 tree sets as described
above resulting in 2430 tree sets in total. The results for
three input trees (n = 3) are presented in Figs. 9, 10 4
6
8
10
12
14
16
0
200
400
600
800
1000
1200
Mean REAL Runtime (s)
89 %
26 %
17 %
100 %
100 %
97 %
PIRN
Hybroscale Fig. 8 An illustration of the cluster degree parameter c = 1. When
inserting an in-going edge e to node v2 that is respecting c, each
node that is marked green or is part of a green marked subnetwork
forms a potential source node Fig. 8 An illustration of the cluster degree parameter c = 1. When
inserting an in-going edge e to node v2 that is respecting c, each
node that is marked green or is part of a green marked subnetwork
forms a potential source node Upper Bound of the Hybridization Number Fig. 9 The figure shows the mean average runtime corresponding to
Hybroscale and PIRNv2.0 grouped by parameter k denoting the
hybridization number of the network that was used to obtain the tree
set T from. Thus, this parameter k acts as an upper bound of the
hybridization number of T . Each percentage indicates the
proportion of tree sets that could be computed within the time limit
of 20 minutes Fig. Additional features The numbers
inside the plot indicate how many tree sets could be computed for
the corresponding hybridization number within the time limit of 20
minutes. Note that for the hybridization numbers 0 to 3 all
corresponding tree sets could be computed by Hybroscale and
PIRNv2.0 within comparable runtimes Fig. 10 The figure shows the mean average runtime of all tree sets
grouped by the computed hybridization numbers. The numbers
inside the plot indicate how many tree sets could be computed for
the corresponding hybridization number within the time limit of 20
minutes. Note that for the hybridization numbers 0 to 3 all
corresponding tree sets could be computed by Hybroscale and
PIRNv2.0 within comparable runtimes Fig. 11 A scatterplot of the runtimes generated by PIRNv2.0 (x-axis)
against the runtimes generated by Hybroscale (y-axis) of all 810 data
sets consisting of three input trees. Note that PIRNv2.0 is not able to
compute the result for 449 tree sets corresponding to each dot in the
figure whose x-value is 1200. From those tree sets just 6 according to
the dots whose y-value is also 1200 could not be computed by
Hybroscale Fig. 11 A scatterplot of the runtimes generated by PIRNv2.0 (x-axis)
against the runtimes generated by Hybroscale (y-axis) of all 810 data
sets consisting of three input trees. Note that PIRNv2.0 is not able to
compute the result for 449 tree sets corresponding to each dot in the
figure whose x-value is 1200. From those tree sets just 6 according to
the dots whose y-value is also 1200 could not be computed by
Hybroscale and 11, whereas the results for four and five input trees
(n = 4, 5) can be found in the Additional file 1. Due to time
limitations, if the hybridization number of a certain tree
set could not be computed within 20 minutes, the compu-
tation of this tree set was aborted. In Figs. 9, 11, 12, and 13
those unfinished tree sets were taken into account with a
runtime of 20 minutes whereas in Fig. 10 these tree sets
were omitted. #Tree Sets
0
200
400
600
800
1000
<1200
<1100
<1000
<900
<800
<700
<600
<500
<400
<300
<200
<100
<50
<10
n=3
Runtime (s)
PIRN
Hybroscale REAL Runtime
Hybroscale USER Runtime
Fig. Additional features 8 An illustration of the cluster degree parameter c = 1. When
inserting an in-going edge e to node v2 that is respecting c, each
node that is marked green or is part of a green marked subnetwork
forms a potential source node Fig. 8 An illustration of the cluster degree parameter c = 1. When
inserting an in-going edge e to node v2 that is respecting c, each
node that is marked green or is part of a green marked subnetwork
forms a potential source node Albrecht BMC Bioinformatics (2015) 16:236 Page 11 of 15 Fig. 11 A scatterplot of the runtimes generated by PIRNv2.0 (x-axis)
against the runtimes generated by Hybroscale (y-axis) of all 810 data
sets consisting of three input trees. Note that PIRNv2.0 is not able to
compute the result for 449 tree sets corresponding to each dot in the
figure whose x-value is 1200. From those tree sets just 6 according to
the dots whose y-value is also 1200 could not be computed by
Hybroscale
#Tree Sets
0
200
400
600
800
1000
<1200
<1100
<1000
<900
<800
<700
<600
<500
<400
<300
<200
<100
<50
<10
n=3
Runtime (s)
PIRN
Hybroscale REAL Runtime
Hybroscale USER Runtime
Fig. 12 The figure shows the number of tree sets that could be
computed within the runtime given on the x-axis by considering the
real-runtime of PIRN and both real- and user-runtime of Hybroscale. Only the rightmost bar group reveals that the massive parallelization
with 16 cores can significantly improve the runtime of Hybroscale in
this case. Note that this is, on the one hand, due to the low time limit
of just 20 minutes and, on the other hand, due to the low
computational complexity of the considered tree sets Fig. 10 The figure shows the mean average runtime of all tree sets
grouped by the computed hybridization numbers. The numbers
inside the plot indicate how many tree sets could be computed for
the corresponding hybridization number within the time limit of 20
minutes. Note that for the hybridization numbers 0 to 3 all
corresponding tree sets could be computed by Hybroscale and
PIRNv2.0 within comparable runtimes Fig. 10 The figure shows the mean average runtime of all tree sets
grouped by the computed hybridization numbers. Additional features 12 The figure shows the number of tree sets that could be
computed within the runtime given on the x-axis by considering the
real-runtime of PIRN and both real- and user-runtime of Hybroscale. Only the rightmost bar group reveals that the massive parallelization
with 16 cores can significantly improve the runtime of Hybroscale in
this case. Note that this is, on the one hand, due to the low time limit
of just 20 minutes and, on the other hand, due to the low
computational complexity of the considered tree sets #Tree Sets
0
200
400
600
800
1000
<1200
<1100
<1000
<900
<800
<700
<600
<500
<400
<300
<200
<100
<50
<10
n=3
Runtime (s)
PIRN
Hybroscale REAL Runtime
Hybroscale USER Runtime Each of the simulation results given in Figs. 9, 10, 11, 12,
and 13, which are now discussed in more detail, clearly
demonstrates that our implementation is much faster than
PIRN. Figure 9 shows that, by increasing the upper bound
of the hybridization number k, the mean average run-
time of the datasets computed by PIRN increases up to
1000 seconds whereas the mean average runtime corre-
sponding to Hybroscale is always below 100 seconds. Note
that, as the runtime of each unfinished dataset was set to
1200 seconds, if we would set the time limit to a higher
value, the maximal mean average runtime produced by
PIRN is expected to be even higher — otherwise, to pro-
duce a reasonable comparison between both programs,
we would have to leave out each dataset, which could
not be computed by one of both programs, which means
that we would end up with only those non representative
datasets that are quite easy to compute. Figure 10 shows
that Hybroscale, in comparison to PIRN, can compute
more datasets within the time limit and datasets having
a significant larger hybridization number. Whereas PIRN Runtime (s) Fig. 12 The figure shows the number of tree sets that could be
computed within the runtime given on the x-axis by considering the
real-runtime of PIRN and both real- and user-runtime of Hybroscale. Only the rightmost bar group reveals that the massive parallelization
with 16 cores can significantly improve the runtime of Hybroscale in
this case. Additional features Note that this is, on the one hand, due to the low time limit
of just 20 minutes and, on the other hand, due to the low
computational complexity of the considered tree sets Fig. 12 The figure shows the number of tree sets that could be
computed within the runtime given on the x-axis by considering the
real-runtime of PIRN and both real- and user-runtime of Hybroscale. Only the rightmost bar group reveals that the massive parallelization
with 16 cores can significantly improve the runtime of Hybroscale in
this case. Note that this is, on the one hand, due to the low time limit
of just 20 minutes and, on the other hand, due to the low
computational complexity of the considered tree sets Albrecht BMC Bioinformatics (2015) 16:236 Page 12 of 15 Fig. 13 Distribution of the speedups of Hybroscale versus PIRN
computed for each tree set of our synthetic dataset. For three input
trees Hybroscale is on mean average about 110 times faster than
PIRN, for four input trees on mean average about 170 times, and for
five input trees on mean average about 190 times higher computational complexity and, on the other hand,
the time limit would be set to a higher value. A possi-
ble explanation for the speedup without taking advan-
tage of parallelization is, on the one hand, the proven
method allMAAFs [18] that is used for solving the NP-
hard problem of computing all maximum acyclic agree-
ment forests. The efficiency of this method has been
indicated recently in the work of Albrecht et al. [9]. On
the other hand, in contrast to our approach, we assume
that PIRN does only apply a subtree reduction and not
additionally a cluster reduction to the set of initial input
trees. Finally, we have computed the speedup of Hybroscale
versus PIRN by comparing its runtimes produced for
each tree set within our synthetic dataset. More pre-
cisely, for each tree set d we have computed the speedup
s(d) = RP(d)/RH(d), where RP and RH denotes the real-
runtime produced by PIRN and Hybroscale, respectively. Application to a grass dataset As mentioned above our algorithm computes all repre-
sentative networks for a set of input trees. In particular,
given only two input trees, this means that Hybroscale
in general outputs multiple networks for each maximum
acyclic agreement forest instead of only one as it is the
case for the method described in the work of Albrecht Thus, it is obvious that our implementation outper-
forms PIRN which becomes even clearer by looking at
Fig. 11 showing a scatterplot of the runtimes produced by
both programs. The figure shows that for each runtime
of a specific data set produced by PIRN the correspond-
ing runtime of Hybroscale is smaller or equal. Moreover,
looking at the bottom right of the figure, there exist a lot
of data sets that could be computed by Hybroscale quite
fast in less than 200 seconds, whereas PIRN is not able to
come up with a result in less than 1200 seconds. Table 1 Output produced by Hybroscale applied to two
phylogenetic trees belonging to a well known grass (Poaceae)
dataset
Genes
Taxa
HNumber
#MAAFs
#HNetworks
ndhf phyB
40
8
459
2079
ndhf rbcl
36
8
72
1488
ndhf rpoc
34
9
144
264
ndhf waxy
19
6
46
599
phyB its
30
8
21
195
phyB rbcl
21
4
4
6
phyB rpoc
21
4
5
9
phyB waxy
14
3
6
10
rbcl rpoc
26
7
18
111
rbcl waxy
12
4
10
84
rpoc its
31
12
12
3480
rpoc waxy
10
2
1
1
waxy its
15
5
6
15 Table 1 Output produced by Hybroscale applied to two
phylogenetic trees belonging to a well known grass (Poaceae)
dataset p
By comparing real- with user-runtimes, Fig. 12 demon-
strates that the better performance of Hybroscale is not
only due to the applied massive parallelization. As the
user-runtime indicates the total CPU time, which means
that the time spent on all available cores is simply added
up, this time indication corresponds to the runtime pro-
duced by a program that is executed on a system only
providing a single core with no parallel execution tak-
ing place. Figure 12 shows the number of tree sets that
could be computed within the runtime given at the x-
axis. Additional features Figure 13, showing the distribution of the speedups corre-
sponding to each of those tree sets, reveals that for three
input trees Hybroscale is on mean average about 110 times
faster than PIRN, for four input trees on mean average
about 170 times, and for five input trees on mean average
about 190 times. Fig. 13 Distribution of the speedups of Hybroscale versus PIRN
computed for each tree set of our synthetic dataset. For three input
trees Hybroscale is on mean average about 110 times faster than
PIRN, for four input trees on mean average about 170 times, and for
five input trees on mean average about 190 times Fig. 13 Distribution of the speedups of Hybroscale versus PIRN
computed for each tree set of our synthetic dataset. For three input
trees Hybroscale is on mean average about 110 times faster than
PIRN, for four input trees on mean average about 170 times, and for
five input trees on mean average about 190 times is just able to compute hybridization numbers up to 5,
Hybroscale is able to compute hybridization numbers up
to 13. Application to a grass dataset set of
representative networks) within 20 minutes (Continued)
Computing HNumbers Computing HNetworks
Genes
#Taxa HNumber
Runtime
#HNetworks Runtime
ndhf
phyB
rbcl
waxy
7
2
2.581
1
2.594
ndhf phyB rpoc
its
19
9
984.937
-
-
ndhf phyB rpoc
waxy
5
0
0.056
1
0.071
ndhf phyB waxy
its
10
5
4.159
8016
26.434
ndhf rbcl rpoc its
24
-
-
-
-
ndhf
rbcl
rpoc
waxy
9
4
3.165
396
14.864
ndhf rbcl waxy its
11
6
54.399
2
159.99
ndhf rpoc waxy
its
10
5
4.213
324
16.663
phyB rbcl rpoc its
14
-
-
-
-
phyB
rbcl
rpoc
waxy
4
0
0.057
1
0.06
phyB rbcl waxy its
6
2
2.574
3
2.589
phyB rpoc waxy
its
5
0
0.064
1
0.065
rbcl rpoc waxy its
9
5
7.205
333
38.471
ndhf
phyB
rbcl
rpoc its
14
-
-
-
-
ndhf
phyB
rbcl
rpoc waxy
4
0
0.066
1
0.084
ndhf
phyB
rbcl
waxy its
6
3
4.232
135
22.506
ndhf phyB rpoc
waxy its
5
0
0.059
1
0.083
ndhf
rbcl
rpoc
waxy its
9
5
35.899
235
587.54
phyB
rbcl
rpoc
waxy its
4
0
0.066
1
0.076
ndhf
phyB
rbcl
rpoc waxy its
4
0
0.062
1
0.083
et al. [9]. As a consequence, the output usually consists of
a huge number of different hybridization networks, which
is demonstrated by Tables 1 and 2 presenting the results
of our software Hybroscale applied to a well known grass
(Poaceae) dataset3 consisting of three nuclear loci and
three chloroplast genes. This dataset, which is also used
in the work of van Iersel et al. [20], was originally pub-
lished by the Grass Phylogeny Working Group (2001) and Table 2 Output produced by Hybroscale applied to
phylogenetic trees belonging to a grass (Poaceae) dataset. Each
runtime given in this table is stated in seconds. A missing result
for a certain tree set means that our software Hybroscale could
not compute the exact hybridization number (resp. Application to a grass dataset set of
representative networks) within 20 minutes (Continued)
Computing HNumbers Computing HNetworks
Genes
#Taxa HNumber
Runtime
#HNetworks Runtime
ndhf
phyB
rbcl
waxy
7
2
2.581
1
2.594
ndhf phyB rpoc
its
19
9
984.937
-
-
ndhf phyB rpoc
waxy
5
0
0.056
1
0.071
ndhf phyB waxy
its
10
5
4.159
8016
26.434
ndhf rbcl rpoc its
24
-
-
-
-
ndhf
rbcl
rpoc
waxy
9
4
3.165
396
14.864
ndhf rbcl waxy its
11
6
54.399
2
159.99
ndhf rpoc waxy
its
10
5
4.213
324
16.663
phyB rbcl rpoc its
14
-
-
-
-
phyB
rbcl
rpoc
waxy
4
0
0.057
1
0.06
phyB rbcl waxy its
6
2
2.574
3
2.589
phyB rpoc waxy
its
5
0
0.064
1
0.065
rbcl rpoc waxy its
9
5
7.205
333
38.471
ndhf
phyB
rbcl
rpoc its
14
-
-
-
-
ndhf
phyB
rbcl
rpoc waxy
4
0
0.066
1
0.084
ndhf
phyB
rbcl
waxy its
6
3
4.232
135
22.506
ndhf phyB rpoc
waxy its
5
0
0.059
1
0.083
ndhf
rbcl
rpoc
waxy its
9
5
35.899
235
587.54
phyB
rbcl
rpoc
waxy its
4
0
0.066
1
0.076
ndhf
phyB
rbcl
rpoc waxy its
4
0
0.062
1
0.083 Table 2 Output produced by Hybroscale applied to
phylogenetic trees belonging to a grass (Poaceae) dataset. Each
runtime given in this table is stated in seconds. A missing result
for a certain tree set means that our software Hybroscale could
not compute the exact hybridization number (resp. set of
representative networks) within 20 minutes (Continued) Table 2 Output produced by Hybroscale applied to
phylogenetic trees belonging to a grass (Poaceae) dataset. Each
runtime given in this table is stated in seconds. A missing result
for a certain tree set means that our software Hybroscale could
not compute the exact hybridization number (resp. set of
representative networks) within 20 minutes not compute the exact hybridization number (resp. Application to a grass dataset For example, the leftmost bar-group shows that PIRN
could only finish 360 of 810 tree sets consisting of three
trees within 1200 seconds whereas Hybroscale could fin-
ish 804 by taking parallelization into account and 793
by not taking parallelization into account. Note that the
difference between both bars corresponding to the real-
and user-runtime of Hybroscale would be even larger if,
on the one hand, the dataset would contain tree sets of Albrecht BMC Bioinformatics (2015) 16:236 Albrecht BMC Bioinformatics (2015) 16:236 Page 13 of 15 Page 13 of 15 Table 2 Output produced by Hybroscale applied to
phylogenetic trees belonging to a grass (Poaceae) dataset. Each
runtime given in this table is stated in seconds. A missing result
for a certain tree set means that our software Hybroscale could
not compute the exact hybridization number (resp. Application to a grass dataset set of
representative networks) within 20 minutes
Computing HNumbers Computing HNetworks
Genes
#Taxa HNumber
Runtime
#HNetworks Runtime
ndhf its
46
17
3.262
-
-
ndhf phyB
40
8
0.199
2079
33.035
ndhf rbcl
36
8
0.175
1488
32.1
ndhf rpoc
34
9
0.197
264
5.353
ndhf waxy
19
6
0.179
599
5.693
phyB its
30
8
0.238
195
8.304
phyB rbcl
21
4
0.083
6
1.65
phyB rpoc
21
4
0.091
9
1.678
phyB waxy
14
3
0.071
10
1.615
rbcl its
29
12
4.41
-
-
rbcl rpoc
26
7
0.147
111
3.836
rbcl waxy
12
4
0.126
84
4.338
rpoc its
31
12
4.5
3480
217.575
rpoc waxy
10
2
0.07
1
1.582
waxy its
15
5
0.118
15
2.712
ndhf phyB its
30
13
243.411
-
-
ndhf phyB rbcl
21
9
7.226
-
-
ndhf phyB rpoc
21
8
6.189
36948
206.114
ndhf phyB waxy
14
4
1.599
54
2.87
ndhf rbcl its
28
-
-
-
-
ndhf rbcl rpoc
26
11
7.223
46946
511.296
ndhf rbcl waxy
12
5
4.198
114
5.577
ndhf rpoc its
31
-
-
-
-
ndhf rpoc waxy
10
3
2.583
14
2.632
ndhf waxy its
15
8
4.213
6490
26.697
phyB rbcl its
17
8
9.437
8661
233.768
phyB rbcl rpoc
15
6
4.652
40
4.867
phyB rbcl waxy
7
2
2.568
11
2.592
phyB rpoc its
19
7
3.774
57
4.633
phyB rpoc waxy
5
0
0.045
1
0.075
phyB waxy its
10
4
3.122
204
3.844
rbcl rpoc its
24
-
-
-
-
rbcl rpoc waxy
9
3
1.585
5
1.62
rbcl waxy its
11
6
6.224
63
7.49
rpoc waxy its
10
4
2.626
4
2.635
ndhf phyB rbcl its
17
-
-
-
-
ndhf
phyB
rbcl
rpoc
15
9
224.934
1517
403.728
Table 2 Output produced by Hybroscale applied to
phylogenetic trees belonging to a grass (Poaceae) dataset. Each
runtime given in this table is stated in seconds. A missing result
for a certain tree set means that our software Hybroscale could
not compute the exact hybridization number (resp. et al. [9]. As a consequence, the output usually consists of
a huge number of different hybridization networks, which
is demonstrated by Tables 1 and 2 presenting the results
of our software Hybroscale applied to a well known grass
(Poaceae) dataset3 consisting of three nuclear loci and
three chloroplast genes. This dataset, which is also used
in the work of van Iersel et al. [20], was originally pub-
lished by the Grass Phylogeny Working Group (2001) and
reanalyzed in Schmidt (2003). Application to a grass dataset Albrecht BMC Bioinformatics (2015) 16:236 Albrecht BMC Bioinformatics (2015) 16:236 Page 14 of 15 Page 14 of 15 Again, we ran Hybroscale on a grid computer providing
16 cores and 40 GB RAM for each tree set within the grass
dataset and summarized the respective results in Table 2. This table shows that Hybroscale is able to calculate the
hybridization number for 50 out of 57 tree sets. This
means, in particular, that for seven tree sets Hybroscale
cannot produce a result within a time limit of 20 minutes. Moreover, even though for 5 tree sets the hybridiza-
tion number could be calculated, the respective entire
set of representative networks could not be calculated as
in this case a time limit of 20 minute is not sufficient
to explore the whole solution space. Consequently, this
biological example demonstrates that, although our algo-
rithm seems to be faster than all so far existing methods,
calculating minimum hybridization networks remains a
computationally hard problem, which is still not solved
sufficiently. the computation of meaningful support values indicat-
ing which of the computed hybridization events might
have played an important role during evolution. Addi-
tionally, in combination with our software Hybroscale, we
improve the interpretation of the reported hybridization
networks by assigning support values to each hybridiza-
tion node and by highlighting the embedding of all input
trees. p
Moreover, even though for 5 tree sets the hybridiza-
tion number could be calculated, the respective entire
set of representative networks could not be calculated as
in this case a time limit of 20 minute is not sufficient
to explore the whole solution space. Consequently, this
biological example demonstrates that, although our algo-
rithm seems to be faster than all so far existing methods,
calculating minimum hybridization networks remains a
computationally hard problem, which is still not solved
sufficiently. Additionally, our reported simulation study indicates
that our algorithm is much faster than the only so far
existing software PIRNv2.0 [7, 8] for computing the exact
hybridization number for more than two binary phyloge-
netic trees on the same set of taxa. As shown in Fig. 12,
the better performance is not only due to paralleliza-
tion but apparently also due to algorithmic issues and,
presumably, due to the application of certain reduction
rules. Application to a grass dataset Availability and requirements Availability and requirements • Project name: Hybroscale • Project home page: www.bio.ifi.lmu.de/
softwareservices/hybroscale • Operating system(s): Platform independent
• Programming language: Java
• Other requirements: Java 7 or higher
A
b
d Conclusion As already discussed in the work of Albrecht et al. [9], it
makes sense to consider hybridization if there is a signif-
icant difference between certain gene trees and if other
effects, as for example incomplete lineage sorting, could
be excluded. The number of genes affected by hybridiza-
tion, however, is of course not limited to a fixed value, e.g.,
two, and, thus, a method computing hybridization net-
works for an arbitrary number of input trees is of high
interest. Application to a grass dataset set of
representative networks) within 20 minutes
Computing HNumbers Computing HNetworks
Genes
#Taxa HNumber
Runtime
#HNetworks Runtime
ndhf its
46
17
3.262
-
-
ndhf phyB
40
8
0.199
2079
33.035
ndhf rbcl
36
8
0.175
1488
32.1
ndhf rpoc
34
9
0.197
264
5.353
ndhf waxy
19
6
0.179
599
5.693
phyB its
30
8
0.238
195
8.304
phyB rbcl
21
4
0.083
6
1.65
phyB rpoc
21
4
0.091
9
1.678
phyB waxy
14
3
0.071
10
1.615
rbcl its
29
12
4.41
-
-
rbcl rpoc
26
7
0.147
111
3.836
rbcl waxy
12
4
0.126
84
4.338
rpoc its
31
12
4.5
3480
217.575
rpoc waxy
10
2
0.07
1
1.582
waxy its
15
5
0.118
15
2.712
ndhf phyB its
30
13
243.411
-
-
ndhf phyB rbcl
21
9
7.226
-
-
ndhf phyB rpoc
21
8
6.189
36948
206.114
ndhf phyB waxy
14
4
1.599
54
2.87
ndhf rbcl its
28
-
-
-
-
ndhf rbcl rpoc
26
11
7.223
46946
511.296
ndhf rbcl waxy
12
5
4.198
114
5.577
ndhf rpoc its
31
-
-
-
-
ndhf rpoc waxy
10
3
2.583
14
2.632
ndhf waxy its
15
8
4.213
6490
26.697
phyB rbcl its
17
8
9.437
8661
233.768
phyB rbcl rpoc
15
6
4.652
40
4.867
phyB rbcl waxy
7
2
2.568
11
2.592
phyB rpoc its
19
7
3.774
57
4.633
phyB rpoc waxy
5
0
0.045
1
0.075
phyB waxy its
10
4
3.122
204
3.844
rbcl rpoc its
24
-
-
-
-
rbcl rpoc waxy
9
3
1.585
5
1.62
rbcl waxy its
11
6
6.224
63
7.49
rpoc waxy its
10
4
2.626
4
2.635
ndhf phyB rbcl its
17
-
-
-
-
ndhf
phyB
rbcl
rpoc
15
9
224.934
1517
403.728 Computing HNumbers Computing HNetworks
Genes
#Taxa HNumber
Runtime
#HNetworks Runtime et al. [9]. As a consequence, the output usually consists of
a huge number of different hybridization networks, which
is demonstrated by Tables 1 and 2 presenting the results
of our software Hybroscale applied to a well known grass
(Poaceae) dataset3 consisting of three nuclear loci and
three chloroplast genes. This dataset, which is also used
in the work of van Iersel et al. [20], was originally pub-
lished by the Grass Phylogeny Working Group (2001) and
reanalyzed in Schmidt (2003). Additional file Additional file 1: Supplementary material. Supplementary Material
contains Supplementary Figures. Endnotes 1www.bio.ifi.lmu.de/softwareservices/hybroscale
2www.bio.ifi.lmu.de/softwareservices/hybroscale
3www.sites.google.com/site/cassalgorithm/data-sets 1www.bio.ifi.lmu.de/softwareservices/hybroscale
2www.bio.ifi.lmu.de/softwareservices/hybroscale While some approaches only focus on reconciling two
binary phylogenetic X -trees [9, 21], in this article, we
present the algorithm ALLHNETWORKS that is able to
cope with multiple input trees. Moreover, instead of
reporting just the hybridization number or only a small
number of hybridization networks, our approach is based
on the first algorithm that is able to output all represen-
tative networks, which is an important feature enabling Application to a grass dataset Finally, we would like to mention that in the mean-
time we have extended the algorithm ALLHNETWORKS
such that it can be applied to multiple rooted multifur-
cating phylogenetic trees sharing an overlapping set of
taxa. Moreover, to make Hybroscale applicable to larger
input sizes, we have added an option allowing to gener-
ate certain constraints for either limiting the search space
of all representative networks before running our algo-
rithm or to filter the set of reported networks after its
computation. This mechanism was motivated by the pre-
vious work of Kelk et. al [22] suggesting to come up with a
general method for generating these constraints, which is
absolutely meaningful in our point of view. In Fig. 7, one out of 324 possible hybridization networks
reconciling four different binary phylogenetic trees corre-
sponding to the sequences ndhf, rpoC, waxy, and ITS is
given. The embedding of the trees is demonstrated by the
four colors blue, red, green, and orange. This means, for
example, that we can simply determine the embedding of
the tree corresponding to rpoC, which is denoted as Tree 1
in this case, by taking the red colored edges into account. h
l
d
h h b d In Fig. 7, one out of 324 possible hybridization networks
reconciling four different binary phylogenetic trees corre-
sponding to the sequences ndhf, rpoC, waxy, and ITS is
given. The embedding of the trees is demonstrated by the
four colors blue, red, green, and orange. This means, for
example, that we can simply determine the embedding of
the tree corresponding to rpoC, which is denoted as Tree 1
in this case, by taking the red colored edges into account. Moreover, the support values assigned to each hybridiza-
tion node reveal that a hybridization event involving the
two species oryza and lygeum occurs in 97 % of all 324 net-
works, which could be a strong signal that this event is
also part of the true underlying evolutionary history. How-
ever, the reader should be aware of the fact that there still
exist other mechanisms explaining such inconsistencies,
as for example incomplete lineage sorting. Hence, such
networks just help to build hypothesis that still have to be
tested by applying further experiments. References 1. Mallet J. Hybrid speciation. Nature. 2007;446:279–83. 2. Rieseberg LH. Hybridization, introgression, and linkage evolution. Plant
Mol Biol. 2000;42:205–24. 3. Soltis P, Soltis D. The role of hybridization in plant speciation. J Comput
Biol. 2009;60:561–88. 4. Schwenk K, Brede N, Streitl B. Introduction. extent, processes and
evolutionary impact of interspecific hybridization in animals. Phil Trans R
Soc B Biol Sci. 2008;363(1505):2805–811. 5. Bordewich M, Semple C. Computing the minimum number of
hybridization events for a consistent evolutionary history. Discrete Appl
Math. 2007;155:914–28. 6. van Iersel L, Linz S. A quadratic kernel for computing the hybridization
number of multiple trees. Inform Process Lett. 2013;113(9):318–23. 7. Wu Y. Close lower and upper bounds for the minimum reticulate network
of multiple phylogenetic trees. Bioinformatics. 2009;26(12):140–8. p
p y g
;
(
)
8. Wu Y. An algorithm for constructing parsimonious hybridization networks
with multiple phylogenetic trees. J Comput Biol. 2013;20(10):792–804. 9. Albrecht B, Scornavacca C, Cenci C, Huson DH. Fast computation of
minimum hybridization networks. Bioinformatics. 2011;28(2):191–7. l
l
b d
l
l p
p y g
8. Wu Y. An algorithm for constructing parsimonious hybridization networks
with multiple phylogenetic trees. J Comput Biol. 2013;20(10):792–804. p
p y g
p
9. Albrecht B, Scornavacca C, Cenci C, Huson DH. Fast computation of
minimum hybridization networks. Bioinformatics. 2011;28(2):191–7. y
10. Baroni M, Semple C, Steel M. Hybrids in real-runtime. Syst Biol. 2006;55(1):46–56. y
10. Baroni M, Semple C, Steel M. Hybrids in real-runtime. Syst Biol. 2006;55(1):46–56. 11. Bordewich M, Semple C. Computing the hybridization number of two
phylogenetic tress is fixed-parameter traceable. IEEE/ACM Trans Comput
Biol Bioinformatics. 2007;4:458–66. 12. Whidden C, Beiko R, Zeh N. Fast FPT algorithms for computing rooted
agreement forests: Theory and experiments In: Festa P, editor. In:
Proceedings of the 9th International Symposium on Experimental
Algorithms, SEA 2010. Heidelberg, Germany: Springer; 2010. p. 141–53. 13. Albrecht B. Computing hybridization networks for multiple rooted binary
phylogenetic trees by maximum acyclic agreement forests. arXiv:1408.3044. 2014. 13. Albrecht B. Computing hybridization networks for multiple rooted binary
phylogenetic trees by maximum acyclic agreement forests. arXiv:1408.3044. 2014. 14. Huson DH, Rupp R, Scornavacca C. Phylogenetic Networks: Concepts,
Algorithm and Applications. Shaftesbury Road, Cambridge: Cambridge
University Press; 2011. doi:ISBN:9780521755962. 14. Huson DH, Rupp R, Scornavacca C. Phylogenetic Networks: Concepts,
Algorithm and Applications. Shaftesbury Road, Cambridge: Cambridge
University Press; 2011. doi:ISBN:9780521755962. 15. Baroni M, Gruenewald S, Moulton V, Semple C. Competing interests
Th
h
d
l
h Competing interests
The authors declare that they have no competing interests. Page 15 of 15 Albrecht BMC Bioinformatics (2015) 16:236 Albrecht BMC Bioinformatics (2015) 16:236 Albrecht BMC Bioinformatics (2015) 16:236 Authors’ contributions
h
h l
k
d The whole work was done by BA involving the development of the algorithm,
the implementation of the software Hybroscale, the conduction of the
simulation study, and finally drafting the manuscript. Acknowledgements We would like to thank Cuong Van Than and Daniel H. Huson for helpful
discussion about certain theoretical issues regarding the presented algorithm. We also gratefully acknowledge the critical review by an anonymous reviewer
on an earlier version of the manuscript. Received: 15 April 2015 Accepted: 3 July 2015 Received: 15 April 2015 Accepted: 3 July 2015 Received: 15 April 2015 Accepted: 3 July 2015 References Bounding the number of
hybridisation events for a consisten evolutionary history. Math Biol. 2005;51:171–82. 15. Baroni M, Gruenewald S, Moulton V, Semple C. Bounding the number of
hybridisation events for a consisten evolutionary history. Math Biol. 2005;51:171–82. 16. Bordewich M, Semple C. On the computational complexity of the rooted
subtree prune and regraft distance. Ann Combinator. 2005;8:409–23. 16. Bordewich M, Semple C. On the computational complexity of the rooted
subtree prune and regraft distance. Ann Combinator. 2005;8:409–23. 17. Linz S. Reticulation in evolution. Germany: PhD thesis,
Mathematisch-Naturwissenschaftliche Fakultät der
Heinrich-Heine-Universität Düsseldorf; 2008. 17. Linz S. Reticulation in evolution. Germany: PhD thesis,
Mathematisch-Naturwissenschaftliche Fakultät der
Heinrich-Heine-Universität Düsseldorf; 2008. Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of: 18. Scornavacca C, Linz S, Albrecht B. A first step towards computing all
hybridization networks for two rooted binary phylogenetic trees. J Comput Biol. 2010;19(11):1227–42. 19. Cardona G, Rossello F, Valiente G. Extended newick: It is time for a
standard representation of phylogenetic networks. BMC Bioinformatics. 2008;9:532. 19. Cardona G, Rossello F, Valiente G. Extended newick: It is time for a
standard representation of phylogenetic networks. BMC Bioinformatics. 2008;9:532. 20. van Iersel L, Kelk S, Rupp R, Huson DH. Phylogenetic networks do not
need to be complex: using fewer reticulations to represent conflicting
clusters. Bioinformatics. 2010;26(12):124–31. 21. Chen ZZ, Wang L. Hybridnet: a tool for constructing hybridization
networks. Bioinformatics. 2010;26:2912–1913. 21. Chen ZZ, Wang L. Hybridnet: a tool for constructing hybridization
networks. Bioinformatics. 2010;26:2912–1913. 22. Kelk S, Linz S, Morrison DA. Fighting network space: it is time for an
sql-type language to filter phylogenetic networks. arXiv:1310.6844. 2013. 22. Kelk S, Linz S, Morrison DA. Fighting network space: it is time for an
sql-type language to filter phylogenetic networks. arXiv:1310.6844. 2013.
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Beneficial Microorganisms to Control the Gray Mold of Grapevine: From Screening to Mechanisms
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To cite this version: Zakaria Amarouchi, Qassim Esmaeel, Lisa Sanchez, Cédric Jacquard, Majida Hafidi, et al.. Beneficial
Microorganisms to Control the Gray Mold of Grapevine: From Screening to Mechanisms. Microor-
ganisms, 2021, 9 (7), 10.3390/microorganisms9071386. hal-03435746 All content following this page was uploaded by Essaïd Ait Barka on 25 June 2021. The user has requested enhancement of the downloaded file. HAL Id: hal-03435746
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Screening to Mechanisms Beneficial Microorganisms to Control the Gray Mold of Grapevine: From
Screening to Mechanisms Beneficial Microorganisms to Control the Gray Mold of
Grapevine: From Screening to Mechanisms Zakaria Amarouchi 1,2, Qassim Esmaeel 1, Lisa Sanchez 1, Cédric Jacquard 1, Majida Hafidi 2,
Nathalie Vaillant-Gaveau 1 and Essaid Ait Barka 1,* Zakaria Amarouchi 1,2, Qassim Esmaeel 1, Lisa Sanchez 1, Cédric Jacquard 1, Majida Hafidi
Nathalie Vaillant-Gaveau 1 and Essaid Ait Barka 1,* 1 Université de Reims Champagne-Ardenne, RIBP EA4707 USC INRAE 1488, SFR Condorcet FR CNRS 3417,
51100 Reims, France; zakariaamarouchi@gmail.com (Z.A.); qassim.esmaeel@univ-reims.fr (Q.E.);
lisa.sanchez@univ-reims.fr (L.S.); cedric.jacquard@univ-reims.fr (C.J.);
nathalie.gaveau@univ-reims.fr (N.V.-G.) 1 Université de Reims Champagne-Ardenne, RIBP EA4707 USC INRAE 1488, SFR Condorcet FR CNRS 3417,
51100 Reims, France; zakariaamarouchi@gmail.com (Z.A.); qassim.esmaeel@univ-reims.fr (Q.E.);
lisa.sanchez@univ-reims.fr (L.S.); cedric.jacquard@univ-reims.fr (C.J.);
nathalie.gaveau@univ-reims.fr (N.V.-G.) 1 Université de Reims Champagne-Ardenne, RIBP EA4707 USC INRAE 1488, SFR Condorcet FR CNRS 3417,
51100 Reims, France; zakariaamarouchi@gmail.com (Z.A.); qassim.esmaeel@univ-reims.fr (Q.E.);
lisa.sanchez@univ-reims.fr (L.S.); cedric.jacquard@univ-reims.fr (C.J.);
nathalie.gaveau@univ-reims.fr (N.V.-G.) nathalie.gaveau@univ-reims.fr (N.V.-G.)
2 Laboratoire de Biotechnologie Végétale et Valorisation des Bio-Ressources, Faculté des Sciences, Université
Moulay Ismail, Meknès B.P 11201, Morocco; hafidimaj@yahoo.fr
* Correspondence: ea.barka@univ-reims.fr; Tel: +33-326913221 2 Laboratoire de Biotechnologie Végétale et Valorisation des Bio-Ressources, Faculté des Sciences, Université
Moulay Ismail, Meknès B.P 11201, Morocco; hafidimaj@yahoo.fr
* Correspondence: ea barka@univ-reims fr; Tel: +33-326913221 2 Laboratoire de Biotechnologie Végétale et Valorisation des Bio-Ressources, Faculté des Sciences, Université
Moulay Ismail, Meknès B.P 11201, Morocco; hafidimaj@yahoo.fr
* Correspondence: ea.barka@univ-reims.fr; Tel: +33-326913221 2 Laboratoire de Biotechnologie Végétale et Valorisation des Bio-Res
Moulay Ismail, Meknès B.P 11201, Morocco; hafidimaj@yahoo.fr y
j y
* Correspondence: ea.barka@univ-reims.fr; Tel: +33-326913221 Abstract: In many vineyards around the world, Botrytis cinerea (B. cinerea) causes one of the most
serious diseases of aerial grapevine (Vitis vinifera L.) organs. The control of the disease relies mainly
on the use of chemical products whose use is increasingly challenged. To develop new sustainable
methods to better resist B. cinerea, beneficial bacteria were isolated from vineyard soil. Once screened
based on their antimicrobial effect through an in vivo test, two bacterial strains, S3 and S6, were able
to restrict the development of the pathogen and significantly reduced the Botrytis-related necrosis. The photosynthesis analysis showed that the antagonistic strains also prevent grapevines from
considerable irreversible PSII photo-inhibition four days after infection with B. cinerea. The 16S
rRNA gene sequences of S3 exhibited 100% similarity to Bacillus velezensis, whereas S6 had 98.5%
similarity to Enterobacter cloacae. On the other hand, the in silico analysis of the whole genome of
isolated strains has revealed the presence of “biocontrol-related” genes supporting their plant
growth and biocontrol activities. Beneficial Microorganisms to Control the Gray Mold of
Grapevine: From Screening to Mechanisms The study concludes that those bacteria could be potentially useful
as a suitable biocontrol agent in harvested grapevine. Citation: Amarouchi, Z.; Esmaeel,
Q.; Sanchez, L.; Jacquard, C.; Hafidi,
M.; Vaillant-Gaveau, N.; Barka, E.A. Beneficial Microorganisms to
Control the Gray Mold of
Grapevine: From Screening to
Mechanisms. Microorganisms 2021, 9,
1386. https://doi.org/10.3390/
microorganisms9071386
Academic Editor: Fred O. Asiegbu
Received: 3 June 2021
Accepted: 23 June 2021
Published: 25 June 2021 Citation: Amarouchi, Z.; Esmaeel,
Q.; Sanchez, L.; Jacquard, C.; Hafidi,
M.; Vaillant-Gaveau, N.; Barka, E.A. Beneficial Microorganisms to
Control the Gray Mold of
Grapevine: From Screening to
Mechanisms. Microorganisms 2021, 9,
1386. https://doi.org/10.3390/
microorganisms9071386 Keywords: gray mold; biocontrol; grapevine Beneficial Microorganisms to Control the Gray Mold of Grapevine: From
Screening to Mechanisms Article in Microorganisms · June 2021 DOI: 10.3390/microorganisms9071386 DOI: 10.3390/microorganisms9071386
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SEE PROFILE 7 authors, including: Some of the authors of this publication are also working on these related projects: BioScreen Project: New biosourced and multifunctional molecules for the control of phytopathogenic agents in crops in the transboundary region View project All content following this page was uploaded by Essaïd Ait Barka on 25 June 2021. The user has requested enhancement of the downloaded file. Citation: Amarouchi, Z.; Esmaeel,
Q.; Sanchez, L.; Jacquard, C.; Hafidi,
M.; Vaillant-Gaveau, N.; Barka, E.A.
Beneficial Microorganisms to
Control the Gray Mold of
Grapevine: From Screening to
Mechanisms. Microorganisms 2021, 9,
1386. https://doi.org/10.3390/
microorganisms9071386 Citation: Amarouchi, Z.; Esmaeel,
Q.; Sanchez, L.; Jacquard, C.; Hafidi,
M.; Vaillant-Gaveau, N.; Barka, E.A.
Beneficial Microorganisms to
Control the Gray Mold of
Grapevine: From Screening to
Mechanisms. Microorganisms 2021, 9,
1386. https://doi.org/10.3390/
microorganisms9071386
Academic Editor: Fred O. Asiegbu
Received: 3 June 2021
Accepted: 23 June 2021
Published: 25 June 2021 1. Introduction The second stage of perception uses the recognition
of microbial effectors, the virulence factors that suppress MTI to initiate effector-triggered
immunity (ETI), triggering a cascade of complex signaling events, leading to suppression
of pathogen assaults. Botrytis cinerea is one of the highest broadly studied necrotrophic fungal pathogens. B. cinerea has no apparent host specificity infecting therefore, more than 1000 plant species
[9]. The gray mold (GM) caused by B. cinerea has a devastating impact on various
economically important crops, including grape, strawberry, and tomato [10] with annual
economic losses exceeding USD 10 to 100 billion worldwide [9,11,12]. Several disease controlling approaches have been implemented in the past and
present to control B. cinerea. Currently, pesticides remain the main method used to fight
the pathogen, and in some instances, the only option, involving significant financial costs. Until lately, the use of chemical fungicides to protect plant was thought to be fairly safe. Nevertheless, more than ever before, chemical fungicides use faces multiple challenges
namely the development of resistance to fungicides resulting in the decline or even failure
of control effect [13–16], increased consumers desire of food free of pesticide residues [17],
enhanced concern regarding environmental pollution, and stricter regulatory policies are
being imposed on the use of synthetic chemical fungicides [17,18] To overcome the difficulties previously stated, it is urgent to search for alternative,
effective, and eco-friendly strategy of disease control [19–21]. Thus, in recent years, the use
of microbes as a biocontrol agent is gaining interest in agriculture [22]. Plant growth promoting rhizobacteria (PGPR) are bacteria that inhabit the
rhizosphere and can improve the extent or quality of plant growth directly and or
indirectly. The direct promotion by PGPR involves either delivering plant with a plant
growth promoting substances or helping plants to mobilize and acquire nutrients from
the rhizosphere. The indirect effect occurs when PGPR prevent the harmful effect of
pathogens. In the last few decades, bacteria including species of Arthobacter, Azospirillum,
Azotobacter, Bacillus, Burkholderia, Enterobacter, Klebsiella, Pseudomonas, and Serratia have
reported to enhance plant fitness. Various mechanisms for antagonism have been implicated, such as competition for
nutrients and space, secretion of cell wall degrading enzymes, siderophores [23,24],
parasitism of the pathogen, biofilm formation, induction of host defenses via production
of various pathogenesis related proteins (PR) [25], and the involvement of reactive oxygen
species (ROS) in the defense response are responsible for their antagonistic activity [18]. 1. Introduction Pathogens cause a devastating impact on crops varying from economic hardship to
poisoning of food supplies (such as ergotism) and horrendous famines such as the Irish
potato famine that lasted from 1845 to 1852. After their assaults, pathogens might trigger
substantial changes to the host physiology, which can occur directly by secreting toxins
and lytic enzymes or indirectly through inducing host responses stimulated by the
pathogen. Among the significant physiological processes, the photosynthesis is the
principal process affected by foliar diseases [1]. The photosynthesis decline might be
proportional to decrease in green leafy tissue. Furthermore, the decline in photosynthesis,
infections can trigger other physiological changes such as limited water use efficiency,
which in turn, excessive water restriction may further induce a lower rate of
photosynthesis (as reviewd in [2]). The pathogen might also impact the net carbon
assimilation rate by enhancing the leaf respiration, which is requested to supply the
demand initiated by the accelerated cells host metabolic activity [3]. The photosynthesis
decreases through the infection process as a result of repression of photosynthetic gene
expression [4,5]. Despite various struggles to decipher mechanisms by which pathogens
can disturb photosynthetic capacity, current knowledges of the subject remain far from
inclusive. Received: 3 June 2021
Accepted: 23 June 2021
Published: 25 June 2021 Publisher’s
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Attribution
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(http://creativecommons.org/licenses
/by/4.0/). www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, 1386. https://doi.org/10.3390/microorganisms9071386 2 of 19 2 of 19 Microorganisms 2021, 9, 1386 Plants do not have a circulatory system and adaptive immune system like animals. To block pathogen progress, plants have evolved a two-layered innate immune system. The first line of plants defense is accomplished via a set of defined receptors, namely
pattern recognition receptors (PRRs), which able to identify conserved microbe-associated
molecular patterns (MAMPs) [6]. Following MAMPs recognition, MAMP-triggered
immunity (MTI) primary defense responses are triggered including mitogen-activated
protein kinase (MAPK) phosphorylation cascades, cell wall alterations, callose deposition,
defense genes expression, and defense-related proteins accumulation [7,8]. When perceived by intracellular immune receptors, pathogen effectors trigger the
effector-triggered immunity (ETI; [8]). 2.2. Isolation, Purification, and Enrichment of Antagonistic Bacteria The isolation of bacteria was carried out according to the protocol previously
described by Nally et al. [29], with some modifications. Portions of 15 g of Rhizospheric
soils were suspended in 250 mL of sterile Luria-Bertani (LB) liquid medium (tryptone 10
g/L; yeast extract 5 g/L; NaCl 10 g/L; pH 7.2). The enrichment culture was incubated on a
rotary shaker (180 rpm) at 28 °C for 24 h. This operation was repeated for the soil recovered
under the vines showing the GM symptoms. After shaking, the cultures were kept
undisturbed for 30 min. Then, isolation of viable bacterial cultures from soil suspensions
was done by serial dilution plate count in phosphate-buffer saline (PBS 10 mM, pH 6.5). Aliquots of 100 μL from the five different dilutions of the sequential enrichment were
spread in triplicate on LB medium and incubated at 28 °C for 24–72 h until colony
development. Colonies with distinct morphologies were picked and purified using the
streaking method. The purified isolates were used to screen antagonistic bacteria against
B. cinerea. Afterwards, pure bacterial cultures were maintained in cryovials containing LB
broth with 25% glycerol and preserved at −80°C. 1. Introduction PR proteins regulated resistance in response to B. cinerea in grapes. Therefore, nitrogen
and carbon metabolisms play critical roles in the resistance of grapes against B. cinerea [26]. However, the main inhibitory action of antagonistic bacteria is to produce antifungal
metabolites and antibiotics [27,28]. Given the wide-ranging and the importance of B. cinerea in agriculture, the control of
GM is of great concern. In this context, our strategy was to develop new biotechnologies
allowing grapevine to better resist parasitic pressures of B. cinerea through the isolation
and screening of beneficial bacteria. Once screened, the mechanisms contributing to the
biocontrol effect of selected bacteria was deciphered by determining their antimicrobial
effect by in vivo test, and finally chlorophyll fluorescence imaging after Botrytis challenge. On the other hand, the in silico analysis of the whole genome of isolated strains has Microorganisms 2021, 9, 1386 3 of 19 revealed the presence of “biocontrol-related” genes supporting their plant growth and
biocontrol activities. revealed the presence of “biocontrol-related” genes supporting their plant growth and
biocontrol activities. 2.1. Soil Sampling Soil samples were collected from different distinct locations of vineyard (Chardonnay
cv.) in Meknes-Morocco. Sampling was carried out at the end of the growing season, after
the occurrence of the asexual multiplication of B. cinerea and the appearance of symptoms
of gray rot detectable on the grapevine. Although isolation of antagonistic bacterial strains
was taken from soil samples of healthy grapevine, the bacteria coming from soil collected
under the infected plants were selected as control, to compare the communities of both
populations. At each sampling points, soil was collected aseptically from healthy and
severely infected grapevines with B. cinerea. Samples were kept in paper bags placed in
ice and processed within 24 h. 2.5. Biochemical Characterization of Biocontrol Isolates Biochemical characteristics of isolated strains such as carbohydrate assimilation and
fermentation were performed using BIOLOG GENIII microtiter plate (Hayward CA, USA)
as recommended by manufacturers. 2.4. Identification of Antagonistic Bacteria All potential antagonistic bacteria were identified by 16S rRNA gene analysis. The
16S rRNA gene was amplified by polymerase chain reaction (PCR) as previously described
by [30]. Briefly, genomic DNA was extracted from a pure colony using the Wizard
Genomic Purification DNA Kit (Promega Corp., Madison, WI, USA), according to the
manufacturer’s instructions. Next, the bacterial 16S rRNA gene was amplified by PCR
using
FD2
(5-
AGAGTTTGATCATGGCTCAG
-3)
and
RP1(5-
ACGGTTACCTTGTTACGACTT -3) primers. The PCR was carried out with a 50-µL final
volume, containing 25-µL of Master Mix (Thermo Scientific Fermentas, Villebon sur
Yvette, France), 2.5 µL of each primer, 15 µL sterile water, and 5 µL template DNA in a
PTC-200 Thermocycler (C1000 touch thermal cycler, Bio-Rad, Hercules, CA, USA). The
PCR conditions used were as follows: an initial denaturation step at 94 ◦C for 5 min,
followed by 30 cycles of denaturation at 94°C for 45 s, primer annealing at 55 °C for 45 s,
and elongation at 72 °C for 1.5 min, and a final elongation step at 72 ◦C for 10 min. g
g
p
The PCR product was subjected to electrophoresis on agarose gel with 0.5X TAE
buffer (Tris Acetate-EDTA) at a ratio of 1% (weight/volume). Gels were stained with
ethidium bromide, visualized under UV light (300 nm), and were then excised and
purified using the Gene JET Gel Extraction Kit (Thermo Scientific Fermentas, Waltham,
USA), as recommended by the manufacturer. A 1000-bp DNA ladder marker served as
the standard size. The PCR product was commercially sequenced by Genewiz Co., Ltd. (Leipzig, Germany). The data output was analyzed, and the sequences were compared
with sequences in the National Center for Biotechnology Information (NCBI) database
using the BLAST search program (http://www.ncbi.nlm.nih.gov/). Alignment of 16S rRNA
gene sequences from GenBank database was performed using ClustalX 1.8.3 with default
settings [31]. Phylogenesis was analyzed by MEGA version 7. Distances were calculated
using the Kimura two parameter distance model. The tree was built by the neighbor-
joining method. The dataset was boot- strapped 1000 times [32]. 2.3. In Vitro Screening of Potential Antagonistic Bacteria Isolated strains were tested for antifungal activity against B. cinerea. Assays were
performed by patching, in the middle of PDA medium (Sigma-Aldrich, MO, USA) plates,
5 mm of agar plug carrying freshly grown culture of the fungal pathogen. After that, a
volume of 5 µL of suspension of each isolate was drooped at four sites approximately 1
cm from the rim of the plate and incubated at 22 °C for five days at which point we start
measurements. Plates inoculated with B. cinerea were also used as control. The plates were
visually inspected for the presence of inhibition zones between the fungus and the colonies
considered to be potential antagonistic bacteria. The antifungal effect was estimated by
calculating the percentage of inhibition (%) of mycelial growth measured as follows: I(%) = (1 − Cn / Co) × 100
(1) (1) where “Cn” is the average diameter of the mycelial in the presence of the antagonists and
“Co” the average diameter of the control. Experiments were conducted in duplicate and
the results reported are averages of three independent experiments. The diameter of the
clear zones depends on the performance of the bacteria. Thus, the screened strains with
high zones of clearing were selected for molecular identification and in vivo assays. Microorganisms 2021, 9, 1386 4 of 19 2.4. Identification of Antagonistic Bacteria 2.6. Cellular Fatty Acid Analysis Cellular fatty acid analysis was carried out at BCCM/LMG (the Belgian Co-ordinated
Collections of Microorganisms, Ghent University, Ghent, Belgium). Bacterial isolates were
grown for 24 h at 30 °C under aerobic conditions on LB medium. Inoculation and
harvesting of the cells, and the extraction and analysis were performed conform to the
recommendations of the commercial identification system MIDI (Microbial Identification
System, Inc., Newark, DE, USA). The whole-cell fatty acid composition was determined
gas chromatographically on an Agilent Technologies 6890N gas chromatograph (Santa
Clara, CA, USA). The peak naming table MIDI TSBA 5.0 was used. 2.10. Inoculation of In Vitro Plantlets with Antagonistic Bacteria and Infection by B. cinerea Isolates with higher percentage of inhibition (%) during in vitro screening were tested
for antagonistic activity against B. cinerea on sterile grapevine plantlets. Briefly, roots of 6-
weeks-old grapevine plantlets were gently removed from the MS agar medium (Sigma-
Aldrich, France) and transferred into magenta boxes containing 120 g of soil. Plantlets
were then inoculated with bacterial inoculum at a concentration of 108 CFU per g of soil. Control was treated with sterile PBS. Bacterized and non-bacterized plantlets were then
grown for an additional 10 days. Thereafter, the upper side of each leaf was inoculated by
a drop of 5 μL of B. cinerea germinated spore suspension. This protocol was used for
measures of necrosis surfaces. The antifungal effect was estimated by calculating the
percentage of inhibition (%) of mycelial growth measured as describes in in vitro tests. For IMAGING-PAM analysis, plantlets were sprayed with the germinated spore
suspension of B. cinerea to have a homogenous application. Plantlets were then placed in
growth chamber at 22 °C.. All the experiments were performed in triplicate. 2.8. Grapevine In Vitro Plantlets Plantlets of Vitis vinifera cv. Chardonnay (clone 7535) were micro-propagated by
nodal explants grown on 15 mL of Murashige-Skoog (MS) agar medium in 25 mm-culture
tubes as described by Ait Barka et al. [36]. Cultures were performed in a growth chamber
under white fluorescent light (200 µmol/m2/s), with 16 h/8 h day/night photoperiod at a
constant temperature of 26 °C. 2.9. Bacterial Isolates and Inoculum Preparation Bacterial suspensions were prepared by inoculating 100 mL of Luria-Bertani (LB)
liquid culture medium and incubated on a rotary shaker at 180 rpm at 28 °C for 18 h at
which point they reached the late exponential growth phase. After incubation, cells were
harvested by centrifugation at 4500 g at 4 °C for 15 min. Each culture was washed three
times and resuspended in 20 mL of phosphate-buffered saline (PBS). The density of
bacterial cultures was determined by spectrophotometry and adjusted at approximately
108 colony-forming units (CFU) mL-1 with an optical density 0.8 at 600 nm (OD600). For the inoculum preparation, the fungal pathogen, B. cinerea strain 630 was grown
on potato dextrose agar (PDA) (Sigma-Aldrich, MO, USA) at 22 °C. The conidia were
collected from 20-day-old culture plates by scratching the Petri dishes surface with sterile
potato dextrose broth (PDB 24 g/L) medium and filtered to remove hyphae. Conidial
concentrations were measured by a hemocytometer and the final density was adjusted to
105 conidia/mL. After incubation during 3 h at 22 °C and 150 rpm, the resulting germinated
spore suspension was used for plant inoculation. 2.7. Whole Genome Sequencing, Assembly, and Annotation 2.7. Whole Genome Sequencing, Assembly, and Annotation The genomic DNA of strains was isolated using the Wizard Genomic Purification
DNA Kit (Promega Corp., Madison, WI, USA) according to the manufacturer’s
instructions. The integrity of extracted DNA was assessed by running the sample on 1%
agarose gel. The genome sequence of the strain was sequenced at MicrobesNG
(http://www.microbesng.uk) using the method summarized in Table S1. The draft genome
sequences were used for annotation using the Rapid Annotation Subsystem Technology
(RAST) server (http://rast.nmpdr.org) [33]. The annotated genes were analyzed using
SEED database [34]. To predict the presence of secondary metabolites (SMs) gene clusters
associated with biocontrol activity, the draft genome sequence was analyzed by Microorganisms 2021, 9, 1386 5 of 19 5 of 19 antiSMASH software online (https://antismash.secondarymetabolites.org/#!/start) [35]. The Whole Genome Shotgun projects of the strains have been deposited in GenBank under
the accession numbers JAFETM000000000 and JAFETL000000000. The version described
in this paper version JAFETM010000000 and JAFETL010000000, respectively. antiSMASH software online (https://antismash.secondarymetabolites.org/#!/start) [35]. The Whole Genome Shotgun projects of the strains have been deposited in GenBank under
the accession numbers JAFETM000000000 and JAFETL000000000. The version described
in this paper version JAFETM010000000 and JAFETL010000000, respectively. 2.12. Statistical Analysis The experimental design used was performed in triplicates. Statistical analyses were
carried out using GraphPad Prism version 5.00 for Windows (GraphPad Software, San
Diego, CA, USA) (www.graphpad.com). For lesion diameter Student’s t-tests (α > 0.05)
was used to compare lesion area between inoculated and non-inoculated plants. 2.11. IMAGING-PAM Analysis Chlorophyll fluorescence parameters were measured with an IMAGING-PAM
measuring system (Heinz Walz, Germany) using the saturation pulse method [37]. Control
and bacterized plantlets were dark adapted for 30 min to establish the minimal
fluorescence level (F0) and the maximal fluorescence (Fm) after a saturating flash (1 s;
13,000 µmol/m2/s). Each leaf was detached from the plantlet then exposed immediately to
an actinic illumination of 79 µmol/m2/s. After fluorescence stabilization, a second
saturating flash was imposed to determine the maximal fluorescence (Fm’) of light-
adapted inflorescences. The effective PSII quantum yield, ΦPSII, is calculated according to
the equation of Genty et al. [38]. The quantum yield of regulated energy dissipation in Microorganisms 2021, 9, 1386 6 of 19 6 of 19 PSII, ΦNPQ, and the quantum yield of nonregulated energy dissipation in PSII, ΦNO, was
calculated according to Kramer et al. (2004) [39]. Please note that ΦPSII + ΦNPQ + ΦNO =
1. The data were collected taking in consideration the entire leaf area including necrosis
area. Measurements were taken 24 h before inoculation of antagonistic bacteria, 24 h
before infection with B. cinerea, and 4 consecutive days after infection. The means ±
standard deviations originated from three independent experiments realized in
duplicates, each replicate consisted of four plantlets. 3.1. Isolation and In Vitro Screening of Antagonistic Bacteria As a result of multiple inoculations and purification, 42 pure cultures of potential
antagonistic bacteria were successfully enriched and isolated from vineyard soil. These
freshly isolated strains were purified on LB plates, selected based on their morphology,
and used as objects of investigation. All isolates were screened for their ability to inhibit
the mycelial growth of B. cinerea by direct confrontation tests in PDA medium plates. Out
of 42 tested strains, only two isolates were estimated as antagonistic potential bacteria
against this fungus. They were nominated S3 and S6 which were the most active against
fungal culture, by showing the strong percentage of inhibition I (%) (Figure 1A). These
isolates were tested for their ability to protect grapevines against GM in planta. 3.2. Disease Symptoms were Significantly Reduced in Root-Bacterized Plantlets To test the ability of S3 and S6 to protect grapevine in our system, we performed
infection on whole potted-plant with B. cinerea strain 630 in control versus root-bacterized
plantlets. Assays performed showed that the two antagonistic bacteria had high inhibiting
ability against B. cinerea in grapevines three days after inoculation of the pathogen. They
significantly reduced Botrytis-related necrosis by approximately 50% at 72 hpi (Figure 1B). In addition, disease symptoms were significantly less developed in bacterized plants,
confirming the protective impact of S3 and S6 against B. cinerea (Figure 1C). Non-bacterized
plantlets showed severe symptoms typical of GM, manifesting as necrosis around the
infection spot (Figure 1C). In contrast, plants treated with the antagonistic bacteria
exhibited a reduction in disease symptoms, displayed by a smaller size of necrosis diameter
compared to the control. These isolates were selected for molecular investigation. 7 of 19 Microorganisms 2021, 9, 1386 Percentage of inhibition I(%)
0%
10%
20%
30%
40%
Bacillus velezensis
Enterobacter cloacae
S3 S6
A
C
B Figure 1. Impact of isolates on B. cinerea growth inhibition. Direct confrontation tests of isolates against B. cinerea
(A). Results indicated are the mean of percent inhibition of mycelial growth of B. cinerea after 72 h of incubation
in PDA medium (A). The Ability of isolate to protect grapevines (cv. Chardonnay), in planta, against B. cinerea (B, C). In
vitro grapevine plantlets inoculated or not with S3 and S6, 72 hours post infection (hpi) with B. cinerea (B). (Control, a; B. cinerea, b; S3 + Bc, c; S6 + Bc, d). Diameter of necrosis measured on leaves infected with B. cinerea (C). Percentage of inhibition I(%)
a
b
c
d
0%
10%
20%
30%
40%
Bacillus velezensis
Enterobacter cloacae
S3 S6
Bc S3+Bc S6+Bc
Diameter of necrosis in cm2
0.1
0.5
0.0
1.5
A
C
B a A b B b c c us ve
S3 C sbacte
S6 S3 S6
Bc S3+Bc S6+Bc
Diameter of necrosis in cm2
0.1
0.5
0.0
1.5
C Figure 1. Impact of isolates on B. cinerea growth inhibition. Direct confrontation tests of isolates against B. cinerea
(A). Results indicated are the mean of percent inhibition of mycelial growth of B. cinerea after 72 h of incubation
in PDA medium (A). The Ability of isolate to protect grapevines (cv. Chardonnay), in planta, against B. cinerea (B, C). 3.2. Disease Symptoms were Significantly Reduced in Root-Bacterized Plantlets In
vitro grapevine plantlets inoculated or not with S3 and S6, 72 hours post infection (hpi) with B. cinerea (B). (Control, a; B. cinerea, b; S3 + Bc, c; S6 + Bc, d). Diameter of necrosis measured on leaves infected with B. cinerea (C). 3.3. Identification of Antagonistic Bacteria The molecular identification using 16S rRNA gene sequences of the antagonistic
rhizobacteria show that S3 and S6 are closely related to Bacillus velezensis (100%), and
Enterobacter cloacae (98.5%), respectively (Figure 2). Strains S3 and S6 also shared sequence
identity with other species including B. subtilis (99%), and Pantoea agglomerans (98%),
respectively (Figure 2). Both bacterial strains (S3 and S6) isolated under healthy canopy,
were used for further characterization. In addition, to calculate the pair-wise average
nucleotide identity (ANI) values of both strains with their closest known relatives, the
draft genome sequences of strains S3 and S6 were compared against all type strain
genomes available in the microbial genomes atlas (MiGA) webserver [40]. Results showed
that strains S3 belongs to Bacillus velezensis (99% ANI). For strain S6, the closest relatives
found was Enterobacter cloacae (98 % ANI). Moreover, digital DDH values of both strains
were compared against all type strain genomes available in the TYGS database [41]. The
analysis generated DDH values more than 70% % for both strains and the closet relatives
were Bacillus velezensis and Enterobacter cloacae for strains S3 and S6, respectively. 8 of 19 Microorganisms 2021, 9, 1386 Figure 2. Neighbor-joining phylogenetic tree based on the 16SrRNA sequences of antagonistic isolates S3, and S6. showing
the relationship with the genus Bacillus (A) and Enterobacter (B). The sequence of B. licheniformis NBRC 12200 and Aeromonas
hydrophila ATCC 7966 were chosen as an out-group. Antagonistic strains are shown in bold. 3.4. Characterization of Biocontrol Isolates
A
B A A B B Figure 2. Neighbor-joining phylogenetic tree based on the 16SrRNA sequences of antagonistic isolates S3, and S6. showing
the relationship with the genus Bacillus (A) and Enterobacter (B). The sequence of B. licheniformis NBRC 12200 and Aeromonas
hydrophila ATCC 7966 were chosen as an out-group. Antagonistic strains are shown in bold. 3.4. Characterization of Biocontrol Isolates The data presented in Table 1 showed some biochemical characteristics of isolated
strains. The selected antagonistic isolates (S3 and S6) were characterized by biochemical
methods. The optimum growth conditions of isolates are at 28°C, pH 6.0, and in the
presence of 1% NaCl. Although cells of strain S3 were sensitive to fusidic acid,
minocycline, naldixic acid, rifamycin SV, lncomycin, and vancomycin, strain S6 was
resistant to them. Isolates were able to grow in the presence of sodium butyrate, guanidine
HCl, lithium chloride, potassium tellurite, and tetrazolium violet, but did not grow in the
presence of sodium bromate, D-serine, and niaproof 4 except E. cloacae, which was
resistant (Table 1). The two isolates failed to hydrolyze gelatin. Furthermore, the strains
showed different abilities to use different carbon sources (Table 1). They were able to
assimilate, D-cellobiose, mannose, mannitol, and N-acetyl-glucosamine, L-glutamic acid,
sucrose, L-aspartic acid, D-maltose, D-fucose (except B. velezensis S3), and L-histidine, as Microorganisms 2021, 9, 1386 9 of 19 9 of 19 sole carbon sources whereas, 3-methylglucose, α-ketobutyric acid, and D-aspartic acid
were not used by both strains. The isolates exhibited different patterns of cellular fatty
acids profile features characterized by different level of C15: 0 iso-anteiso, C17: 0 iso-
anteiso, summed feature 2 (comprising C12: 0 aldehyde, C14: 0 3-OH/ C16: 1 iso I and/or
unknown ECL 10.928), C18:1 ω7c, C16: 0, C17:0 cyclo, C14:0, summed feature 3
(comprising C16:1 ω7c/15 iso 2OH), C12: 0, and C19: 0 cyclo ω8c. Isolate S3 was
characterized by a fatty acid profile dominated to an unusual extent (> 98%) by saturated
fatty acids (Figure S1). Hence, cells had less iso odd-numbered fatty acid and more anteiso
odd-numbered fatty acid, with the major fatty acid being anteiso-C15:0(37.18%) (Table 1). Cells of strain S6 exhibited only 37,88% of saturated fatty acids but revealed other structure
as branched chain (29,51%), cyclopropane (16,90%), and hydroxy unsaturated (14.68%)
fatty acids that was deficient in strain S3 (Figure S2). The major fatty acid for S6 were C16:0
(18.07%) (Table 2). Table 1. Biochemical characteristics for the antagonistic strains based on BIOLOG GENIII microtiter plate
(Hayward CA, USA). 3.4. Characterization of Biocontrol Isolates Oxidation of
Oxidation of
Oxidation of
S6
S3
S6
S3
S6
S3
3-methylglucose
-
-
D-melibiose
+
+
L-lacticacid
+
+
Aceticacid
+
-
D-raffinose
+
-
L-malicacid
+
+
acetoaceticacid
-
-
D-saccharicacid
+
±
L-pyroglutamicacid
±
±
bromo-succinicacid
+
-
D-salicin
±
-
L-rhamnose
+
-
Citricacid
+
±
D-serine
-
-
L-serine
+
-
D-arabitol
±
-
D-sorbitol
+
+
Methylpyruvate
+
±
D-asparticacid
-
-
D-trehalose
+
+
mucicacid
+
±
D-cellobiose
+
+
D-turanose
±
±
myo-inositol
+
±
Dextrin
±
+
Formicacid
±
-
N-acetylneuraminicacid
-
±
D-fructose
+
+
Gelatin
-
-
N-acetyl-D-galactosamine
+
±
D-fructose-6-PO4
+
±
Gentiobiose
+
-
N-acetyl-D-glucosamine
+
+
D-fucose
±
-
Glucuronamide
+
+
N-acetyl-β-D-mannosamine
+
+
D-galactose
+
-
Glycerol
+
±
Pectin
±
±
D-galacturonicacid
+
+
Glycyl-L-proline
+
-
p-hydroxyphenylaceticacid
±
-
D-gluconicacid
+
+
Inosine
+
±
propionicacid
-
-
D-glucose-6-PO4
+
±
L-alanine
+
+
Quinicacid
-
±
D-glucuronicacid
+
+
L-arginine
±
+
Stachyose
+
-
D-lacticacidmethylester
±
±
L-asparticacid
+
+
Sucrose
+
+
D-malicacid
-
-
L-fucose
±
-
Tween 40
-
±
D-maltose
+
+
L-galactonicacid lactone
+
+
Α-D-glucose
+
+
D-mannitol
+
+
L-glutamicacid
+
+
D-mannose
+
+
L-histidine
+
+
Growth in the presence of
S6
S3
Growth in the presence of
S6
S3
Growth in the presence of
S6
S3
1% Nacl
+
+
Lincomycin
+
-
Rifamycin SV
+
-
4% Nacl
+
+
Lithiumchloride
+
+
Sodium bromate
-
-
8% Nacl
±
±
Minocycline
±
-
Sodium butyrate
+
+
1% sodium lactate
+
+
Nalidixicacid
±
-
Tetrazoliumblue
+
-
Aztreonam
±
-
Niaproof 4
+
-
Tetrazolium violet
+
±
D-serine
-
-
pH 5
±
-
Troleandomycin
+
-
Fusidicacid
±
-
pH 6
+
+
Vancomycin
+
-
Guanidinehcl
+
+
Potassium tellurite
+
+ Table 1. Biochemical characteristics for the antagonistic strains based on BIOLOG GENIII microtiter plate
(Hayward CA, USA). emical characteristics for the antagonistic strains based on BIOLOG GENIII microtiter plate
USA). Table 1. Biochemical characteristics for the antagonistic strains based on BIOLOG GENIII microtiter plate
(Hayward CA, USA). 10 of 19 Microorganisms 2021, 9, 1386 Table 2. Major cellular fatty acid content of S3 and S6 strains. 3.4. Characterization of Biocontrol Isolates Structure
Fatty acid
Systematic name Saturated
% In isolated strains
S3
S6
Saturated
C12: 0
Dodecanoic
4.23
7.52
C13: 0
Tridecanoic
-
1.68
C13: 0 ANTEISO
0.40
-
C14: 0
Tetradecanoic
1.01
8.23
C14: 0 ISO
1,11
-
C15: 0 ISO
11.95
-
C15: 0 ANTEISO
37.18
-
C16: 0
Hexadecanoic
17.16
18.07
C16: 0 ISO
2.41
-
C17: 0
Heptadecanoic
0.57
2.38
C17: 0 ISO
9.44
-
C17: 0 ANTEISO
11.64
-
C18: 0
Octadecenoic
1.21
-
Hydroxy
Unsaturated
C15: 0 3-OH
3- Hydroxy- pentadecenoic
-
0.58
C16: 1 ω 5c
cis-11-Hexadecenoic
-
-
C16: 1 ω 11c
1.69
-
C17:1 ω 8c
-
0.60
C18: 1 ω 7c
cis-11- Octadecenoic
-
13.49
Cyclopropane
C17: 0 cyclo
Cyclo-heptadecanoic
-
15.04
C19: 0 cyclo ω 8c
9-(2-eptylcyclopropyl) Nonanoic
-
1.86
Branched chain
Summed feature 1
C 15:1 ISO H/ C 13:0 3OH C 15:1 ISO I/ C 13:0 3OH
-
3.28
Summed feature 2
C12: 0 aldehyde, C 16:1 ISO I/ C 14:0 3OH and/or unknown ECL
10.928
-
17.85
Summed feature 3
C 16:1 ω7c/ 15 iso 2OH
-
8.38 Table 2. Major cellular fatty acid content of S3 and S6 strains. 3.5. Genomic Feature and In Silico Analysis 3.5. Genomic Feature and In Silico Analysis As summarized in Table 3, the draft genome sequence of the strain S3, assembled into
21 contigs, consists of 4,157, 680p with a 46.35% G+C. Strain S6, grouped into 30 contigs
exhibited genome size of 4, 604, 658bp, and the GC content was 55.90%. The total number
of predicted protein-coding sequences and RNAs was 4316 and 98 of RNAs for S3 versus
4228 and 116 of RNAs in S6, respectively. The genome characteristics of each isolate were
detailed in Table 3. The predicted coding sequences (CDS) of S6 were classified into 560
subsystems, most of which were involved in amino acids and derivatives synthesis,
carbohydrate and protein metabolism, cofactors, vitamins, prosthetic groups and pigment
formations, and stress response (Figure S2). Based on phylogenic analysis, chemical
characteristics, and genotypic data described in this report, the isolate S3 belongs to B. velezensis while S6 is attributed to E. cloacae species. The draft genome sequence of each
isolate was subject to multiple genomic analyses with the aim to identify all the genes
potentially responsible for its antimicrobial activity especially those produced by non-
ribosomal peptides synthetases (NRPSs). The in silico analysis using antiSMASH server
revealed the presence of different secondary metabolites gene clusters (SMGCs) involved
in biocontrol. The draft genome of S3 predicted the presence of many SMGCs, including
bacillibactin, fengycin, surfactin and bacillaene. In our analysis of S6 genome, we detected
the presence of non-ribosomal peptide (NRPs) and bacteriocin. Microorganisms 2021, 9, 1386 11 of 19 Table 3. Genomic future and in silico analysis of draft genome sequences of bacterial isolates. Attribute
S3
S6
Size (bp)
4.15 Mb
4.60 Mb
G+C content (%)
46.35
55.90
RNA genes
98
101
Protein-coding genes
3983
4258
N50
959,830
319,447
L50
2
4
Number of Subsystems
328
560
Most frequently species
B. velezensis
E. cloacae
Number of contigs
21
30
3.6. Antagonistic Strains Prevent Plants from Considerable Photo-Inhibition of PSII after
Pathogen Challenge Table 3. Genomic future and in silico analysis of draft genome sequences of bacterial isolates. 3.6. Antagonistic Strains Prevent Plants from Considerable Photo-Inhibition of PSII after
Pathogen Challenge To evaluate the effect of root inoculation with B. velezensis S3 and E. cloacae S6 on
photosynthesis before and 24, 48, 72, and 96 h post-infection with B. cinerea, changes in
excitation flux at PSII were monitored. 3.5. Genomic Feature and In Silico Analysis Photosynthetic parameters including effective PSII
quantum yield Y(II), quantum yield of nonregulated energy dissipation Y(NO), quantum
yield of regulated energy dissipation Y(NPQ), and maximum PSII quantum yield (Fv/Fm)
were evaluated. The false-color scales shown at the bottom of the fluorescence images
indicate the amplitude of the particular parameter (Figure 3a). Before infection with the
pathogen, no significant difference between bacterized and non-bacterized plantlets was
observed regarding the monitored photosynthetic parameters. The value Fv/Fm were
around 0.8 before the infection (Figure 3b). Furthermore, no significant fluctuation was
occurred in Fv/Fm value in plantlets during 3 days after infection. However, considerable
PSII photo-inhibition was observed when plants were not treated with bacteria and were
exposed to a prolonged infection with B. cinerea (Figure 3b), while at the same time, in
response to B. cinerea, bacterized plantlets exhibit indistinct symptoms (Figure 3a), and
Fv/Fm value remain constant during kinetics (Figure 3b). Best results were displayed by
the two strains. In the case of bacterized plantlets, the higher effective quantum yield of
photochemical energy conversion in PSII Y(II) was maintained before as well at 96 hpi
with B. cinerea in comparison to the non-bacterized plant, which decreased. The quantum
yield of regulated energy n PSII Y(NPQ) was down-regulated before infection, compared
to control. In contrast, four days after infection, the bacterized plantlets dissipated a higher
Y(NPQ) than control (Figure 3c). Although no difference in the quantum yield of
nonregulated energy loss in PSII Y(NO) was observed between plantlets before infection,
this response resulted in a lower Y(NO) in bacterized plantlets after a prolonged infection
with B. cinerea. 12 of 19 Microorganisms 2021, 9, 1386 Figure 3. B. velezensis S3 and E. cloacae S6 prevent grapevines from PSII photo-inhibition four days after infection with B. cinerea. Images of the effective PSII quantum yield Y(II), the quantum yield of regulated energy dissipation Y(NPQ) and
of nonregulated energy dissipation Y(NO), from grapevine leaves inoculated or not with B. velezensis S3 and E. cloacae S6
0, 24, 48, 72, and 96 hpi with B. cinerea. The pixel value display is based on a false-color scale ranging from black (0.000) via
red, yellow, green, blue, to purple (ending at 1.00). The figure shows representative images of one from two independent
experiments (A). Evolution of the maximum PSII quantum yield (Fv/Fm) from grapevine leaves inoculated or not with B. velezensis S3 and E. Y(II)
Y(NPQ)
Y(NO) Figure 3. B. velezensis S3 and E. cloacae S6 prevent grapevines from PSII photo-inhibition four days after infection with B. cinerea. Images of the effective PSII quantum yield Y(II), the quantum yield of regulated energy dissipation Y(NPQ) and
of nonregulated energy dissipation Y(NO), from grapevine leaves inoculated or not with B. velezensis S3 and E. cloacae S6
0, 24, 48, 72, and 96 hpi with B. cinerea. The pixel value display is based on a false-color scale ranging from black (0.000) via
red, yellow, green, blue, to purple (ending at 1.00). The figure shows representative images of one from two independent
experiments (A). Evolution of the maximum PSII quantum yield (Fv/Fm) from grapevine leaves inoculated or not with B. velezensis S3 and E. cloacae S6 0, 24, 48, 72, and 96 hpi with B. cinerea (B). Changes in chlorophyll fluorescence parameters
(Y(II), Y(NO), and Y(NPQ)) from grapevine leaves inoculated or not with B. velezensis S3 and E. cloacae S6 0, 24, 48, 72, and
96 hpi with B. cinerea. Excitation flux at PSII in infected leaves (C). Values shown are means ± SD of two independent
repetitions (each repetition was realized in triplicates). 3.5. Genomic Feature and In Silico Analysis cloacae S6 0, 24, 48, 72, and 96 hpi with B. cinerea (B). Changes in chlorophyll fluorescence parameters
(Y(II), Y(NO), and Y(NPQ)) from grapevine leaves inoculated or not with B. velezensis S3 and E. cloacae S6 0, 24, 48, 72, and
96 hpi with B. cinerea. Excitation flux at PSII in infected leaves (C). Values shown are means ± SD of two independent
repetitions (each repetition was realized in triplicates). Control
Control + Bc 630
B. velesenzis + Bc 630
E. cloacea + Bc 630
YII
YNPQ
YNO
YII
YNPQ
YNO
YII
YNPQ
YNO
YII
YNPQ
YNO
24hPI
72hPI
96hPI
0%
20%
40%
60%
80%
100%
Control + Bc
B. velezensis+ Bc
E. cloacae+ Bc
Control + Bc
B. velezensis+ Bc
E. cloacae+ Bc
Control + Bc
B. velezensis+ Bc
E. cloacae+ Bc
Control + Bc
B. velezensis+ Bc
E. cloacae+ Bc
Control + Bc
B. velezensis+ Bc
E. cloacae+ Bc
0 hpi
24 hpi
48 hpi
72 hpi
96 hpi
Y(II)
Y(NPQ)
Y(NO)
0.5
0.55
0.6
0.65
0.7
0.75
0.8
0
24
48
72
96
Fv/Fm
Hours after infection
Control + Bc
B. velezensis + Bc
E. cloacae + Bc
0
1
A
B
C Control
Control + Bc 630
B. velesenzis + Bc 630
E. cloacea + Bc 630
YII
YNPQ
YNO
YII
YNPQ
YNO
YII
YNPQ
YNO
YII
YNPQ
YNO
24hPI
72hPI
96hPI
A A 0%
20%
40%
60%
80%
100%
Control + Bc
B. velezensis+ Bc
E. cloacae+ Bc
Control + Bc
B. velezensis+ Bc
E. cloacae+ Bc
Control + Bc
B. velezensis+ Bc
E. cloacae+ Bc
Control + Bc
B. velezensis+ Bc
E. cloacae+ Bc
Control + Bc
B. velezensis+ Bc
E. cloacae+ Bc
0 hpi
24 hpi
48 hpi
72 hpi
96 hpi
1
C 0.5
0.55
0.6
0.65
0.7
0.75
0.8
0
24
48
72
96
Fv/Fm
H
ft
i f
ti
0
B 1 C B 4. Discussion Rhizosphere is an extremely competitive environment, where diverse genera of
microorganisms are constantly competing for resources and with each other to survive
[42]. This work was undertaken to screen efficient competitive strains from vineyard to
control grapevine GM. Among 42, two strains (S3 and S6) have been showed the best
performance against the B. cinerea. When plantlets were previously bacterized with S3 or
S6 then infected with the B. cinerea, the symptoms of gray mold were reduced compared
to control, confirming therefore the beneficial effect of these strains as reported in in vitro
confrontation test. Similar results were reported by Miotto-Vilanova et al., [37] when
grapevine plantlets were previously bacterized with Burkholderia phytofirmans PsJN before
infection with B. cinerea. Microorganisms 2021, 9, 1386 13 of 19 13 of 19 The 16S rRNA gene sequences of S3 exhibited 100% similarity to B. velezensis, while
the strain S6 showed 98.5% similarity to E. cloacae. B. velezensis and E. cloacae have been
frequently reported as plant growth promoting bacteria and/or biocontrol agents [43–50]. The fact that many Bacilus species have very close phenotypic and physiological properties
as well as 16S rRNAs gene sequences makes their classification very difficult [51]. The
Bacillus genus encompasses a large genetic biodiversity [52], and in addition to the
“original members” B. subtilis, B. licheniformis, and B. pumilus, earlier described by Gordon
et al. [53], many novel species belonging to the B. subtilis species complex have been
described in recent decades, among them B amyloliquefaciens [54], B. velezensis [55], and B. methylotrophicus [56]. Recently, these Bacillus species have been reclassified by genome
comparisons and phylogenomic analyses [57]. In particular, B. methylotrophicus and B. amyloliquefaciens subsp. plantarum were later confirmed as heterotypic synonyms of B. velezensis [58]. Hence, many studies have suggested that B. amyloliquefaciens subsp. Plantarum, B. methylotrophicus, and B. velezensis formed a monophyletic group [59,60]. y
p
p y
g
p
The plant growth-promoting ability has been related to distinctive physiological
activities and molecular changes that might have an intense impact on the fitness (growth
and/or health) of plants. Both B. velezensis S3 and E. cloacae S6 have triggered the resistance
of grapevine toward GM disease. B. velezensis is a heterotypic synonym of B. methylotrophicus, B. amyloliquefaciens subsp. plantarum, and B. oryzicola, and is used to
control plant fungal diseases [61] such as B. cinerea [62–64]. 4. Discussion Bacillus species are promising
agent for the biological control of postharvest diseases [52], particularly B. velezensis,
which is widely used in agriculture [65]. Additionally, Morales-Cedeño et al. [66] showed
that B. velezensis BLE7 showed similar activities to thiabendazole, a commonly used
fungicide for B. cinerea [67]. In cells of B. velezensis, the composition of iso and anteiso fatty
acids was higher. The a-C15:0 became even more prominent component of the fatty acids. Furthermore, the major changes observed were a sharp decrease in a-C17:0 content in
parallel with a significant increase in a-C15:0. The shift to a fatty acid profile dominated
by a-C15:0 draws attention to the critical role of this fatty acid in low temperatures (4 °C)
growth [68], presumably due to its physical properties and their effects in maintaining a
fluid, liquid-crystalline state of membrane lipids [69], making this bacterium a potential
effective biocontrol agent in extreme environments. Enterobacter cloacae is perhaps best known as an opportunistic human pathogen that
is commonly found in hospitals causing a wide range of infections, although some lineages
have also been described as plant endophytes [49]. Indeed, several Enterobacter species can
colonize internal plant tissues, improve plant growth and prevent from pathogens attacks
[70–74]. Thus, Entrobacter cloacae was found to halt fungal phytopathogens growth such as
Phytium debaryanum by 35.13% and Rhizoctonia solani with pathogen growth inhibition up
to 60% [75]. Additionally, E. cloacae inhibited the development of Pythium myriotyum,
Gaeumannomyces graminis and Heterobasidion annosum [52]. y
g
The observed inhibition is due to production of several antifungal metabolites such
as H2S, ammonia and volatile compounds such as phenylethyl alcohol, 4,5-dimethy l-1-
hexene, and butyl acetate that halt the growth of fungal phytopathogens [50,76]. Recently,
it has been reported that E. cloacae is able to produce inorganic volatile substances such as
ammonia, IAA and hydroxamate siderophore, hydrogen cyanide and salicylic acid [74,77],
in addition to chitinase, cellulase, and beta-glucosidase enzyme all of which may
participate to the biocontrol activity [73,78–80]. Furthermore, Chaouachi et al. [81]
reported for the first time volatile organic compounds (VOCs) with antifungal activity
produced by E. cloacae against B. cinerea decay on tomato fruit. Similarly, Bacillus species
such as B. velezensis [63,82] are known to produce antifungal VOCs against several
phytopathogens including B. cinerea. In addition, Patel et al. [83] and Agbodjato et al. 4. Discussion [84] reported that AIA with ammonia
production prevents the development of various plant pathogenic fungi and enhance the
plant growth. Additionally, solubilization of phosphate and potassium occurs due to the
production of protons and oxalic, tartaric acid and polysaccharidic capsules by bacteria Microorganisms 2021, 9, 1386 14 of 19 14 of 19 [76,85]. Romero et al. [86] have reported that antimicrobial compounds produced by
Bacillus spp. are mainly classified into two categories: ribosome-synthesized peptides such
as bacteriocin, and small microbial peptides enzymatically synthesized by non-ribosomal
pathways, mainly cyclic lipopeptides (CLPs). Since B. velezensis is not pathogenic to
humans, different strains of B. velezensis, which is a typical PGPR, have received significant
attention in the last decade. [76,85]. Romero et al. [86] have reported that antimicrobial compounds produced by
Bacillus spp. are mainly classified into two categories: ribosome-synthesized peptides such
as bacteriocin, and small microbial peptides enzymatically synthesized by non-ribosomal
pathways, mainly cyclic lipopeptides (CLPs). Since B. velezensis is not pathogenic to
humans, different strains of B. velezensis, which is a typical PGPR, have received significant
attention in the last decade. So far, B. velezensis was described to halt the growth of many pathogenic fungi, such
as Aspergillus flavus [87], Cylindrocladium quinqueseptatum, Cryphonectria parasitica and
Helicobasidium purpureum [88], Fusarium oxysporum and Ralstonia solanacearum [89], and
Rhizoctonia solani AG1-IB [90], by the biosynthesis of β-1,3-1,4-glucanase, lipopeptide
antibiotics (surfactin, iturin, and fengycin, for example), polyketides (actinomycin D,
bacitracin, and cyclosporin A, for example), siderophores, and NH3 [44,91–93]. These data
are in line with our in silico analysis of both bacteria B. velezensis S3 and E. cloacae S6. Thus,
analysis of E. cloacae S6 genome pointed out the presence of non-ribosomal peptide
synthetase (NRPS) and bacteriocin, while the genome of strain B. velezensis S3 revealed
different SMGCs, such as bacillibactin, fengycin, surfactin and bacillaene. Hence, it was
hypothesized that these strains might produce a variety of antifungal compounds that
participate in the control of the grapevine GM disease. p
p
g
p
The photosynthesis provides near 90–95 % of plants dry matter and the metabolic
energy needed for plant’s development. Pathogen attack not only impact plant defenses
reactions but can also lead to changes in the rate of photosynthesis and therefore the
carbohydrates metabolism [37]. 4. Discussion Hence, four days after infection, it appears that under
Botrytis exposure, thermal dissipation in control was down-regulated not due to an
increased PSII quantum efficiency, but due to an increased nonregulated energy loss in
PSII, suggesting that both photochemical energy conversion and protective regulatory
mechanism were insufficient [94]. Consequently, the larger portion of absorbed light
energy is allocated to nonregulated energy loss in PSII [95]. The latter parameter indicates
an irreversible damage of photosynthetic apparatus as confirmed by the decreased Fv/Fm
ratio, since the Y(NO) leads to the formation of singlet oxygen via the triplet state of
chlorophyll (3chl*) [96,97]. In line with our results, several reports on photosynthesis have
indicated that photosynthesis rates are altered after infection with several plant pathogens
[4,95]. We also have observed that the regulation of mechanisms involved in
nonphotochemical dissipation of energy was blocked, making grapevine plantlets unable
to protect themselves against damage from excessed illumination [37]. A significant
decline of effective quantum yield of PSII(Y(II)) complemented by a quantum yield of
regulated energy dissipation nonphotochemical chlorophyll fluorescence quenching
[Y(NPQ)] increase was observed after the B. cinerea infection. The [Y(NPQ)] is a molecular
adaptation that represents the fastest response of the photosynthetic membrane to excess
light. It is a protective process in which excess absorbed light energy is dissipated into heat
[98,99] and prevent the photosynthetic apparatus from oxidative damage [100]. However,
the irreversible damages described above were prevented/attenuated when grapevine
plantlets were bacterized either with B. velezensis S3 and E. cloacae S6, probably by
restricting mycelial development, and therefore protecting photosynthesis apparatus. Therefore, finding safe and eco-friendly alternatives to synthetic fungicides is urgently
needed to control postharvest diseases of fruit [101]. Hence, this study provided new
biotechnologies by reporting for the first time that following root inoculation, B. velezensis
S3 and E. cloacae S6 allowing grapevine to better resist aerial parasitic pressures of B. cinerea while at the same time prevent grapevine from considerable photo-inhibition. 5. Conclusions and Z.A.; writing—review and editing, E.A.B., Q.E., N.V.-
G., Z.A., and M.H.; supervision, E.A.B., N.V.-G., C.J., and M.H.; project administration, E.A.B., N.V.-
G. and M.H.; funding acquisition, E.A.B., N.V.-G. and M.H. All authors have read and agreed to the
published version of the manuscript. Author Contributions: Conceptualization, E.A.B., N.V.-G. and M.H.; methodology, E.A.B., N.V.-G.,
M.H., L.S. and Q.E.; software, Q.E.; validation, E.A.B., N.V.-G., and M.H.; formal analysis, E.A.B.,
N.V.-G., L.S., Z.A., and Q.E.; investigation, E.A.B., Q. E., N.V.-G., and M.H.; resources, E.A.B., Q.E.,
N.V.-G. and M.H.; data curation, E.A.B., N.V.-G, Z.A., and M.H.; writing—original draft
preparation, E.A.B., Q.E., N.V.-G., M.H. and Z.A.; writing—review and editing, E.A.B., Q.E., N.V.-
G., Z.A., and M.H.; supervision, E.A.B., N.V.-G., C.J., and M.H.; project administration, E.A.B., N.V.-
G. and M.H.; funding acquisition, E.A.B., N.V.-G. and M.H. All authors have read and agreed to the
published version of the manuscript. Funding: This work was supported by the University of Reims Champagne-Ardenne, and the
University Moulay Ismail-Faculty of Sciences via the attribution of the doctoral grant. Conflicts of Interest: The authors declare no conflict of interest. 5. Conclusions In conclusion, both bacteria screened as efficient anti-Botrytis were identified as B. velezensis S3 and E. cloacae S6. In silico analysis of draft genome sequences of B. velezensis
S3 indicates the presence of gene clusters involved in amino acids and derivatives
synthesis, carbohydrates and proteins metabolism, cofactors, vitamins, prosthetic groups Microorganisms 2021, 9, 1386 15 of 19 15 of 19 and pigment formations, and stress response; while that of the E. cloacae S6 revealed the
presence of different secondary metabolites gene clusters, including bacillibactin,
fengycin, surfactin and bacillaene and the presence of non-ribosomal peptide synthetase
and bacteriocin. Further work is obviously required to characterize how these bacteria trigger the
biotic stress resistance in plants to establish a set of biotic stress biomarkers that might
help to predict efficacy of induced resistance for different crops. In addition, it would also
be very interesting to analyze the plant’s response to colonization, i.e., lipid content, fatty
acid composition as well as secondary metabolites profiles. p
y
p
Nevertheless, genus Enterobacter is a member of the ESKAPE group, which contains
the major opportunistic and multi-resistant bacterial pathogens for humans during recent
decades in hospital wards. Therefore, a deeper progress in genome sequencing of E. cloacae
S6 is critical before a potential use of this strain for plant protection. Additionally,
deciphering the mechanisms of horizontal gene transfer that may occur in specific
microhabitats may be a key step in the development of a regulatory framework for the
environmental release of these bacteria. Supplementary
Materials:
Supplementary
materials
can
be
found
at
www.mdpi.com/article/10.3390/microorganisms9071386/s1. Table S1. Project information of the
draft genome sequences of the strains. Figure S1. Proportion of different structure in a fatty acid
profile of isolated strains. Figure S2. Subsystem information of the strain B. velezensis S3 and E. cloacae
S6 and predicted by SEED viewer, most of which were involved in amino acids and derivatives
synthesis, carbohydrate metabolism, cofactors, vitamins, prosthetic groups and pigment formations,
and stress response. Author Contributions: Conceptualization, E.A.B., N.V.-G. and M.H.; methodology, E.A.B., N.V.-G.,
M.H., L.S. and Q.E.; software, Q.E.; validation, E.A.B., N.V.-G., and M.H.; formal analysis, E.A.B.,
N.V.-G., L.S., Z.A., and Q.E.; investigation, E.A.B., Q. E., N.V.-G., and M.H.; resources, E.A.B., Q.E.,
N.V.-G. and M.H.; data curation, E.A.B., N.V.-G, Z.A., and M.H.; writing—original draft
preparation, E.A.B., Q.E., N.V.-G., M.H. References Weiberg, A.; Wang, M.; Lin, F.-M.; Zhao, H.; Zhang, Z.; Kaloshian, I.; Huang, H.; Jin, H. Fungal Small RNAs Suppress Plant
Immunity by Hijacking Host RNA Interference Pathways. Science (80-) 2013, 342, 118–123, doi:10.1126/science.1239705. 12. Boddy, L. Chapter 8-Pathogens of Autotrophs, 3rd ed.; Watkinson, S.C., Boddy, L., Money, N.P., Eds.; Academic Press: Boston, MA,
USA, 2016; pp. 245–292. 13. Fernández-Ortuño, D.; Torés, J.A.; Chamorro, M.; Pérez-García, A.; de Vicente, A. Characterization of resistance to six chemical
classes of site-specific fungicides registered for gray mold control on strawberry in Spain. Plant Dis. 2016, 100, 2234–2239,
doi:10.1094/PDIS-03-16-0280-RE. 14. Rupp, S.; Weber, R.W.S.; Rieger, D.; Detzel, P.; Hahn, M. Spread of Botrytis cinerea strains with multiple fungicide resistance in
german horticulture. Front. Microbiol. 2017, 7, doi:10.3389/fmicb.2016.02075. 15. Yin, W.X.; Adnan, M.; Shang, Y.; Lin, Y.; Luo, C.X. Sensitivity of Botrytis cinerea from nectarine/cherry in China to six fungicides
and characterization of resistant isolates. Plant Dis. 2018, 102, 2578–2585, doi:10.1094/PDIS-02-18-0244-RE. 16. Sautua, F.J.; Baron, C.; Pérez-Hernández, O.; Carmona, M.A. First report of resistance to carbendazim and procymidone in
Botrytis
cinerea
from
strawberry,
blueberry
and
tomato
in
Argentina. Crop
Prot. 2019,
125,
2017–2020,
doi:10.1016/j.cropro.2019.104879. j
p
17. Reeves, W.R.; McGuire, M.K.; Stokes, M.; Vicini, J.L. Assessing the Safety of Pesticides in Food: How Current Regulations Protect
Human Health. Adv. Nutr. 2019, 10, 80–88, doi:10.1093/advances/nmy061. y
18. Liu, J.; Sui, Y.; Wisniewski, M.; Droby, S.; Liu, Y. Review: Utilization of antagonistic yeasts to manage postharvest fungal diseases
of fruit. Int. J. Food Microbiol. 2013, 167, 153–160, doi:10.1016/j.ijfoodmicro.2013.09.004. 19. Liu, Y.; Wang, W.; Zhou, Y.; Yao, S.; Deng, L.; Zeng, K. Isolation, identification and in vitro screening of Chongqing orangery
yeasts for the biocontrol of Penicillium digitatum on citrus fruit. Biol. Control 2017, 110, 18–24, doi:10.1016/j.biocontrol.2017.04.002. 20. Romanazzi, G.; Feliziani, E.; Bautista-Baños, S.; Sivakumar, D. Shelf Life Extension of Fresh Fruit and Vegetables by Chitosan
Treatment. Crit. Rev. Food Sci. Nutr. 2017, 57, doi:10.1080/10408398.2014.900474. T.; Zhu, J.; Zhang, L.; Mu, W.; Liu, F. Evaluation of the antifungal and biochemical activities of
against Botrytis cinerea. Pestic. Biochem. Physiol. 2021, 104784, doi:10.1016/j.pestbp.2021.104784. 21. Li, T.; Li, H.; Liu, T.; Zhu, J.; Zhang, L.; Mu, W.; Liu, F. Evaluation of the antifungal and b
mefentrifluconazole against Botrytis cinerea. Pestic. Biochem. Physiol. 2021, 104784, doi:10.1016/j.pestbp. 22. Pertot, I.; Caffi, T.; Rossi, V.; Mugnai, L.; Hoffmann, C.; Grando, M.S.; Gary, C.; Lafond, D.; Duso, C.; Thiery, D.; et al. References A critical
review of plant protection tools for reducing pesticide use on grapevine and new perspectives for the implementation of IPM
in viticulture. Crop Prot. 2017, 97, 70–84, doi:10.1016/j.cropro.2016.11.025. j
p
ey, P.; Kang, S.C.; Maheshwari, D.K. Rhizobia as a biological control agent against soil borne plan
ian J. Exp. Biol. 2003, 41, 1160–1164. p
j
p
23. Deshwal, V.K.; Pandey, P.; Kang, S.C.; Maheshwari, D.K. Rhizobia as a biological control agent
pathogenic fungi. Indian J. Exp. Biol. 2003, 41, 1160–1164. 24. Lugtenberg, B.; Kamilova, F. Plant-Growth-Promoting Rhizobacteria. Annu. Rev. Microbiol. 2009, 63, 541–556,
doi:10.1146/annurev.micro.62.081307.162918. 25. Kumar, H.; Bajpai, V.K.; Dubey, R.C.; Maheshwari, D.K.; Kang, S.C. Wilt disease management and enhancement of growth and
yield of Cajanus cajan (L) var. Manak by bacterial combinations amended with chemical fertilizer. Crop Prot. 2010, 29, 591–598,
doi:10.1016/j.cropro.2010.01.002. j
p
26. Peian, Z.; Haifeng, J.; Peijie, G.; Sadeghnezhad, E.; Qianqian, P.; Tianyu, D.; Teng, L.; Huanchun, J.; Jinggui, F. Chitosan induces
jasmonic acid production leading to resistance of ripened fruit against Botrytis cinerea infection. Food Chem. 2021, 337, 127772,
doi:10.1016/j.foodchem.2020.127772. 27. Platania, C.; Restuccia, C.; Muccilli, S.; Cirvilleri, G. Efficacy of killer yeasts in the biological control of Penicillium digitatum on
Tarocco orange fruits (Citrus sinensis). Food Microbiol. 2012, 30, 219–225, doi:10.1016/j.fm.2011.12.010. J.W.; Ryu, C.M. Rhizosphere bacteria help plants tolerate abiotic stress. Trends Plant Sci. 2009, 14, 1–4
.2008.10.004. 28. Yang, J.; Kloepper, J.W.; Ryu, C.M. Rhizosphere bacteria help plants tolerate abiotic stress. Trends
doi:10.1016/j.tplants.2008.10.004. 29. Nally, M.C.; Pesce, V.M.; Maturano, Y.P.; Muñoz, C.J.; Combina, M.; Toro, M.E.; de Figueroa, L.C.; Vazquez, F. Biocontrol of
Botrytis cinerea in table grapes by non-pathogenic indigenous Saccharomyces cerevisiae yeasts isolated from viticultural
environments in Argentina. Postharvest Biol. Technol. 2012, 64, 40–48, doi:10.1016/j.postharvbio.2011.09.009. 30. Esmaeel, Q.; Jacquard, C.; Clément, C.; Sanchez, L.; Ait, E.; Barka, E.A. Genome sequencing and traits analysis of Burkholderia
strains reveal a promising biocontrol effect against grey mould disease in grapevine (Vitis vinifera L.). World J. Microbiol. Biotechnol. 2019, 35, doi:10.1007/s11274-019-2613-1. ,
,
31. Thompson, J.D.; Gibson, T.J.; Plewniak, F.; Jeanmougin, F.; Higgins, D.G. The CLUSTAL X windows interface: Flexible strategies
for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997, 25, 4876–4882,
doi:10.1093/nar/25.24.4876. 32. Tamura, K.; Peterson, D.; Peterson, N.; Stecher, G.; Nei, M.; Kumar, S. MEGA5: Molecular evolutionary genetics analysis using
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 2011, 28, 2731–2739,
doi:10.1093/molbev/msr121. 33. References 1. Rojas, C.M.; Senthil-Kumar, M.; Tzin, V.; Mysore, K.S. Regulation of primary plant metabolism during plant-pathogen
interactions and its contribution to plant defense. Front. Plant Sci. 2014, 5, 1–12, doi:10.3389/fpls.2014.00017. 1. Rojas, C.M.; Senthil-Kumar, M.; Tzin, V.; Mysore, K.S. Regulation of primary plant metabolism during plant-pathogen
interactions and its contribution to plant defense. Front. Plant Sci. 2014, 5, 1–12, doi:10.3389/fpls.2014.00017. 1. Rojas, C.M.; Senthil-Kumar, M.; Tzin, V.; Mysore, K.S. Regulation of primary plant metabolism during plant-pathogen
interactions and its contribution to plant defense. Front. Plant Sci. 2014, 5, 1–12, doi:10.3389/fpls.2014.00017. 2. Verdenal, T.; Dienes-Nagy, Á.; Spangenberg, J.E.; Zufferey, V.; Spring, J.; Viret, O.; Marin-Carbonne, J.; van Leeuwen, C. Understanding and managing nitrogen nutrition in grapevine: A review. OENO ONE 2021, 55, 1–43, doi:10.20870/oeno-
one.2021.55.1.3866. 3. Júnior, A.F.N.; Tränkner, M.; Ribeiro, R.V.; von Tiedemann, A.; Amorim, L. Photosynthetic Cost Associated With Induced
Defense to Plasmopara viticola in Grapevine. Front. Plant Sci. 2020, 11, 1–14, doi:10.3389/fpls.2020.00235. 4. Bonfig, K.B.; Schreiber, U.; Gabler, A.; Roitsch, T.; Berger, S. Infection with virulent and avirulent P. syringae strains differentially
affects photosynthesis and sink metabolism in Arabidopsis leaves. Planta 2006, 225, 1–12, doi:10.1007/s00425-006-0303-3. 5. Nascimento, R.; Maia, M.; Ferreira, A.E.N.; Silva, A.B.; Freire, A.P.; Cordeiro, C.; Silva, M.S.; Figueiredo, A. Early stage metabolic
events associated with the establishment of Vitis vinifera–Plasmopara viticola compatible interaction. Plant Physiol. Biochem. 2019,
137, 1–13, doi:10.1016/j.plaphy.2019.01.026. j p p y
6. Jones, D.J.L.; Dangl, J.L. The plant immune system. Nature 2006, 444, 323–329. j p p y
6. Jones, D.J.L.; Dangl, J.L. The plant immune system. Nature 2006, 444, 323–329. p
y
y, K.J. Modelling signaling networks underlying plant defence. Curr. Opin. Plant Biol. 2015, 27, 165–171
5.07.007. 7. Windram, O.; Denby, K.J. Modelling signaling networks underlying plant defence. Curr. Opin. doi:10.1016/j.pbi.2015.07.007. 8. De Lorenzo, G.; Ferrari, S.; Cervone, F.; Okun, E. Extracellular DAMPs in Plants and Mammals: Immunity, Tissue Damage and
Repair. Trends Immunol. 2018, 39, 937–950, doi:10.1016/j.it.2018.09.006. p
,
,
,
/j
9. Fillinger, S.; Yigal, E. Botrytis–the Fungus, the Pathogen and its Management in Agricultural Systems; Springer:
Berlin/Heidelberg, Germany, 2016. Microorganisms 2021, 9, 1386 16 of 19 16 of 19 10. Dean, R.; Van-Kan, J.A.; Pretorius, Z.A.; Hammond-Kosack, K.E.; Di-Pietro, A.; Spanu, P.D; Rudd, J.J.; Dickman, M.; Kahmann,
R.; Ellis, J.; et al. The Top 10 fungal pathogens in molecular plant pathology. Mol. Plant Pathol. 2012, 13, 414–430,
doi:10.1111/j.1364-3703.2011.00783.x. j
11. References Aziz, R.K.; Bartels, D.; Best, A.A.; DeJongh, M.; Disz, T.; Edwards, R.A.; Formsma, K.; Gerdes, S.; Glass, E.M.; Kubal, M.; et al. The RAST Server: Rapid annotations using subsystems technology. BMC Genom. 2008, 9, 1–15, doi:10.1186/1471-2164-9-75. p
g
y
gy
34. Disz, T.; Akhter, S.; Cuevas, D.; Olson, R.; Overbeek, R.; Vonstein, V.; Stevens, R.; Edwards, R.A. Accessing the SEED Genome
Databases via Web Services API: Tools for Programmers. BMC Bioinform. 2010, 11, doi:10.1186/1471-2105-11-319. 17 of 19 17 of 19 Microorganisms 2021, 9, 1386 35. Weber, T.; Blin, K.; Duddela, S.; Krug, D.; Kim, H.U.; Bruccoleri, R.; Lee, S.Y.; Fischbach, M.A.; Müller, R.; Wohlleben, W.; et al. AntiSMASH 3.0-A comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res. 2015, 43,
W237–W243, doi:10.1093/nar/gkv437. g
36. Barka, E.A.; Nowak, J.; Clément, C. Enhancement of chilling resistance of inoculated grapevine plantlets with a plant growth-
promoting rhizobacterium, Burkholderia phytofirmans strain PsJN. Appl. Environ. Microbiol. 2006, 72, 7246–7252,
doi:10.1128/AEM.01047-06. 37. Miotto-Vilanova, L.; Jacquard, C.; Courteaux, B.; Wortham, L.; Michel, J.; Clément, C.; Barka, E.A.; Sanchez, L. Burkholderia
phytofirmans PsJN Confers Grapevine Resistance against Botrytis cinerea via a Direct Antimicrobial Effect Combined with a Better
Resource Mobilization. Front. Plant Sci. 2016, 7, 1–15, doi:10.3389/fpls.2016.01236. p
38. Genty, B.; Briantais, J.-M.; Baker, N.R. The relationship between the quantum yield of photosynthetic electron transport and
quenching of chlorophyll fluorescence. Biochim. Biophys. Acta-Gen. Subj. 1989, 990, 87–92, doi:10.1016/S0304-4165(89)80016-9. 39. Kramer, D. M.; Johnson, G.; Kiirats, O.; Edwards, G. E. New fluorescence parameters for the determination of QA redox state
and excitation energy fluxes. Photosynth. Res., 2004, 79, 209–218, doi: 10.1023/B:PRES.0000015391.99477.0d. 40. Rodriguez-R, L. M.; Gunturu, S.; Harvey, W. T.; Rosselló-Mora, R.; Tiedje, J. M.; Cole, J. R.; Konstantinidis, K. T. The Microbial
Genomes Atlas (MiGA) webserver: taxonomic and gene diversity analysis of Archaea and Bacteria at the whole genome level. Nucleic Acids Res. 2018, 46, W282–W288, doi: 10.1093/nar/gky467. g y
41. Meier-Kolthoff, J. P. & Göker, M. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat. Commun, 2019, 10, 1–10, doi: 10.1038/s41467-019-10210-3. 42. Sasse, J.; Martinoia, E.; Northen, T. Feed Your Friends: Do Plant Exudates Shape the Root Microbiom
23, 25–41, doi:10.1016/j.tplants.2017.09.003. E.; Northen, T. Feed Your Friends: Do Plant Exudates Shape the Root Microbiome? Trends Plant Sci. 2018
6/j.tplants.2017.09.003. j p
43. Wang, C.; Zhao, D.; Qi, G.; Mao, Z.; Hu, X.; Du, B.; Liu, K.; Ding, Y. References Effects of Bacillus velezensis FKM10 for Promoting the
Growth of Malus hupehensis Rehd. and Inhibiting Fusarium verticillioides. Front. Microbiol. 2020, 10, 2889,
doi:10.3389/fmicb.2019.02889. 44. Meng, Q.; Jiang, H.; Hao, J.J. Effects of Bacillus velezensis strain BAC03 in promoting plant growth. Biol. Control 2016, 98, 18–26,
doi:10.1016/j.biocontrol.2016.03.010. 45. Taghavi, S.; van der Lelie, D.; Hoffman, A.; Zhang, Y.; Walla, M.D.; Vangronsveld, J.; Newman, L.; Monchy, S. Genome sequence
of
the
plant
growth
promoting
endophytic
bacterium
Enterobacter
sp. 638. PLoS
Genet. 2010,
6,
19,
doi:10.1371/journal.pgen.1000943. j
pg
46. Macedo-Raygoza, G.M.; Valdez-Salas, B.; Prado, F.M.; Prieto, K.R.; Yamaguchi, L.F.; Kato, M.J.; Canto-Canché, B.B.; Carrillo-
Beltrán, M.; di Mascio, P.; White, J.F.; Beltrán-García, M.J. Enterobacter cloacae, an endophyte that establishes a nutrient-transfer
symbiosis with banana plants and protects against the black sigatoka pathogen. Front. Microbiol. 2019, 10, 804,
doi:10.3389/fmicb.2019.00804. 47. Toral, L.; Rodríguez, M.; Béjar, V.; Sampedro, I. microorganisms Crop Protection against Botrytis cinerea by Rhizhosphere
Biological Control Agent Bacillus velezensis XT1. Microorganisms 2020, 8, 992, doi:10.3390/microorganisms8070992. g
g
g
g
48. Chen, L.; Wang, X.; Ma, Q.; Bian, L.; Liu, X.; Xu, Y.; Zhang, H.; Shao, J.; Liu, Y. Bacillus velezensis CLA178-Induced Systemic
Resistance of Rosa multiflora Against Crown Gall Disease. Front. Microbiol. 2020, 11, 587667, doi:10.3389/fmicb.2020.587667. 49. Shastry, R.P.; Welch, M.; Rai, V.R.; Ghate, S.D.; Sandeep, K.; Rekha, P.D. The whole-genome sequence analysis of Enterobacter
cloacae strain Ghats1: Insights into endophytic lifestyle-associated genomic adaptations. Arch Microbiol. 2020, 202, 1571–1579,
doi:10.1007/s00203-020-01848-5. 50. Shi, J.F.; Sun, C.Q. Isolation, identification, and biocontrol of antagonistic bacterium against Botrytis cinerea after tomato harvest. Braz. J. Microbiol. 2017, 48, 706–714, doi:10.1016/j.bjm.2017.03.002. j j
51. Pan, H.Q.; Li, Q.L.; Hu, J.C. The complete genome sequence of Bacillus velezensis 9912D reveals its biocontrol mechanism as a
novel commercial biological fungicide agent. J. Biotechnol. 2017, 247, 25–28, doi:10.1016/j.jbiotec.2017.02.022. 52. Wang, F.; Xiao, J.; Zhang, Y.; Li, R.; Liu, L.; Deng, J. Biocontrol ability and action mechanism of Bacillus halotolerans against
Botrytis cinerea causing grey mould in postharvest strawberry fruit. Postharvest Biol. Technol. 2021, 174, 111456,
doi:10.1016/j.postharvbio.2020.111456. j p
53. Gordon, R.E.; Haynes, W.C.; Pang, C.H.-N.; Smith, N.R. The Genus Bacillus; Agricultural Research Service, U.S. Department of
Agriculture: Washington, DC, USA, 1973; Available online: http://books.google.com/books?id=lXMpXssWFD0C (accessed on 3
June 2021). )
54. Priest, F.G.; Goodfellow, M. Bacillus amyloliquefaciens sp. nov. norn. rev. Microbiol. Soc. 1987, doi:10.1099/00207713-37-1-69. 54. Priest, F.G.; Goodfellow, M. Bacillus amyloliquefaciens sp. nov. norn. rev. Microbiol. Soc. 1987, doi:10.1099/00207713-37-1-69. 55. 57.
Dunlap, C.A.; Bowman, M.J.; Schisler, D.A.; Rooney, A.P. Genome analysis shows Bacillus axarquiensis is not a later heterotypic
synonym of Bacillus mojavensis; reclassification of Bacillus malacitensis and Brevibacterium halotolerans as heterotypic synonyms of
Bacillus axarquiensis. Int. J. Syst. Evol. Microbiol. 2016, 66, 2438–2443, doi:10.1099/ijsem.0.001048. References Identification of endophytic Bacillus velezensis ZSY-1 strain and antifungal activity
of
its
volatile
compounds
against
Alternaria
solani
and
Botrytis
cinerea. Biol. Control
2017,
105,
27–39,
doi:10.1016/j.biocontrol.2016.11.007. j
65. Xu, Z.; Zhang, H.; Sun, X.; Liu, Y.; Yan, W.; Xun, W.; Shen, Q.; Zhang, R. Bacillus velezensis wall teichoic acids are required for
biofilm. Appl. Environ. Microbiol. 2019, 85, 1–14. 66. Morales-Cedeño, L.R.; Orozco-Mosqueda, M.D.; Loeza-Lara, P.D.; Parra-Cota, F.I.; de los Santos-Villalobos, S.; Santoyo, G. Plant
growth-promoting bacterial endophytes as biocontrol agents of pre- and post-harvest diseases: Fundamentals, methods of
application and future perspectives. Microbiol. Res. 2021, 242, 12, doi:10.1016/j.micres.2020.126612. 67. Lima, G.; de Curtis, F.; Piedimonte, D.; Spina, A.M.; de Cicco, V. Integration of biocontrol yeast and thiabendazole protects
stored apples from fungicide sensitive and resistant isolates of Botrytis cinerea. Postharvest Biol. Technol. 2006, 40, 301–307,
doi:10.1016/j.postharvbio.2006.01.017. j p
68. Püttmann, M.; Ade, N.; Hof, H. Dependence of fatty acid composition of Listeria spp. on growth tem
1993, 144, 279–283, doi:10.1016/0923-2508(93)90012-Q. N.; Hof, H. Dependence of fatty acid composition of Listeria spp. on growth temperature. Res. Microbiol
doi:10.1016/0923-2508(93)90012-Q. 69. Annous, B.A.; Becker, L.A.; Bayles, D.O.; Labeda, D.P.; Wilkinson, B.J. Critical role of anteiso-C(15:0) fatty acid in the growth of
Listeria monocytogenes at low temperatures. Appl. Environ. Microbiol. 1997, 63, 3887–3894, doi:10.1128/aem.63.10.3887-3894.1997. 70. Fernández-González, A.J.; Martínez-Hidalgo, P.; Cobo-Díaz, J.F.; Villadas, P.J.; Martínez-Molina, E.; Toro, N.; Tringe, S.G.;
Fernández-López, M. The rhizosphere microbiome of burned holm-oak: Potential role of the genus Arthrobacter in the recovery
of burned soils. Sci. Rep. 2017, 7, 1–12, doi:10.1038/s41598-017-06112-3. p
71. Tsuda, K.; Kosaka, Y.; Tsuge, S.; Kubo, Y.; Horino, O. Evaluation of the Endophyte Enterobacter cloacae SM10 Isolated from
Spinach Roots for Biological Control against Fusarium Wilt of Spinach. J. Gen. Plant Pathol. 2001, 67, 78–84,
doi:10.1007/PL00012993. rthikeyan, M.; Rajeswari, E. Plant Health under Biotic Stress; Springer: Berlin/Heidelberg, Germany, 2019. 72. Latha, P.; Karthikeyan, M.; Rajeswari, E. Plant Health under Biotic Stress; Springer: Berlin/Heidelber 73. Guo, D.J.; Singh, R.K.; Singh, P.; Li, D.P.; Sharma, A.; Xing, Y.X.; Song, X.P.; Yang, L.T.; Li, Y.R. Complete Genome Sequence of
Enterobacter roggenkampii ED5, a Nitrogen Fixing Plant Growth Promoting Endophytic Bacterium with Biocontrol and Stress
Tolerance Properties, Isolated From Sugarcane Root. Front. Microbiol. 2020, 11, 28, doi:10.3389/fmicb.2020.580081. p
g
74. Mohamed, B.F.F.; Sallam, N.M.A.; Alamri, S.A.M.; Abo-Elyousr, K.A.M.; Mostafa, Y.S.; Hashem, M. References Ruiz-García, C.; Béjar, V.; Martínez-Checa, F.; Llamas, I.; Quesada, E. Bacillus velezensis sp. nov., a surfactant-producing
bacterium isolated from the river Vélez in Málaga, southern Spain. Int. J. Syst. Evol. Microbiol. 2005, 55, 191–195,
doi:10.1099/ijs.0.63310-0. j
56. Madhaiyan, M.; Poonguzhali, S.; Kwon, S.W.; Sa, T.M. Bacillus methylotrophicus sp. nov., a methanol-utilizing, plant-growth-
promoting bacterium isolated from rice rhizosphere soil. Int. J. Syst. Evol. Microbiol. 2010, 60, 2490–2495, doi:10.1099/ijs.0.015487-
0. 57. Dunlap, C.A.; Bowman, M.J.; Schisler, D.A.; Rooney, A.P. Genome analysis shows Bacillus axarquiensis is not a later heterotypic
synonym of Bacillus mojavensis; reclassification of Bacillus malacitensis and Brevibacterium halotolerans as heterotypic synonyms of
Bacillus axarquiensis. Int. J. Syst. Evol. Microbiol. 2016, 66, 2438–2443, doi:10.1099/ijsem.0.001048. Microorganisms 2021, 9, 1386 18 of 19 18 of 19 58. Dunlap, C.A.; Kim, S.J.; Kwon, S.W.; Rooney, A.P. Bacillus velezensis is not a later heterotypic synonym of Bacillus
amyloliquefaciens; Bacillus methylotrophicus, Bacillus amyloliquefaciens subsp. Plantarum and ‘Bacillus oryzicola’ are later heterotypic
synonyms of Bacillus velezensis based on phylogenom. Int. J. Syst. Evol. Microbiol. 2016, 66, 1212–1217, doi:10.1099/ijsem.0.000858. y
y
p y
g
J
y
j
59. Dunlap, C.A.; Kim, S.-J.; Kwon, S.-W.; Rooney, A.P. Phylogenomic analysis shows that Bacillus amyloliquefaciens subsp. plantarum is a later heterotypic synonym of Bacillus methylotrophicus. Int. J. Syst. Evol. Microbiol. 2015, 65, 2104–2109,
doi:10.1099/ijs.0.000226. 60. Wu, L.; Wu, H.J.; Qiao, J.; Gao, X.; Borriss, R. Novel routes for improving biocontrol activity of Bacillus based bioinoculants. Front. Microbiol. 2015, 6, 1–13, doi:10.3389/fmicb.2015.01395. , J.; Klenk, H.P.; Borriss, R. Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamensis Form an ‘Ope
yloliquefaciens’ within the B. subtilis species complex. Front. Microbiol. 2017, 8, 22, doi:10.3389/fmicb.2017.000 61. Fan, B.; Blom, J.; Klenk, H.P.; Borriss, R. Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamen
Group B. amyloliquefaciens’ within the B. subtilis species complex. Front. Microbiol. 2017, 8, 22, doi:10.33 p
y
q
f
p
p
62. Myo, E.M.; Liu, B.; Ma, J.; Shi, L.; Jiang, M.; Zhang, K.; Ge, B. Evaluation of Bacillus velezensis NKG-2 for bio-control activities
against fungal diseases and potential plant growth promotion. Biol. Control 2019, 134, 23–31, doi:10.1016/j.biocontrol.2019.03.017. g
g
p
p
g
p
j
63. Jiang, C.H.; Liao, M.J.; Wang, H.K.; Zheng, M.Z.; Xu, J.J.; Guo, J.H. Bacillus velezensis, a potential and efficient biocontrol agent
in control of pepper gray mold caused by Botrytis cinerea. Biol. Control 2018, 126, 147–157, doi:10.1016/j.biocontrol.2018.07.017. 64. Gao, Z.; Zhang, B.; Liu, H.; Han, J.; Zhang, Y. References Approving the biocontrol
method of potato wilt caused by Ralstonia solanacearum (Smith) using Enterobacter cloacae PS14 and Trichoderma asperellum T34. Egypt. J. Biol. Pest Control 2020, 30, 13, doi:10.1186/s41938-020-00262-9. gyp
75. Abdeljalil, N.O.; Vallance, J.; Gerbore, J.; Yacoub, A.; Daami-Remadi, M.; Rey, P. Combining potential oomycete and bacterial
biocontrol
agents
as
a
tool
to
fight
tomato
Rhizoctonia
root
rot. Biol. Control
2021,
155,
104521,
doi:10.1016/j.biocontrol.2020.104521. j
76. Chen, P.-S.; Peng, Y.-H. Inhibition of Penicillium digitatum and Citrus Green Mold by Volatile Compounds Produced by
Enterobacter cloacae. J. Plant Pathol. Microbiol. 2016, 7, doi:10.4172/2157-7471.1000339. 77. Singh, R.P.; Nalwaya, S.; Jha, P.N. The draft genome sequence of the plant growth promo
Enterobacter cloacae SBP-8. Genom. Data 2017, 12, 81–83, doi:10.1016/j.gdata.2017.03.006. ya, S.; Jha, P.N. The draft genome sequence of the plant growth promoting rhizospheric bacterium
SBP-8. Genom. Data 2017, 12, 81–83, doi:10.1016/j.gdata.2017.03.006. 78. Cho, S.T.; Chang, H.H.; Egamberdieva, D.; Kamilova, F.; Lugtenberg, B.; Kuo, C.H. Genome analysis of Pseudomonas fluorescens
PCL1751: A rhizobacterium that controls root diseases and alleviates salt stress for its plant host. PLoS ONE 2015, 10,
doi:10.1371/journal.pone.0140231. j
p
79. Luo, Y.; Cheng, Y.; Yi, J.; Zhang, Z.; Luo, Q.; Zhang, D.; Li, Y. Complete genome sequence of industrial biocontrol strain
Paenibacillus polymyxa HY96-2 and further analysis of its biocontrol mechanism. Front. Microbiol. 2018, 9, 1–14,
doi:10.3389/fmicb.2018.01520. 80. Yang, E.; Sun, L.; Ding, X.; Sun, D.; Liu, J.; Wang, W. Complete genome sequence of Caulobacter flavus RHGG3T, a type species
of the genus Caulobacter with plant growth-promoting traits and heavy metal resistance. 3 Biotech 2019, 9, 42,
doi:10.1007/s13205-019-1569-z. 19 of 19 Microorganisms 2021, 9, 1386 19 of 19 81. Chaouachi, M.; Marzouk, T.; Jallouli, S.; Elkahoui, S.; Gentzbittel, L.; Ben, C.; Djébali, N. Activity assessment of tomato
endophytic bacteria bioactive compounds for the postharvest biocontrol of Botrytis cinerea. Postharvest Biol. Technol. 2021, 172,
18, doi:10.1016/j.postharvbio.2020.111389. j p
82. Gao, H.; Li, P.; Xu, X.; Zeng, Q.; Guan, W. Research on volatile organic compounds from Bacillus subtilis CF-3: Biocontrol effects
on fruit fungal pathogens and dynamic changes during fermentation. Front. Microbiol. 2018, 9, doi:10.3389/fmicb.2018.00456. g
p
g
y
g
g
83. Patel, P.; Shah, R.; Modi, K. Isolation and characterization of plant growth promoting potential of Acinetobacter sp. RSC7 isolated
from Saccharum officinarum cultivar Co 671. J. Exp. Biol. Agric. Sci. 2017, 5, 483–491, doi:10.18006/2017.5(4).483.491. 84. References Agbodjato, N.A.; Noumavo, P.A.; Baba-Moussa, F.; Salami, H.A.; Sèzan, A.; Bankolé, H.; Adjanohoun, A.; Baba-Moussa, L. Characterization of potential plant growth promoting rhizobacteria isolated from Maize (Zea mays L.) in central and Northern
Benin (West Africa). Appl. Environ. Soil Sci. 2015, 2015, doi:10.1155/2015/901656. pp
85. Bashan, Y.; Kamnev, A.A.; de-Bashan, L.E. Tricalcium phosphate is inappropriate as a universal selection factor for isolating
and testing phosphate-solubilizing bacteria that enhance plant growth: A proposal for an alternative procedure. Biol. Fertil. Soils
2013, 49, 465–479, doi:10.1007/s00374-012-0737-7. 86. Romero, D.; de Vicente, A.; Rakotoaly, R.H.; Dufour, S.E.; Veening, J.W.; Arrebola, E.; Cazorla, F.M.; Kuipers, O.P.; Paquot, M.;
Pérez-García, A. The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward
Podosphaera fusca. Mol. Plant-Microbe Interact. 2007, 20, 430–440, doi:10.1094/MPMI-20-4-0430. p
87. Chen, L.; Shi, H.; Heng, J.; Wang, D.; Bian, K. Antimicrobial, plant growth-promoting and genomic properties of the peanut
endophyte Bacillus velezensis LDO2. Microbiol. Res. 2019, 218, 41–48, doi:10.1016/j.micres.2018.10.002. p y
j
88. Xu, T.; Zhu, T.; Li, S. β-1,3-1,4-glucanase gene from Bacillus velezensis ZJ20 exerts antifungal effect on
World J. Microbiol. Biotechnol. 2016, 32, 26, doi:10.1007/s11274-015-1985-0. J
89. Cao, Y.; Pi, H.; Chandrangsu, P.; Li, Y.; Wang, Y.; Zhou, H.; Xiong, H.; Helmann, J.D.; Cai, Y. Antagonism of Two Plant-Growth
Promoting Bacillus velezensis Isolates Against Ralstonia solanacearum and Fusarium oxysporum. Sci. Rep. 2018, 8, 1–14,
doi:10.1038/s41598-018-22782-z. 90. Chowdhury, S.P; Dietel, K.; Rändler, M.; Schmid, M.; Junge, H.; Borriss, R.; Hartmann, A.; Grosch, R. Effects of Bacillus
amyloliquefaciens FZB42 on Lettuce Growth and Health under Pathogen Pressure and Its Impact on the Rhizosphere Bacterial
Community. PLoS ONE 2013, 8, 1–10, doi:10.1371/journal.pone.0068818. 91. Kim, S.Y.; Song, H.; Sang, M.K.; Weon, H.Y.; Song, J. The complete genome sequence of Bacillus velezensis strain GH1-13 reveals
agriculturally beneficial properties and a unique plasmid. J. Biotechnol. 2017, 259, 221–227, doi:10.1016/j.jbiotec.2017.06.1206. 92
Adeniji A A ; Loots D T ; Babalola O O Bacillus velezensis: Phylogeny useful applications and avenues for exploitation Appl 91. Kim, S.Y.; Song, H.; Sang, M.K.; Weon, H.Y.; Song, J. The complete genome sequence of Bacillus velezensis strain GH1-13 reveals
agriculturally beneficial properties and a unique plasmid. J. Biotechnol. 2017, 259, 221–227, doi:10.1016/j.jbiotec.2017.06.1206. 92. Adeniji, A.A.; Loots, D.T.; Babalola, O.O. Bacillus velezensis: Phylogeny, useful applications, and avenues for exploitation. Appl. Microbiol. Biotechnol. 2019, 103, 3669–3682, doi:10.1007/s00253-019-09710-5. agriculturally beneficial properties and a unique plasmid. J. Biotechnol. 2017, 259, 221–227, doi:10.1016/j.jbiotec.2017.06.1206. 92. References Adeniji, A.A.; Loots, D.T.; Babalola, O.O. Bacillus velezensis: Phylogeny, useful applications, and avenues for exploitation. Appl. Microbiol. Biotechnol. 2019, 103, 3669–3682, doi:10.1007/s00253-019-09710-5. 93. Fan, B.; Wang, C.; Song, X.; Ding, X.; Wu, L.; Wu, H.; Gao, X.; Borriss, R. Bacillus velezensis FZB42 in
Model Strain for Plant Growth Promotion and Biocontrol. Front. Microbiol. 2018, 9, 1–14, doi:10.3389/fm 94. Calatayud, A.; Roca, D.; Martínez, P.F. Spatial-temporal variations in rose leaves under water stress conditions studied by
chlorophyll fluorescence imaging. Plant Physiol. Biochem. 2006, 44, 564–573, doi:10.1016/j.plaphy.2006.09.015. 95. Sperdouli, I.; Moustakas, M. A better energy allocation of absorbed light in photosystem II and less photooxidative damage
contribute to acclimation of Arabidopsis thaliana young leaves to water deficit. J. Plant Physiol. 2014, 171, 587–593,
doi:10.1016/j.jplph.2013.11.014. j jp p
96. Gawroński, P.; Witoń, D.; Vashutina, K.; Bederska, M.; Betliński, B.; Rusaczonek, A.; Karpiński, S. Mitogen-activated protein
kinase 4 is a salicylic acid-independent regulator of growth but not of photosynthesis in Arabidopsis. Mol. Plant 2014, 7, 1151–
1166, doi:10.1093/mp/ssu060. 97. Moustaka, J.; Ouzounidou, G.; Sperdouli, I.; Moustakas, M. Photosystem II Is More Sensitive than Photosystem I to Al3+ Induced
Phytotoxicity. Materials 2018, 11, 1772, doi:10.3390/ma11091772. .K.; Roitsch, T. Plant physiology meets phytopathology: Plant primary metabolism and plant-pathogen
Bot. 2007, 58, 4019–4026, doi:10.1093/jxb/erm298. 98. Berger, S.; Sinha, A.K.; Roitsch, T. Plant physiology meets phytopathology: Plant primary met
interactions. J. Exp. Bot. 2007, 58, 4019–4026, doi:10.1093/jxb/erm298. p
j
99. Demmig-Adams, B.; Adams, W.W. Photoprotection in an ecological context: The remarkable complexity of thermal energy
dissipation. New Phytol. 2006, 172, 11–21, doi:10.1111/j.1469-8137.2006.01835.x. 100. Jusović, M.; Velitchkova, M.Y.; Misheva, S.P.; Börner, A.; Apostolova, E.L.; Dobrikova, A.G. Photosynthetic Responses of a
Wheat Mutant (Rht-B1c) with Altered DELLA Proteins to Salt Stress. J. Plant Growth Regul. 2018, 37, 645–656, doi:10.1007/s00344-
017-9764-9. 101. Guo, J.; Sun, K.; Zhang, Y.; Hu, K.; Zhao, X.; Liu, H.; Wu, S.; Hu, Y.; Zhang, Y.; Wang, Y. SlMAPK3, a key mitogen-activated
protein kinase, regulates the resistance of cherry tomato fruit to Botrytis cinerea induced by yeast cell wall and β-glucan. Postharvest Biol. Technol. 2021, 171, 111350, doi:10.1016/j.postharvbio.2020.111350. View publication stats
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Genome-wide identification of microsatellite markers from cultivated peanut (Arachis hypogaea L.)
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Lu et al. BMC Genomics (2019) 20:799
https://doi.org/10.1186/s12864-019-6148-5 Lu et al. BMC Genomics (2019) 20:799
https://doi.org/10.1186/s12864-019-6148-5 Open Access © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Genome-wide identification of
microsatellite markers from cultivated
peanut (Arachis hypogaea L.) Qing Lu1†, Yanbin Hong1†, Shaoxiong Li1, Hao Liu1, Haifen Li1, Jianan Zhang2, Haofa Lan2, Haiyan Liu1, Xingyu Li1,
Shijie Wen1, Guiyuan Zhou1, Rajeev K. Varshney3, Huifang Jiang4, Xiaoping Chen1* and Xuanqiang Liang1* Abstract Background: Microsatellites, or simple sequence repeats (SSRs), represent important DNA variations that are widely
distributed across the entire plant genome and can be used to develop SSR markers, which can then be used to
conduct genetic analyses and molecular breeding. Cultivated peanut (A. hypogaea L.), an important oil crop
worldwide, is an allotetraploid (AABB, 2n = 4× = 40) plant species. Because of its complex genome, genomic marker
development has been very challenging. However, sequencing of cultivated peanut genome allowed us to develop
genomic markers and construct a high-density physical map. Results: A total of 8,329,496 SSRs were identified, including 3,772,653, 4,414,961, and 141,882 SSRs that were
distributed in subgenome A, B, and nine scaffolds, respectively. Based on the flanking sequences of the identified
SSRs, a total of 973,984 newly developed SSR markers were developed in subgenome A (462,267), B (489,394), and
nine scaffolds (22,323), with an average density of 392.45 markers per Mb. In silico PCR evaluation showed that an
average of 88.32% of the SSR markers generated only one in silico-specific product in two tetraploid A. hypogaea
varieties, Tifrunner and Shitouqi. A total of 39,599 common SSR markers were identified among the two A. hypogaea varieties and two progenitors, A. duranensis and A. ipaensis. Additionally, an amplification effectiveness of
44.15% was observed by real PCR validation. Moreover, a total of 1276 public SSR loci were integrated with the
newly developed SSR markers. Finally, a previously known leaf spot quantitative trait locus (QTL), qLLS_T13_A05_7,
was determined to be in a 1.448-Mb region on chromosome A05. In this region, a total of 819 newly developed
SSR markers were located and 108 candidate genes were detected. Conclusions: The availability of these newly developed and public SSR markers both provide a large number of
molecular markers that could potentially be used to enhance the process of trait genetic analyses and improve
molecular breeding strategies for cultivated peanut. Keywords: Genome sequence, Simple sequence repeats, Molecular breeding, Peanut (Arachis hypogaea L.) Background fighting malnutrition and ensuring food security. In China,
peanut accounts for almost half of the total output of all
oil crops and is increasingly important as an oil and pro-
tein crop. Therefore, it is critical to improve peanut pro-
duction and quality to ensure an edible oil supply. Cultivated peanut or groundnut (Arachis hypogaea L.) is a
globally important legume that is widely planted in Asia,
Africa, America, and other areas because it is rich in seed
oil and protein. Thus, peanut has great significance for However, peanut production is often constrained by
factors such as drought, salinity, and disease [1]. During
the past 10 years, with the development of peanut gen-
omics, there have been several successful achievements
in peanut trait mapping [2–5] and molecular breeding
[6, 7]. Owing to the limited available genetic markers * Correspondence: chenxiaoping@gdaas.cn; liangxuanqiang@gdaas.cn
†Qing Lu and Yanbin Hong contributed equally to this work. 1Crops Research Institute, Guangdong Academy of Agricultural Sciences,
South China Peanut Sub-Center of National Center of Oilseed Crops
Improvement, Guangdong Provincial Key Laboratory for Crop Genetic
Improvement, Guangzhou 510640, China
Full list of author information is available at the end of the article Whole genome identification of SSRs In this study, the whole genome sequence of A. hypo-
gaea cv. Fuhuasheng was used to identify SSRs with dif-
ferent repeat motifs, from mono- to hexa-nucleotide. A
total of 8,329,496 SSRs were obtained, with a density of
~ 3264.31 SSRs per Mb (Table 1; Additional file 1: Table S1). Penta-nucleotide was the most common type, accounting for
more than half of all identified SSRs (57.75%), followed by
hexa-nucleotide (26.61%) (Table 1). In addition, more SSRs
were identified in subgenome B (4,414,961) than in subge-
nome A (3,772,653), and 141,882 SSRs were identified on
the nine scaffolds of the peanut reference genome assembly
(Table 1). Analysis of SSR distribution on each chromosome re-
vealed that the largest number of SSRs was present on
chromosome A07 (538,928), followed by chromosome B09
(532,326) (Additional file 1: Table S1; Additional file 2: Fig-
ure S1). Pearson correlation analysis revealed that chromo-
some length was significantly positively associated with the
number of SSRs of each chromosome (r = 0.996, p < 0.01)
(Additional file 1: Table S1). Furthermore, the average dens-
ity of SSRs was 3283.89 SSRs per Mb, ranging from 3164.75
SSRs per Mb on chromosome A10 to 3843.36 SSRs per Mb
on chromosome A08 (Additional file 1: Table S1; Additional
file 2: Figure S1). Moreover, the densities of SSRs on chro-
mosomes A08 and B03 were the highest in subgenomes A
and B, respectively (Additional file 2: Figure S1). g
Cultivated peanut is an allotetraploid (AABB, 2n =
4× = 40) that probably derived from hybridization be-
tween two diploids, A. duranensis and A. ipaensis [18,
19]. Recently, breakthroughs have been made in peanut
genome sequencing. From 2016 to 2018, the genomes of
the two diploid progenitors and one allotetraploid wild
species, A.monticola, were successfully sequenced [20–
23]. Based on the genomic sequences of the two diploid
progenitors, genome-wide g-SSRs were identified and
developed, and a high-density SSR physical map of wild
peanut species was constructed [24]. Importantly, in
2019,
cultivated
peanut
genomics
research
experi-
enced a substantial milestone when genome sequen-
cing was completed for three cultivated peanut :
Fuhuasheng [25], Shitouqi [26], and Tifrunner [27]. The
high-quality
genome
assemblies
provide
the
opportunity
for
developing
genome-wide
g-SSR
markers in cultivated peanut. p
y (
g
)
A total of 501 types of SSR motifs were identified in
the peanut genome (Additional file 1: Table S2). Lu et al. BMC Genomics (2019) 20:799 Lu et al. BMC Genomics (2019) 20:799 Page 2 of 9 novel, newly developed g-SSR markers could be helpful
for advancing agronomic trait mapping, gene cloning, and
molecular breeding of cultivated peanut in the future. and low-density genetic maps, those studies could not
provide optimal resolution of trait dissection and identify
candidate genes. Therefore, the development of a high-
density genetic map is particularly urgent for peanut
trait mapping and breeding. Whole genome identification of SSRs The re-
peat number of all of these motifs ranged from 2 to 335,
most of which were concentrated in the top of 50s
(Additional file 2: Figure S2a). The proportions of penta-
and hexa-nucleotides with two repeats were much
greater than those of other types in both subgenome A
and B, and ranged from ~ 20 to 50% (Additional file 2:
Figure S2b). Of the SSR motif types, the penta-nucleotide
type, AAAAT/ATTTT, had the highest occurrence, and
accounted for 9.55% of all types, followed by AAATT/
AATTT and AAAAG/CTTTT, which accounted for 4.97
and 4.85%, respectively (Additional file 2: Figure S3). For
each type of SSR motif, from mono- to hexa-nucleotide, the
richest motifs were T (1.48%), TA (1.25%), AAT (0.37%),
AAAT (0.34%), AAAAT (1.44%), and ACGCGT (0.64%). Here, we identified genome-wide g-SSRs and devel-
oped g-SSR markers from the genome assembly of A. hypogaea cv. Fuhuasheng, a landrace from North China
that was sequenced in our previous work [25]. The aims
of this study were to: (1) identify genome-wide SSRs and
show the distribution of motif length, type, and repeat
number between the two subgenomes (A and B); (2) de-
velop g-SSR markers and construct a high-density SSR
physical map of cultivated peanut; and (3) evaluate the
application of these SSR markers and validate the poly-
morphisms in different cultivated peanut species. These Results and discussion Simple sequence repeats (SSRs) are genomic fragments
that consist of tandemly repeated units that are present
in both coding and non-coding regions of the genome
[8, 9]. SSR markers, designed by flanking sequences, are
useful for and widely applied in plant genetic analyses
and marker-assisted selection breeding. SSRs derived
from expressed sequence tags (ESTs), transcriptome se-
quences, and genomic DNA sequences are referred to as
EST-SSRs, transcriptome-SSRs, and g-SSRs, respectively. In the past decade, several hundred EST-SSR markers
were developed by investigating ESTs [10, 11], and thou-
sands of transcriptome-SSR markers were identified
based on different transcriptome libraries of cultivated
peanut [12–14]. Meanwhile, two integrated consensus
genetic maps with thousands of different types of
markers, such as EST-SSRs, transcriptome-SSRs, and g-
SSRs, were constructed [15, 16]. Limited by the poly-
morphisms, these available SSRs were insufficient for
constructing high-density genetic maps and enhancing
molecular breeding [17]. Genome-wide SSR marker development The flanking sequences of all identified SSRs were used
to design suitable forward and reverse primer pairs. A
total of 973,984 SSR markers were successfully devel-
oped on 20 chromosomes and nine scaffolds in peanut Lu et al. BMC Genomics (2019) 20:799 Page 3 of 9 Table 1 Different types of SSRs identified in A. hypogaea L
SSR types
SSR Number
Proportion (%)
Subgenome A
Proportion (%)
Subgenome B
Proportion (%)
Scaffolds
Mono-nucleotide
255,774
3.07
112,466
2.98
138,624
3.14
Di-nucleotide
376,173
4.52
167,574
4.44
202,132
4.58
Tri-nucleotide
337,026
4.05
155,918
4.13
175,197
3.97
Tetra-nucleotide
334,132
4.01
143,250
3.8
185,051
4.19
Penta-nucleotide
4,810,032
57.75
2,189,218
58.03
2,538,767
57.5
Hexa-nucleotide
2,216,359
26.61
1,004,227
26.62
1,175,190
26.62
Total
8,329,496
3,772,653
4,414,961
141,882 Table 1 Different types of SSRs identified in A. hypogaea L density in the middle of each chromosome except for
chromosome B07 (Figs. 1 and 2). In addition, for all SSR
markers in subgenome A, subgenome B, and the scaf-
folds, penta-nucleotide SSRs were the most abundant
(491,927) (Additional file 2: Figure S4a) and accounted
for more than half of all SSRs (~ 50.51%) (Additional file 2:
Figure S4b). Hexa-nucleotide SSRs were the second most
abundant (199,408),and represented 20.47% of the SSRs in
the two subgenomes and scaffolds, followed by compound
and di-nucleotide SSRs. Of the repeat motif types, TA/AT
was most abundant, accounting for more than 4.0% of all
repeat motifs (Additional file 2: Figure S5). The second
highest number of repeat motifs was AAAAT/TTTTA,
which represented 3.4% of all repeat motifs. These analyses
showed that the AT repeat patterns were the dominant re-
peat motifs of SSR markers, whereas GC repeat patterns
were rare. (Additional file 1: Table S1), which accounted for 11.69%
of all identified SSRs. Of these newly developed SSR
markers, a total of 462,267 and 489,394 SSR markers were
located in subgenomes A (Additional file 3: Table S3 and
Additional file 4: Table S4) and B (Additional file 5: Table
S5 and Additional file 6: Table S6), which accounted for
47.46
and
50.25% of all
SSR
markers,
respectively
(Additional file 1: Table S1). In addition, 22,323 SSR markers
were mined on the nine scaffolds, and only accounted for
2.29% of all SSR markers (Additional file 7: Table S7). Based on the start positions of SSR markers, we suc-
cessfully anchored these markers to the reference gen-
ome physical map (Fig. 1). Genome-wide SSR marker development Currently, g-SSR markers are common and popular
for such analyses, and they have wide applications in mo-
lecular genetics and breeding, because they have multiple
advantages, such as simplicity, abundance, ubiquity, vari-
ation, co-dominance, and multi-allelism [28]. Recently,
several studies were devoted to developing different types
of SSR markers in peanut, such as EST-SSRs [11, 29],
transcriptome-SSRs
[12–14],
and
g-SSRs
[30],
even
though the peanut genome had not yet been resolved. With the recent completion of genome sequencing of pea-
nut and two diploid progenitor species, A. duranensis and
A. ipaensis, a large number of genome-wide g-SSRs were
identified [20–22]. Furthermore, tens of thousands of g-
SSR markers (51,354 for A. duranensis and 60,893 for A. ipaensis) were also developed from the two progenitor
species in 2017 [24]. However, there was limited reports
on the development of a large number of genome-wide g-
SSR markers from allotetraploid cultivated peanut because
of the challenging in its genome sequencing. Fortunately,
the genomes of allotetraploid A. hypogaea cv. Fuhuasheng,
Shitouqi, and Tifrunner were successfully sequenced
[25–27]. Here, large-scale genome-wide g-SSR markers
were developed from the A. hypogaea cv. Fuhuasheng
genome to help enhance genetic and genomic analyses
and molecular breeding of peanut. crops. Currently, g-SSR markers are common and popular
for such analyses, and they have wide applications in mo-
lecular genetics and breeding, because they have multiple
advantages, such as simplicity, abundance, ubiquity, vari-
ation, co-dominance, and multi-allelism [28]. Recently,
several studies were devoted to developing different types
of SSR markers in peanut, such as EST-SSRs [11, 29],
transcriptome-SSRs
[12–14],
and
g-SSRs
[30],
even
though the peanut genome had not yet been resolved. With the recent completion of genome sequencing of pea-
nut and two diploid progenitor species, A. duranensis and
A. ipaensis, a large number of genome-wide g-SSRs were
identified [20–22]. Furthermore, tens of thousands of g-
SSR markers (51,354 for A. duranensis and 60,893 for A. ipaensis) were also developed from the two progenitor
species in 2017 [24]. However, there was limited reports
on the development of a large number of genome-wide g-
SSR markers from allotetraploid cultivated peanut because
of the challenging in its genome sequencing. Fortunately,
the genomes of allotetraploid A. hypogaea cv. Fuhuasheng,
Shitouqi, and Tifrunner were successfully sequenced
[25–27]. Here, large-scale genome-wide g-SSR markers
were developed from the A. hypogaea cv. Fuhuasheng
genome to help enhance genetic and genomic analyses
and molecular breeding of peanut. Genome-wide SSR marker development For the physical map, the
marker density significantly differed among chromo-
somes, and ranged from 338.38 per Mb on chromosome
B01 to 699.92 per Mb on chromosome A08, with an
average density of 392.45 per Mb (Additional file 1:
Table S1; Fig. 2). Moreover, the distribution of each
chromosome showed that there was lower marker Development of new markers can help improve genetic
analysis, gene/QTL mapping, and molecular breeding of Fig. 1 Overview of the high-density SSR physical map in peanut (A. hypogaea L.).The bar represents the number of SSR markers within a
1-Mb window Fig. 1 Overview of the high-density SSR physical map in peanut (A. hypogaea L.).The bar represents the number of SSR markers within a
1-Mb window Lu et al. BMC Genomics (2019) 20:799 Page 4 of 9 Fig. 2 Genome-wide distribution of SSR markers on different chromosomes of the A. hypogaea genome. From the outer edge inward, circles
represent the (a) gene position; (b) distribution of all genes; (c) gene density per Mb; (d) distribution of the 1276 integrated public SSR markers;
(e) distribution of the 188 SSRs tested by PCR amplification; (f) distribution of all newly developed SSR markers; and (g) SSR marker density per
Mb. Blue and red lines represent homologous loci in different and the same chromosomes, respectively Fig. 2 Genome-wide distribution of SSR markers on different chromosomes of the A. hypogaea genome. From the outer edge inward, circles
represent the (a) gene position; (b) distribution of all genes; (c) gene density per Mb; (d) distribution of the 1276 integrated public SSR markers;
(e) distribution of the 188 SSRs tested by PCR amplification; (f) distribution of all newly developed SSR markers; and (g) SSR marker density per
Mb. Blue and red lines represent homologous loci in different and the same chromosomes, respectively In silico evaluation of the newly developed SSR markers
To evaluate the amplification specificity of the newly de-
veloped SSR markers, the forward and reverse primers
of each SSR marker were used for in silico analysis based
on the cultivated peanut genome sequences of A. hypo-
gaea cv. Tifrunner and Shitouqi, and genome sequences
of its two progenitors, A. duranensis and A. ipaensis
(Table 2). crops. Integration of publicly available SSR markers g
p
y
Previously, an integrated consensus genetic map, which
contained 5874 markers, was successfully constructed
with 20 linkage groups [16]. Based on forward and re-
verse primer sequences, a total of 5125 markers were
mapped to the reference genome, A. hypogaea cv. Fuhuasheng. Finally, 3304 markers with 6838 loci were
mined from 20 chromosomes (6699 loci) and nine scaf-
folds (139 loci) (Additional file 2: Figure S7). In addition,
most of the markers (2927), which accounted for 88.6%
of all markers, only matched one or two loci. For ex-
ample, approximately 1832 markers only matched a sin-
gle locus (Additional file 7: Table S10), and a total of
1095 markers were double loci identification on the refer-
ence genome (Additional file 2: Figure S7). The remaining
markers had more than three loci, and one marker even
matched 258 loci (Additional file 2: Figure S7). Further-
more, most of the multiple loci of these markers were lo-
cated in the corresponding subgenomic chromosomes,
such as A01 vs. B01 and A02 vs. B02 (Fig. 2). This may
have been caused by co-evolution of the two peanut
subgenomes. After clumped, a total of 39,599 SSR markers were
shared among the four Arachis species, which indicates
that these common markers were very conservative in
different Arachis (Fig. 3; Additional file 7: Table S8). Moreover, 9334 and 210,058 markers overlapped be-
tween the two progenitors and two cultivated varieties,
respectively. In
particular,
numerous
specific
SSR
markers were also obtained for each genome, which in-
dicates genomic polymorphism among the four different
Arachis species. Genome-wide SSR marker development The number of in silico products indicated that
87.35% (855,422) and 89.29% (874,384) of the SSR
markers generated only one in silico-specific product in
the twotetraploids A. hypogaea cv. Tifrunner and Shi-
touqi, respectively. Approximately 4% of markers were
mismatched in the two cultivated varieties, and less than
8% of markers generated two products. In addition, less
than 0.8% of SSR markers generated more than two
products; in particular, only 0.08 and 0.11% of the
markers generated ≥6 in silico products. For the two
progenitors, a total of 356,778 and 410,764 SSR markers,
which accounted for 36.43 and 41.59% of all SSR
markers, respectively, generated only one in silico-
specific product. However, more than half of total
markers were mismatched in the two progenitors. Ap-
proximately 3% of all markers generated two in silico
products, and less than 0.5% of the markers generated Lu et al. BMC Genomics (2019) 20:799 Page 5 of 9 Table 2 In silico PCR products in A. hypogaea and its two progenitors
specise
0
1
2
3
4
5
≥6
A. hypogaea cv. Tifrunner
40,116 (4.10%)
855,422 (87.35%)
72,710 (7.42%)
7789 (0.80%)
1850 (0.19%)
600 (0.06%)
804 (0.08%)
A. hypogaea cv. Shitouqi
40,210 (4.11%)
874,384 (89.29%)
53,994 (5.51%)
7162 (0.73%)
1777 (0.18%)
667 (0.07%)
1097 (0.11%)
A. duranensis
581,706 (59.4%)
356,778 (36.43%)
34,490 (3.52%)
4668 (0.48%)
933 (0.10%)
293 (0.03%)
423 (0.04%)
A. ipaensis
532,523 (54.38%)
410,764 (41.59%)
30,814 (3.15%)
4120 (0.42%)
607 (0.06%)
199 (0.02%)
264 (0.03%) more than two in silico products (Table 2). In total, 1,
729,806 and 767,542 markers could generate only one in
silico product in cultivated peanut and its two progeni-
tors, respectively. These SSR markers that only gener-
ated one in silico product would be potentially useful for
molecular breeding in the future. varieties. These results indicated that the newly developed
SSR markers were available and could be useful for molecu-
lar breeding strategies, such as true hybrid F1 offspring selec-
tion, in the future. PCR validation of the newly developed SSR markers in
different species To validate amplification of the newly developed SSR
markers, a total of 188 SSR markers with motifs ≥5 repeats
were arbitrarily and evenly selected for PCR amplification in
two cultivars, Fuhuasheng and Yueyouhei4hao (Additional
file 7: Table S9). In total, 83 of the SSR markers (44.15%)
could amplify clear target products in at least one of the two
varieties (Additional file 2: Figure S6). Moreover, 25 of the
83 SSR markers displayed polymorphism between the two Fig. 3 Venn diagram of SSR markers in different Arachis species Fig. 3 Venn diagram of SSR markers in different Arachis species Page 6 of 9 Lu et al. BMC Genomics (2019) 20:799 Lu et al. BMC Genomics (2019) 20:799 Page 6 of 9 Page 6 of 9 Of these 1832 unique loci that matched markers, a
total of 1276 SSR markers were located in the corre-
sponding genetic linkage groups (Additional file 7: Table
S10). Based on the physical positions of the 1276 pub-
licly
available
SSR
markers,
they
were
successfully
merged with the physical map and anchored to the draft
genome sequence of tetraploid cultivated peanut (Fig. 2). This map, which has the highest marker density and the
most uniform genetic background, will be of great bene-
fit to molecular breeding, and gene and QTL mining of
peanut in the future. near this QTL, GNB464 and TC40D04 were successfully
anchored in a 1.448-Mb region on chromosome A05 in the
A. hypogaea cv. Fuhuasheng reference genome (Additional
file 2: Figure S8d). Searching this region in the high-density
SSR physical map revealed a total of 819 newly developed
markers (Additional file 2: Figure S8d). Gene identification
of this region indicated that there were 108 genes, and
functional annotation showed that multiple genes were re-
lated to disease resistance, such as the TMV resistance pro-
tein and NAC domain (Additional file 7: Table S11). Genetic linkage mapping is an effective strategy for
QTL identification in crops. However, a high-density gen-
etic map is a prerequisite for QTL fine mapping of mul-
tiple phenotypic traits. In peanut, most QTLs were only
preliminarily mapped because of the lack of sufficiently
available genetic markers in the last decade [33–37]. Therefore, the development of molecular markers has be-
come increasingly important. Then, high-density inte-
grated consensus maps with thousands of markers were
constructed in 2013 and 2018 [15, 16]. PCR validation of the newly developed SSR markers in
different species However, in culti-
vated
peanut,
development
of
high-density
physical
markers was rare. Here, we developed millions of SSR
markers in cultivated peanut, and this large number of
newly developed SSR markers, such as multiple QTLs on
chromosome A05 or even on the region of qLLS_T13_
A05_7, could provide more possibilities for further fine
mapping or even gene cloning. A high-density physical map with uniform genomic
positions and coverage is necessary for conducting high-
resolution gene/QTL mapping in crops. During the past
years, several available QTLs for yield, disease resistance,
and other traits were detected in peanut (https://www. peanutbase.org/). However, most of the QTLs were de-
tected across different genetic backgrounds and environ-
ments, and only a few QTLs have been used for
molecular breeding; therefore, the use of the QTLs in
molecular breeding has been limited [31]. Thus, consen-
sus genetic map construction was needed to improve the
use of QTLs in molecular breeding. Therefore, two inte-
grated
consensus
genetic
maps
with
thousands
of
markers were constructed in 2013 and 2018 [15, 16],
and multiple independent consensus QTLs were identi-
fied [16]. Here, to obtain a comprehensive consensus
map, we integrated the published public SSR markers
with the newly developed SSR physical map (Fig. 1). The
availability of a high-density physical map would provide
an opportunity to generate high-throughput genotyping
data for different types of populations, and accelerate
mapping and breeding applications of different traits. Application of SSR markers in peanut High-density SSR physical maps can be used to screen
more markers for QTL fine mapping. For example, a
previous study reported that multiple novel QTLs for re-
sistance to leaf spots and tomato spotted wilt virus were
identified in peanut based on an improved genetic link-
age map with a total of 418 markers, and multiple resist-
ance QTL clusters were detected on linkage A05, which
only contained 24 markers [32]. Based on the physical
position of the marginal linkage markers (PM179–
TC40D04), a total of 6804 new SSR markers were ob-
tained in this region (Additional file 2: Figure S8a). On
the A05 linkage, a total of 11 leaf spot QTLs were de-
tected, especially for the QTL qLLS_T13_A05_7, which
had the largest LOD value (5.26) and phenotypic vari-
ation explanation (15.55%) (Additional file 2: Figure
S8b). Moreover, this QTL overlapped with our previous
meta-QTL
MQTL_LLS_A05.1,
sharing
the
common
marker GNB464 (Additional file 2: Figure S8b and c)
[16]. Based on the sequences of the flanking markers Conclusions Development of new genome-wide markers and construc-
tion of a physical map with uniformly distributed genome-
wide physical markers would aid in the elucidation of
complex traits and improvement of molecular breeding. In this study, we identified a total of 8,329,496 genomic
SSRs and developed 973,984 newly genomic SSR markers
from the cultivated peanut reference genome “Fuhua-
sheng” using MISA software with default parameters. Moreover, we mined a number of 39,599 common SSR
markers from two A. hypogaea cv. Tifrunner and Shitouqi
and two progenitors, A. duranensis and A. ipaensis. In
addition, we integrated 1276 public SSR loci with the
newly developed physical map, and obtained the first
high-density genomic physical map for peanut. Finally, we
fine mapped a leaf spot quantitative trait locus to a 1.448-
Mb region by marker encryption and identified 108 candi-
date genes. In summary, these newly developed and inte-
grated public SSR markers are an important genomic
resource for both accelerating genetic analyses of complex
traits and molecular breeding applications in peanut. Supplementary information
S
l
i f
i Supplementary information
Supplementary information accompanies this paper at https://doi.org/10. 1186/s12864-019-6148-5. pp
y
Supplementary information accompanies this paper at https://doi.org/10. 1186/s12864-019-6148-5. Additional file 1: Table S1. Number of SSRs and SSR markers identified
on different chromosomes. Table S2. Summary of SSR motifs and repeats. Additional file 1: Table S1. Number of SSRs and SSR markers identified
on different chromosomes. Table S2. Summary of SSR motifs and repeats. y
Additional file 2: Figure S1. Chromosome-wide distribution of SSRs in A. hypogaea cv. Fuhuasheng genome. Figure S2. Number of SSR repeat motifs. Figure S3. Abundance of the top 30 different types of SSR motifs. Figure S4. Number (A) and percentage (B) of different types of SSR markers. Figure S5. Summary of SSR types of the developed SSR markers. Figure S6. Product size
of 188 SSR markers tested by PCR amplification. Figure S7. Number of loci in
the public SSR markers as determined by e-PCR remapping. Figure S8. Comparison of known QTLs in genetic and physical maps. An R package (http://www.R-project.org), Cmplot, was
used to draw the high-density physical map of the newly
developed SSR markers, and another R package, Rcircos,
was used to visualize various features of each chromo-
some, such as gene position and distribution, the density
of all SSR markers, and the distribution of the tested
SSR markers. Additional file 3: Table S3. Summary of SSR markers in subgenome
A01–A05. Additional file 3: Table S3. Summary of SSR markers in subgenome
A01–A05. Additional file 4: Table S4. Summary of SSR markers in subgenome
A06–A10. Additional file 4: Table S4. Summary of SSR markers in subgenome
A06–A10. In silico evaluation and PCR validation of SSR markers
Four whole genome sequences, including those of two
cultivated peanut landraces, A. hypogaea cv. Shitouqi
[26] and Tifrunner [27], and their two diploid progeni-
tors, A. duranensis and A. ipaensis [20], were used as
templates for in silico evaluation of the designed SSR
markers using electronic PCR (e-PCR) with the follow-
ing parameters: 4-bp mismatch, 1-bp gap, and 0–2000-
bp product size [40]. The paired primers should meet Additional file 5: Table S5. Summary of SSR markers in subgenome
B01–B05. Additional file 5: Table S5. Summary of SSR markers in subgenome
B01–B05. Additional file 6: Table S6. Summary of SSR markers in subgenome
B06–B10. Additional file 7: Table S7. Summary of SSR markers on nine scaffolds. Table S8. Plant materials and DNA isolation Plant materials and DNA isolation A cultivated peanut that was de novo sequenced in our
previous study [25], A. hypogaea cv. Fuhuasheng, was Page 7 of 9 Page 7 of 9 Page 7 of 9 Lu et al. BMC Genomics (2019) 20:799 the following criteria: (1) only one in silico PCR product
generated from any one template; (2) the SSR basic
motif was as expected; and (3) the PCR product length
was also as expected. used as the reference genome for SSR identification. Two peanut varieties, Fuhuasheng and Yueyouhei4hao,
were used for amplification and validation of the newly
developed SSR markers by polymerase chain reaction
(PCR). All of the materials were planted in a field in the
summer season (July to November) at the experimental
station of Guangdong Academy of Agricultural Sciences,
Guangzhou, China. High-quality genomic DNA was ex-
tracted from young leaves using a Plant Genomic DNA
Extraction Kit (BioTeke Corporation, Beijing, China) ac-
cording to the manufacturer’s handbook (http://www. bioteke.com/en/?c=show&m=view&id=99). p
A total of 188 newly developed SSR primer pairs were
arbitrarily selected and then synthesized for validation
by PCR amplification. The PCR mixture was prepared in
a 10-μl volume that contained 1 μl template DNA (~
100 ng), 0.4 μl of each primer, 3.2 μl ddH2O, and 5 μl 2×
Power Taq PCR Master Mix (BioTeke Corporation,
Beijing, China). The PCR amplification program was as
follows: (1) 95 °C for 5 min; (2) total of 35 cycles, each
cycle was as below: 30 s at 95 °C, 30s at 55 °C for anneal-
ing, 30 s at 72 °C for extension; (3)10 min at 72 °C, and
then storage at 4 °C. PCR products were separated and
tested in Fragment Analyzer™Automated CE System
(Advanced Analytical Technologies, Inc., Beijing, China),
and PROSize 2.0 was used to analyze and visualize the
data (https://www.aati-us.com/support/software/). Identification of SSRs and design for SSR markers Identification of SSRs and design for SSR markers
The reference genome sequence of A. hypogaea cv. Fuhuasheng was downloaded from GenBank under ac-
cession number SDMP00000000 [25]. Perl scripts from
MISA were used to perform SSR identification with the
default parameters (http://pgrc.ipk-gatersleben.de/misa/
). The identification criteria were as follows: mono-
nucleotide repeat motifs with at least 12 repeats, di-
nucleotide repeat motifs with five repeats, tri-nucleotide
repeat motifs with four repeats, tetra-nucleotide repeat
motifs
with
three
repeats,
and
penta-
and
hexa-
nucleotide repeat motifs with two repeats. Compound
SSRs were defined as those with a < 100-nt interval be-
tween two repeat motifs [24, 38]. Integrating new and previously published SSR markers
In a previous study, we constructed an integrated con-
sensus genetic map with published markers, including
EST-SSR, transcriptome-SSR, and g-SSR markers [16]. Based on the forward and reverse primer sequences of
each marker, these public SSR markers were mapped to
the A. hypogaea cv. Fuhuasheng reference genome for
integration with the newly developed SSR markers using
e-PCR software with default parameters [40]. The forward and reverse primers of each SSR were de-
signed based on the flanking sequences of SSR repeat
motifs using Primer 3 [39]. Two Perl scripts, p3_in.pl
and
p3_out.pl,
were
used
for
the
programmer-to-
programmer data interchange between MISA and Pri-
mer
3.0
(http://pgrc.ipk-gatersleben.de/misa/primer3. html). The primer design criteria were as follows: primer
length was between 18 and 27 bp, melting temperature
was 57 to 63 °C, GC content ranged from 30 to 70%, and
product size was from 100 to 300 bp. Funding 10. Guo B, Chen X, Hong Y, Liang X, Dang P, Brenneman T, et al. Analysis of
gene expression profiles in leaf tissues of cultivated peanuts and
development of EST-SSR markers and gene discovery. Int J Plant Genomics. 2009;715605. https://doi.org/10.1186/1471-2229-9-35. This work was supported by the National Natural Science Foundation of
China (31771841, 31801401), the National key research and development
program (2018YFD0201009), the Science and Technology Program of
Guangzhou, China (201904010190), the Open Fund of Guangdong Key
Laboratory of Crop Genetic Improvement (2017B030314090), the Natural
Science Foundation of Guangdong Province (2017A030311007), the Modern
Agro-industry Technology Research System (CARS-13), the teamwork projects
funded by Guangdong Natural Science Foundation (No.2017A030312004),
“Jinying Star” (2018) and “Youth Associate Research Fellow” (2019) Talent Pro-
ject of Guangdong Academy of Agricultural Sciences. These funding bodies
had no role in the design of the study, extraction, analysis, and interpretation
of data, or in writing the manuscript. 11. Liang X, Chen X, Hong Y, Liu H, Zhou G, Li S, et al. Utility of EST derived SSR
in cultivated peanut (Arachis hypogaea L.) and Arachis wild species. BMC
Plant Biol. 2009;9:35. 12. Zhang J, Liang S, Duan J, Wang J, Chen S, Cheng Z, et al. De novo assembly
and characterization of the transcriptome during seed development, and
generation of genic-SSR markers in Peanut (Arachis hypogaea L.). BMC
Genomics. 2012;13:90. 13. Huang L, Wu B, Zhao J, Li H, Chen W, Zheng Y, et al. Characterization and
transferable utility of microsatellite markers in the wild and cultivated
Arachis species. PLoS One. 2016;11:e0156633. Supplementary information
S
l
i f
i Summary of common SSR markers in four Arachis species. Table S9. Summary of 188 SSR markers tested by PCR amplification. Table S10. Summary of the public SSR markers with only a single locus
match to the reference genome. Table S11. Candidate gene
identification and annotation for the target QTL, qLLS_T13_A05_7. Page 8 of 9 Page 8 of 9 Page 8 of 9 Lu et al. BMC Genomics (2019) 20:799 Lu et al. BMC Genomics (2019) 20:799 Lu et al. BMC Genomics Acknowledgments 7. Pandey MK, Roorkiwal M, Singh VK, Ramalingam A, Kudapa H, Thudi M,
et al. Emerging genomic tools for legume breeding: current status and
future prospects. Front Plant Sci. 2016;7:455. Consent for publication Consent for publication
Not applicable. 16. Lu Q, Liu H, Hong YB, Li H, Liu HY, Li XY, et al. Consensus map integration
and QTL meta analysis narrowed a locus for yield traits to 0.7 cM and
refined a region for late leaf spot resistance traits to 0.38 cM on linkage
group A05 in peanut (Arachis hypogaea L.). BMC Genomics. 2018;19:887. Not applicable. group A05 in peanut (Arachis hypogaea L.). BMC Genomics. 2018;19 Received: 27 July 2019 Accepted: 29 September 2019 Received: 27 July 2019 Accepted: 29 September 2019 22. Lu Q, Li H, Hong Y, Zhang G, Wen S, Li X, et al. Genome sequencing and
analysis of the peanut B-genome progenitor (Arachis ipaensis). Front Plant
Sci. 2018;9:1099. Abbreviations 5. Mondal S, Badigannavar AM. Mapping of a dominant rust resistance gene
revealed two R genes around the major rust_QTL in cultivated peanut
(Arachis hypogaea L.). Theor Appl Genet. 2018;131:1671–81. ESTs: Expressed sequence tags; PCR: Polymerase chain reaction;
QTL: Quantitative trait locus; SSR: Simple sequence repeats 6. Varshney RK, Mohan SM, Gaur PM, Gangarao NV, Pandey MK, Bohra A, et al
Achievements and prospects of genomics-assisted breeding in three
legume crops of the semi-arid tropics. Biotechnol Adv. 2013;31:1120–34. Authors’ contributions XPC and XQL designed the experiments. QL, YBH, JNZ and HFL1 analyzed
the data. HL and HFL2 developed SSR markers. HYL, XYL and SW performed
PCR experiments. QL and YBH and HFJ drafted the manuscript. SXL, GYZ and
Varshney RK revised the manuscript, respectively. All authors have read and
approved the final manuscript. 1 Haofa Lan; 2 Haifen Li. 8. Gupta PK, Balyan HS, Sharma PC, Ramesh B. Microsatellites in plants: a new
class of molecular markers. Curr Sci. 1996;70:45–54. 9. Haq SU, Jain R, Sharma M, Kachhwaha S, Kothari SL. Identification and
characterization of microsatellites in expressed sequence tags and their
cross transferability in different plants. Int J Genomics. 2014; 2014:863948. See: https://www.ncbi.nlm.nih.gov/pubmed/25389527. Author details
1 18. Kochert G, Halward T, Branch WD, Simpson CE. RFLP variability in peanut (Arachis
hypogaea L.) cultivars and wild species. Theor Appl Genet. 1991;81:565–70. 1Crops Research Institute, Guangdong Academy of Agricultural Sciences,
South China Peanut Sub-Center of National Center of Oilseed Crops
Improvement, Guangdong Provincial Key Laboratory for Crop Genetic
Improvement, Guangzhou 510640, China. 2MolBreeding Biotechnology Co.,
Ltd., Shijiazhuang, China. 3Center of Excellence in Genomics & Systems
Biology, International Crops Research Institute for the Semi-Arid Tropics
(ICRISAT), Hyderabad, India. 4Key Laboratory of Biology and Genetic
Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research
Institute of the Chinese Academy of Agricultural Sciences, Wuhan 430062,
China. 19. Moretzsohn MC, Gouvea EG, Inglis PW, Leal-Bertioli SC, Valls JF, Bertioli DJ. A
study of the relationships of cultivated peanut (Arachis hypogaea) and its
most closely related wild species using intron sequences and microsatellite
markers. Ann Bot. 2013;111:113–26. 20. Bertioli DJ, Cannon SB, Froenicke L, Huang G, Farmer AD, Cannon EK, et al. The genome sequences of Arachis duranensis and Arachis ipaensis, the
diploid ancestors of cultivated peanut. Nat Genet. 2016;48:438–46. 21. Chen X, Li H, Pandey MK, Yang Q, Wang X, Garg V, et al. Draft genome of
the peanut A-genome progenitor (Arachis duranensis) provides insights into
geocarpy, oil biosynthesis, and allergens. Proc Natl Acad Sci U S A. 2016;113:
6785–90. Competing interests p
g
The authors declare that they have no competing interests. 17. Cho YG, Ishii T, Temnykh S, Chen X, Lipovich L, McCouch SR, et al. Diversity
of microsatellites derived from genomic libraries and GenBank sequences in
rice (Oryza sativa L.). Theor Appl Genet. 2000;100:713–22. 25.
Chen X, Lu Q, Liu H, Zhang J, Hong Y, Lan H, et al. Sequencing of cultivated
peanut, Arachis hypogaea, yields insights into genome evolution and oil
improvement. Mol Plant. 2019;12:920–34. 3.
Chen Y, Ren X, Zheng Y, Zhou X, Huang L, Yan L, et al. Genetic mapping of
yield traits using RIL population derived from Fuchuan Dahuasheng and
ICG6375 of peanut (Arachis hypogaea L.). Mol Breed. 2017;37:17. 4.
Shasidhar Y, Vishwakarma MK, Pandey MK, Janila P, Variath MT, Manohar SS,
et al. Molecular mapping of oil content and fatty acids using dense genetic
maps in groundnut (Arachis hypogaea L.). front. Plant Sci. 2017;8:794. Availability of data and materials 14. Zhong R, Zhou M, Zhao C, Hou L, Li C, Wang X, et al. SSR marker
development from peanut gynophore transcriptome sequencing. Plant
Breed. 2016;135:111–7. The datasets supporting the conclusions of this article are included within
the article and its additional files. 15. Shirasawa K, Bertioli DJ, Varshney RK, Moretzsohn MC, Leal-Bertioli SC, Thudi
M, et al. Integrated consensus map of cultivated peanut and wild relatives
reveals structures of the a and B genomes of Arachis and divergence of the
legume genomes. DNA Res. 2013;20:173–84. Ethics approval and consent to participate
Not applicable. Not applicable. 3.
Chen Y, Ren X, Zheng Y, Zhou X, Huang L, Yan L, et al. Genetic mapping of
yield traits using RIL population derived from Fuchuan Dahuasheng and
ICG6375 of peanut (Arachis hypogaea L.). Mol Breed. 2017;37:17.
4.
Shasidhar Y, Vishwakarma MK, Pandey MK, Janila P, Variath MT, Manohar SS,
et al. Molecular mapping of oil content and fatty acids using dense genetic
maps in groundnut (Arachis hypogaea L.). front. Plant Sci. 2017;8:794. References Genome-wide
characterization of microsatellites in Triticeae species: abundance,
distribution and evolution. Sci Rep. 2016;6:32224. 38. Deng P, Wang M, Feng K, Cui L, Tong W, Song W, et al. Genome-wide
characterization of microsatellites in Triticeae species: abundance,
distribution and evolution. Sci Rep. 2016;6:32224. 39. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, et al. Primer3—new capabilities and interfaces. Nucleic Acids Res. 2012;40:e115. 40. Schuler GD. Electronic PCR: bridging the gap between genome mapping
and genome sequencing. Trends Biotechnol. 1998;16:456–9. References 1. Krishna G, Singh BK, Kim EK, Morya VK, Ramteke PW. Progress in genetic
engineering of peanut (Arachis hypogaea L.)—a review. Plant Biotechnol J. 2015;13:147–62. 1. Krishna G, Singh BK, Kim EK, Morya VK, Ramteke PW. Progress in genetic
engineering of peanut (Arachis hypogaea L.)—a review. Plant Biotechnol J. 2015;13:147–62. 23. Yin D, Ji C, Ma X, Li H, Zhang W, Li S, et al. Genome of an allotetraploid wild
peanut Arachis monticola: a de novo assembly. Gigascience. 2018;7:1–9. 24. Zhao C, Qiu J, Agarwal G, Wang J, Ren X, Xia H, et al. Genome-wide
discovery of microsatellite markers from diploid progenitor species, Arachis
duranensis and A. ipaensis, and their application in cultivated peanut (A. hypogaea). Front Plant Sci. 2017;8:1209. 2. Pandey MK, Wang ML, Qiao L, Feng S, Khera P, Wang H, et al. Identification
of QTLs associated with oil content and mapping FAD2 genes and their
relative contribution to oil quality in peanut (Arachis hypogaea L.). BMC
Genet. 2014;15:133. 25. Chen X, Lu Q, Liu H, Zhang J, Hong Y, Lan H, et al. Sequencing of cultivated
peanut, Arachis hypogaea, yields insights into genome evolution and oil
improvement. Mol Plant. 2019;12:920–34. 3. Chen Y, Ren X, Zheng Y, Zhou X, Huang L, Yan L, et al. Genetic mapping of
yield traits using RIL population derived from Fuchuan Dahuasheng and
ICG6375 of peanut (Arachis hypogaea L.). Mol Breed. 2017;37:17. 26. Zhuang W, Chen H, Yang M, Wang J, Pandey MK, Zhang C, et al. The
genome of cultivated peanut provides insight into legume karyotypes,
polyploid evolution and crop domestication. Nat Genet. 2019;51:865–76. 4. Shasidhar Y, Vishwakarma MK, Pandey MK, Janila P, Variath MT, Manohar SS,
et al. Molecular mapping of oil content and fatty acids using dense genetic
maps in groundnut (Arachis hypogaea L.). front. Plant Sci. 2017;8:794. Page 9 of 9 Lu et al. BMC Genomics (2019) 20:799 Lu et al. BMC Genomics (2019) 20:799 27. Bertioli DJ, Jenkins J, Clevenger J, Dudchenko O, Gao D, Seijo G, et al. The
genome sequence of segmental allotetraploid peanut Arachis hypogaea. Nat Genet. 2019;51:877–84. 28. Powell W, Machray GC, Provan J. Polymorphism revealed by simple
sequence repeats. Trends Plant Sci. 1996;1:215–22. 29. Song GQ, Li MJ, Xiao H, Wang XJ, Tang RH, Xia H, et al. EST sequencing and
SSR marker development from cultivated peanut (Arachis hypogaea L.). ElectrJ Biotechnol. 2010;13:7–8. 30. References Wang H, Penmetsa RV, Yuan M, Gong L, Zhao Y, Guo B, et al. Development
and characterization of BAC-end sequence derived SSRs, and their
incorporation into a new higher density genetic map for cultivated peanut
(Arachis hypogaea L.). BMC Plant Biol. 2012;12:10. 31. Zhao T, Wang J, Xu G, Weng B. Advance on peanut breeding of high-
yielding and good-quality and the application of biological technology. Chin J Trop Crop. 2011;32:2187–95 (in Chinese with English abstract). 32. Pandey MK, Wang H, Khera P, Vishwakarma MK, Kale SM, Culbreath AK, et al. Genetic dissection of novel QTLs for resistance to leaf spots and tomato
spotted wilt virus in peanut (Arachis hypogaea L.). Front Plant Sci. 2017;8:25. 33. Hong YB, Liang XQ, Chen XP, Liu HY, Zhou GY, Li SX, et al. Construction of
Genetic Linkage Map Based on SSR Markers in Peanut (Arachis hypogaea L.). Agr Sci China. 2008;7:915–21. 34. Varshney RK, Bertioli DJ, Moretzsohn MC, Vadez V, Krishnamurthy L, Aruna R,
et al. The first SSR-based genetic linkage map for cultivated groundnut
(Arachis hypogaea L.). Theor Appl Genet. 2009;118:729–39. 35. Khedikar YP, Gowda MV, Sarvamangala C, Patgar KV, Upadhyaya HD,
Varshney RK. A QTL study on late leaf spot and rust revealed one major QTL
for molecular breeding for rust resistance in groundnut (Arachis hypogaea L. ). Theor Appl Genet. 2010;121:971–84. 35. Khedikar YP, Gowda MV, Sarvamangala C, Patgar KV, Upadhyaya HD,
Varshney RK. A QTL study on late leaf spot and rust revealed one major QTL
for molecular breeding for rust resistance in groundnut (Arachis hypogaea L. ). Theor Appl Genet. 2010;121:971–84. 36. Sarvamangala C, Gowda MVC, Varshney RK. Identification of quantitative
trait loci for protein content, oil content and oil quality for groundnut
(Arachis hypogaea L.). Field Crop Res. 2011;122:49–59. 37. Sujay V, Gowda MVC, Pandey MK, Bhat RS, Khedikar YP, Nadaf HL, et al. Quantitative trait locus analysis and construction of consensus genetic map
for foliar disease resistance based on two recombinant inbred line
populations in cultivated groundnut (Arachis hypogaea L.). Mol Breeding. 2012;30:773–88. 37. Sujay V, Gowda MVC, Pandey MK, Bhat RS, Khedikar YP, Nadaf HL, et al. Quantitative trait locus analysis and construction of consensus genetic map
for foliar disease resistance based on two recombinant inbred line
populations in cultivated groundnut (Arachis hypogaea L.). Mol Breeding. 2012;30:773–88. 38. Deng P, Wang M, Feng K, Cui L, Tong W, Song W, et al. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
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English
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How does climate change adaptation affect public budgets? Development of an assessment framework and a demonstration for Austria
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Mitigation and adaptation strategies for global change
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How does climate change adaptation affect public budgets?
Development of an assessment framework
and a demonstration for Austria Gabriel Bachner1
& Birgit Bednar-Friedl1,2 & Nina Knittel1 Received: 24 September 2018 /Accepted: 15 January 2019
# The Author(s) 2019
/Published online: 4
2019
February ORIGINAL ARTICLE
How does climate change adaptation affect public budgets?
Development of an assessment framework
and a demonstration for Austria ORIGINAL ARTICLE * Gabriel Bachner
gabriel.bachner@uni-graz.at https://doi.org/10.1007/s11027-019-9842-3
Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 https://doi.org/10.1007/s11027-019-9842-3
Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11027-019-
9842-3) contains supplementary material, which is available to authorized users. 1 Introduction In the last decade, national strategies for adaptation to climate change have been developed and
implemented in numerous countries (Lesnikowski et al. 2015). It is widely held that, unlike
mitigation, adaptation is a private good and therefore should be carried out by private actors
(Tol 2005), whereas the role of the public sector is limited to providing the right incentives
(Jones et al. 2013) and to correct for market failure (Osberghaus et al. 2010). However, the
state owns public infrastructure, acts as service provider (e.g., by means of early warning
systems), and plays a significant role as information broker (e.g., to coordinate extreme
weather event preparedness and response) (Eakin and Patt 2011). This view is also supported
when looking at national adaptation strategies, since a considerable share of adaptation is
initiated by legislation, as well as financed and implemented by the public sector (Urwin and
Jordan 2008; Biesbroek et al. 2010; McDonald 2011; Mees et al. 2012). Motivated by that, this
paper takes a closer look at the direct and indirect consequences of public adaptation for
federal budgets and develops a general modeling framework for quantification. The public household is affected by climate change impacts and public adaptation through
various channels (Bräuer et al. 2009; Bachner and Bednar-Friedl 2018). The impacts of climate
change lead to direct costs for the public household, such as higher expenditures for disaster
relief payments to private households or reconstruction of damaged public infrastructure. The
benefit of adaptation is that it reduces some of these direct impact costs. Additionally, indirect
effects on public budgets arise from both impacts and adaptation measures. This is because of
changes in the tax base (e.g., due to changed economic activity and structure) and thus also tax
revenues (cf. Lis and Nickel 2010; Schinko et al. 2016). Public adaptation is usually integrated into the government’s general practices to Bclimate
proof^ investment decisions and to mainstream adaptation into other policy fields (Bierbaum
et al. 2013). However, the economic literature on adaptation until now has mostly ignored this
complexity and modeled adaptation in a very stylized way, e.g., in the form of generic
adaptation capital formation to identify an optimal adaptation level (de Bruin et al. 2009;
Agrawala et al. 2011a, b; Bosello et al. 2013). Abstract Public adaptation to climate change affects government budgets directly on the expenditure side, but
also indirectlyviachangesin the tax base and governmentconsumption patterns. While such indirect
effects have been analyzed intensively for mitigation policies, similarly detailed model-based frame-
works and studies for adaptation policy are still missing. The objectives of the present paper are (i) to
fill this gap by proposing a general modeling framework that allows for a comprehensive analysis of
effects of adaptation on federal budgets, both on the expenditure and the revenue side, as well as of
macroeconomiceffectsand(ii)todemonstrateitsusefulnessbyapplyingthisframeworktothecaseof
Austria.Wefindthatpublicadaptationcanleadtosubstantialpositivemacroeconomiceffectsongross
domesticproduct(GDP),welfare,andemployment.Theresultsarerobustwithrespecttoassumptions
abouttheeffectivenessofadaptation.Also,wedemonstratethatitisessentialforanalysistocoverboth
the expenditure and revenue side, as overall government revenues can increase due to adaptation,
offsetting additional direct public expenses for adaptation, thus increasing the budget balance. This is
becauseoflesssevereclimatechangeimpactsandthecorrespondinglowerpaymentsforpost-disaster
relief and unemployment benefits as well as higher tax revenues. We thus strongly recommend
makinguseofeconomy-widemodelingframeworkswhenplanningfor adaptation,astheyshedlight
on the true costs and benefits of adaptation. Keywords Climate change . Public adaptation . Indirect effects . Public budgets . Computable
general equilibrium Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11027-019-
9842-3) contains supplementary material, which is available to authorized users. * Gabriel Bachner
gabriel.bachner@uni-graz.at * Gabriel Bachner
gabriel.bachner@uni-graz.at Birgit Bednar-Friedl
birgit.friedl@uni-graz.at 1
Wegener Center for Climate and Global Change, University of Graz, Brandhofgasse 5, 8010 Graz,
Austria 2
Institute of Economics, University of Graz, Universitätsstraße 15, 8010 Graz, Austria Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1326 1 Introduction For practical decision-making on public
adaptation, however, these generalized insights from integrated assessment models are insuf-
ficient in that they do not allow for assessing the effects and usefulness of specific adaptation
measures. This paper intends to fill this gap by developing a modeling framework that allows
for a more detailed analysis of the economy-wide and budgetary effects of public adaptation. As shown in the literature, climate change leads to significant losses in a broad range of
economic sectors and of overall economic performance (Ciscar et al. 2011, 2012; Bosello et al. 2012; Sussman et al. 2014; Bosello and De Cian 2014; OECD 2015; Steininger et al. 2015;
Dellink et al. 2017). Economy-wide effects can be much larger than direct impacts within sectors,
due to indirect effects (Hallegatte et al. 2007). As argued above, adaptation also influences public
budgets indirectly via sectoral spillovers and macroeconomic feedback effects, which affect the
tax base and thereby alter the budget balance and the fiscal position of a country. Yet while the fiscal implications of mitigation have been addressed (see Siegmeier et al. 2018 for a review), there is little known about the implications of public adaptation on
government budgets. We argue that there is an essential difference between mitigation and
adaptation, which requires a separate and detailed analysis to better understand the effects of
adaptation on government budgets. While mitigation policies have the potential to generate
revenues (e.g., via carbon taxes), the direct effects of adaptation arise mostly on the expendi-
ture side. Mitigation therefore allows for cutting distortionary taxes or increasing expenditures Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1327 (such as on carbon-free technologies) but financing adaptation requires either cutting other
public expenditures or raising taxes in order to ensure sustainable government finances. (such as on carbon-free technologies) but financing adaptation requires either cutting other
public expenditures or raising taxes in order to ensure sustainable government finances. In general, the literature on budgetary implications of climate change impacts and adapta-
tion is scarce. Regarding the direct consequences of climate change impacts for public budgets,
some papers describe effects qualitatively and provide empirical estimates for changes in
expenditures (Bräuer et al. 2009; Margulis and Narain 2010; Osberghaus and Reif 2010; Jones
et al. 2013; Gilmore and St. Clair 2018). 1 Introduction Complementary to these are econometric studies that
estimate the effects of climate variability and extreme weather events on public finances (Lis
and Nickel 2010; Ouattara and Strobl 2013; Melecky and Raddatz 2015; Leppänen et al. 2015); however, they are limited to direct effects on public expenditures, disregarding indirect
effects. Indirect effects to public budgets have been analyzed for climate change impacts—but
not for public adaptation—in a computable general equilibrium framework by Bachner and
Bednar-Friedl (2018). Since the quantification of adaptation-induced effects on public budgets is still underde-
veloped, the objectives of this paper are as follows. First, we present a general modeling
framework that allows for a comprehensive analysis of effects of adaptation on federal
budgets, both on the expenditure and the revenue side, as well as of macroeconomic effects. The proposed framework can be applied to any country or region, even though adaptation is
very location specific. Second, we demonstrate its usefulness by applying this framework to
the case of Austria. Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1328 Baseline
Impact
Adaptation
Tax revenue loss
Other transfers
Unemployment benefits
Additional adaptation costs
Reconstruction and relief payments
Non-climate related public services
Government
consumption
Increased government
consumption with adaptation
Decrease in
other transfers to
balance revenue
and expenditure
Total government expenditure
Fig. 1 Conceptual model of effects of climate change impacts and adaptation on government expenditures. Magnitudes of expenditure categories in the Baseline are set to reflect the EU-wide public expenditure structures
(EUROSTAT 2018). The effects in the Impact and Adaptation scenarios are depicted exaggeratedly for
illustrative purposes Baseline
Impact
Adaptation
Government
consumption
Increased government
consumption with adaptation
Decrease in
other transfers to
balance revenue
and expenditure
Total government expenditure Fig. 1 Conceptual model of effects of climate change impacts and adaptation on government expenditures. Magnitudes of expenditure categories in the Baseline are set to reflect the EU-wide public expenditure structures
(EUROSTAT 2018). The effects in the Impact and Adaptation scenarios are depicted exaggeratedly for
illustrative purposes Fig. 1 Conceptual model of effects of climate change impacts and adaptation on government expenditures. Magnitudes of expenditure categories in the Baseline are set to reflect the EU-wide public expenditure structures
(EUROSTAT 2018). The effects in the Impact and Adaptation scenarios are depicted exaggeratedly for
illustrative purposes for expenditure but also increasing the need for unemployment benefits (lower economic
activity implies higher unemployment). To ensure no increase of the public deficit, govern-
ment expenditures need to be re-distributed within the now smaller total available budget. To
achieve this aim, two adjustments are possible: cutting either transfers to households or other
non-climate-related government consumption, or both. It is plausible to assume for many
countries that the level of government consumption is fixed by multi-annual budgeting rules. As a consequence, the total level of government consumption would remain constant but is
restructured towards more disaster relief payments and less other government consumption,
leaving other transfers to balance revenue and expenditure (as indicated in Fig. 1). for expenditure but also increasing the need for unemployment benefits (lower economic
activity implies higher unemployment). To ensure no increase of the public deficit, govern-
ment expenditures need to be re-distributed within the now smaller total available budget. To
achieve this aim, two adjustments are possible: cutting either transfers to households or other
non-climate-related government consumption, or both. 2.1 Scenarios and conceptual model In the modeling framework, we distinguish between three types of scenarios for 2050: (i) the
Baseline scenario, describing general socioeconomic development; (ii) the Impact scenario,
which adds climate change impacts to the Baseline; and (iii) the Adaptation scenario, which
adds adaptation measures to the Impact scenario, reducing impacts but also inducing new costs
for adaptation. By comparing these scenarios to each other, we are able isolate effects of
climate change impacts (Impact versus Baseline scenario) and of adaptation (Adaptation
versus Impact scenario). In the Impact and Adaptation scenarios, the federal government’s
budget is affected both on the expenditure and on the revenue side, however, in different ways. Figure 1 conceptually illustrates the effects of climate change impacts and adaptation on
government expenditures. In the Baseline scenario, expenditures consist of four major items:
(i) non-climate-related government consumption of goods and services (including wages of
public workers), (ii) expenditures on reconstruction (investment) and relief payments for
damages caused by extreme weather events, (iii) unemployment benefits to be paid as social
security, and (iv) other transfers to households. The first two items represent general govern-
ment consumption. With climate change, i.e., in the Impact scenario, the composition, and the level of
government expenditures change. As a direct effect of climate change, reconstruction and
relief payments increase due to higher damages, but several indirect effects also emerge. Depending on the sectoral and macroeconomic impact of climate change, tax revenues change. As most macroeconomic climate change impact studies find a negative effect on general
economic activity (GDP), it is plausible to assume a loss in tax revenues, reducing the means 2.2 Quantitative model and analysis To analyze and quantify the economy-wide and budgetary effects of climate change impacts
and adaptation, including indirect effects, we suggest a stepwise approach. Step 1 aims to
isolate adaptation (relevant) expenditures from total public expenditures. To do so, budget and
government expenditure reports need to be collected and screened for adaptation-relevant
keywords that indicate adaptation actions. The selection of adaptation-relevant keywords can
be supported by reviewing national adaptation strategies, implementation plans, or submis-
sions to national climate assessments (Bierbaum et al. 2013). After the identification of
adaptation-relevant budgetary items, it is necessary to narrow down adaptation-relevant
expenditure to adaptation expenditure. This can be done by applying shares, which is not an
easy endeavor, especially when adaptation is not the primary objective but a co-benefit
(Gilmore and St. Clair 2018). Experts in ministerial departments often have valuable knowl-
edge concerning the exact purposes of budgetary items and can thus aid in estimating the
shares of adaptation. Alternatively, the OECD Development Assistance Committee (2016) and
the European Commission (2016) developed a set of attribution rules that indicate which share
of total costs should be assigned to adaptation expenditures for specific budgetary items. In step 2, starting from these current expenditures, a realistic adaptation pathway is co-
developed for the future (e.g., 2050) together with experts and stakeholders, ideally from key
ministries engaged in adaptation action as well as finance ministries. Often, however, the
capacity and the willingness to engage in such a process is limited. An alternative procedure is
therefore to develop this pathway based on general recommendations from the literature, such
as the concept of adaptation phasing (Watkiss et al. 2015). To deal with uncertainties and limits
to specific adaptation actions, such a pathway should not be regarded as set in stone, but rather
be regularly re-assessed in an iterative manner, such as is proposed by Haasnoot et al. (2013)
who present the approach of Bdynamic adaptive policy pathways.^ In step 3, the direct and indirect consequences of the adaptation pathway for the public
budget are analyzed. In principle, several approaches could be used to assess these impacts:
projections based on econometric models (Lis and Nickel 2010; Ouattara and Strobl 2013;
Leppänen et al. 2015), government financial risk simulation models like CATSIM (Hochrainer-
Stigler et al. 2014b), or multi-sectoral computable general equilibrium (CGE) models. Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 It is plausible to assume for many
countries that the level of government consumption is fixed by multi-annual budgeting rules. As a consequence, the total level of government consumption would remain constant but is
restructured towards more disaster relief payments and less other government consumption,
leaving other transfers to balance revenue and expenditure (as indicated in Fig. 1). In the Adaptation scenario, direct and indirect effects again impact government expendi-
tures. The direct effect is caused by additional public adaptation costs. We assume that multi-
annual budgeting accounts for public (planned) adaptation and thus adaptation increases total
government consumption. The indirect effects arise again via macroeconomic feedback effects. Impacts are reduced by adaptation and economic activity increases; therefore, total tax revenue
losses are less severe compared to the Impact scenario. Since reconstruction and relief payments
are smaller with adaptation, other government consumption is expanded as compared to the
Impact scenario (however, not reaching the Baseline level due to residual damages also in the
Adaptation scenario). Due to higher employment, which can be caused by both higher
economic activity and typically high labor intensity of government consumption, expenditure
for unemployment benefits is lower compared to the Impact scenario. Depending on the size of
the of tax revenue loss and the effect on employment, other transfers can then either be
increased (as shown in Fig. 1) or cut to keep the balance between revenue and expenditure. In this context, it is important to consider government budgeting rules. Many countries have
committed themselves to avoiding excessive deficits and pursuing fiscal sustainability. For
instance, the European Stability and Growth Pact (European Union 2008, Art. 121 and 126)
requires that all member states keep the government deficit below 3% of GDP, with govern-
ment debt not exceeding 60% of GDP. In our analysis and as illustrated in Fig. 1, we therefore
assume that government consumption in the Impact scenario is fixed at the Baseline level, and
that this fixation is established via adjustments in transfers to households. Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1329 2.2 Quantitative model and analysis Econo-
metric models are based on time-series or panel data, which allows for an assessment of
budgetary effects over time, but they usually neglect macroeconomic feedback effects that alter
the tax base. Financial risk simulation models are particularly adept at studying loss distribu-
tions and effects on budgetary gaps, but the representation of the overall economy is highly
aggregated. CGE models have the advantage of capturing these macroeconomic and cross-
sectoral feedback effects as they distinguish between different economic sectors and agents
(Fisher-Vanden et al. 2013) and also capture the revenue side. However, CGE models are more
suitable for capturing long-term equilibrium effects, not short-term effects. 3 Model implementation and data In the following analysis, we apply the described modeling framework to the case of Austria. We focus on the impact fields with the highest federal budgetary importance and with
potentially strong impacts for the Austrian economy (Bednar-Friedl et al. 2017): Agriculture,
Forestry, and Catastrophe Management (including protection from natural hazards). Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1330 Agriculture is heavily subsidized and thus public resources indirectly fund adaptation in this
sector. Forestry is of high relevance since its expected contribution to macroeconomic damages
is relatively high (−0.8% GDP loss in 2050; Bachner et al. 2015). In addition, the government
owns a large share of the protection forests in Austria. Catastrophe Management is closely
connected to the public domain, since the Austrian disaster fund is fully financed out of tax
revenue (Schinko et al. 2016). Our scenarios are in line with the RCP-SSP framework, which
is standard in climate change research. Representative Concentration Pathways (RCPs) de-
scribe different global emission trajectories, while Shared Socioeconomic Pathways (SSPs)
describe different narratives for socioeconomic development. By combining RCPs and SSPs,
different states of the world emerge in which climate change impacts materialize (Moss et al. 2010; O’Neill et al. 2014). For the Baseline scenario, we choose a Bmiddle-of-the-road^ shared
socioeconomic pathway (SSP2; O’Neill et al. 2014). For details on the Baseline calibration,
see Appendix A.1. 1 This co-development took place within the research project PACINAS (http://anpassung.ccca.at/pacinas/en/),
funded by the Austrian Climate and Energy Fund. 3.1 Current and future public adaptation at the federal level in Austria To identify current federal expenditures on public adaptation (step 1), we screened the federal
budget in the base year (2016) for adaptation-relevant expenditure items and categorized them
into gray, green, or soft measures (see Appendix A.2 for details). Soft measures comprise
information measures such as early warning systems; gray measures are comprised of struc-
tural protection, for example, flood protection dams; and green measures are ecosystem-based
measures such as natural flood retention areas or forest management. Additionally, we include
a separate category for research and development (R&D). The result of the screening is shown in the base year (2016) in Fig. 2, with the largest
expenditure items being gray measures in the Catastrophe Management impact field (CATM, €
471 million p.a.) and green measures in the Agriculture impact field (AGRI, € 257 million
p.a.). Expenditures on soft measures and R&D play only a minor role. In total, current annual
adaptation-relevant expenditures sum up to € 908 million (15% of the screened budgetary
subdivisions). Starting from 2016, we co-developed1 an indicative adaptation expenditure pathway until
2050 (step 2). This pathway combines expert judgment on the additional resources needed for
single adaptation measures, on limits to further increase gray adaptation measures and
international recommendations on the phasing of gray, green, and soft measures (Watkiss
et al. 2015). For the Baseline expenditure path, the growth rate of the mid-term budget forecast
for the Federal State of Austria is used (1.65% annually; assuming the same shares as in 2016)
(BMF 2015). Figure 2 illustrates the pathway by type of adaptation measure and impact field
in which adaptation is carried out and funded. On average, expenditures of this indicative
Adaptation scenario rise by 3% p.a. over the period 2016–2050, which is above the 1.65%
assumed in the Baseline. For details on this co-developed pathway, see Appendix A.2. 3.2 Macroeconomic model and scenario implementation For the macroeconomic analysis (step 3), we use a single-country, comparative static CGE
model of Austria (Bachner et al. 2015; Steininger et al. 2016; Bachner and Bednar-Friedl Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1331 -
250
500
750
1,000
1,250
1,500
1,750
2,000
2,250
2,500
2,750
Mio. €
R&D: R&D
gray: CATM
gray: FORE
green: CATM
green: FORE
green: AGRI
soft: CATM
soft: FORE
soft: AGRI
Baseline 1.65% p.a. Fig. 2 Indicative adaptation pathway for the Austrian federal budget until 2050 (R&D= Research and Devel-
opment; CATM= Catastrophe Management; FORE= Forestry; AGRI= Agriculture) Fig. 2 Indicative adaptation pathway for the Austrian federal budget until 2050 (R&D= Research and Devel-
opment; CATM= Catastrophe Management; FORE= Forestry; AGRI= Agriculture) 2018). The model covers 40 economic sectors and one representative private household, which
is endowed with labor and capital. The respective factor income is spent for consumption or is
saved. In addition, there is a public household providing public services, financed by the
following taxes: sales taxes on output, tax on capital gains, labor tax, value added tax, and
export tax. All tax rates are fixed, thus determining flexible government income, which in turn
gives the total amount of available public budget to be spent. The model includes classical
unemployment and international trade is depicted via the Armington (1969) assumption,
where domestic goods are imperfect substitutes for imports. The foreign balance is fixed at
the share of the model’s benchmark year (2008). Regarding the development of the budget
deficit, we assume a constant deficit-to-GDP ratio, which is an empirically well-supported
assumption for the structural deficit and also in accordance with the criteria of the EU Stability
and Growth Pact. For more details and the algebraic formulation, see Bachner (2017). 3.2.1 Implementation of the impact scenario Climate change is implemented as average changes for the future 30-year climatic period
2036–2065 (i.e., 2050), relative to the average of the reference climatic period 1981–2010. Specifically, we use the SRES (Special Report on Emissions Scenarios) A1B emissions
scenario (Nakićenović and Swart 2000), which corresponds to the RCP6.0 scenario with +
2.5 °C global mean temperature by the end of the century (Knutti and Sedláček 2013). For
details, see Appendix A.1. Climate change impacts are implemented in ten impact fields. For each of these fields,
different types of impacts are quantified using a range of (bio)physical models. These impacts
are implemented into the macroeconomic CGE model by (i) changes in production cost
structures (e.g., a different production process in Agriculture), (ii) changes in productivity
(e.g., yield changes in Agriculture and Forestry), (iii) changes in investments (e.g., reconstruc-
tion of infrastructure after flood events), and/or (iv) changes in public expenditures (e.g., more
post-disaster relief payments in Catastrophe Management). Table 1 summarizes the impacts for
the three impact fields under consideration (for all other, see Appendix A.1). As explained in
Section 2, to keep expenditures on public service provision (i.e., government consumption) at
the same level as in the Baseline scenario, we assume in the Impact scenario that transfers to
private households are adjusted accordingly. Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1332 Table 1 Summary of impacts and (bio-)physical impact models
Impact field
Impacts
(Bio)physical impact model used f
quantifying impact
Agriculture
Crop productivity of main crops (grain maize,
winter wheat, winter rape, soybean,
temporary grassland) and grassland due to
changes in temperature and precipitation
Regression analysis (Mitter et al. 2
based on simulations with the b
process model EPIC (Izaurralde
2006) and the farm optimizatio
PASMA (Schmid 2004)
Forestry
Biomass productivity in commercial
production forests due to changed
precipitation and temperature, bark beetle
disturbances on productivity of
commercial forests and protection
functionality of protection forests
Estimation of productivity change
forestry revenue model PICUS
(Schörghuber et al. 2010) and o
from spruce bark beetles with F
scenario model (Seidl et al. 200
impact of bark beetle disturbanc
tection functionality based on e
(Lexer et al. 2015)
Catastrophe
Management
Building damages due to riverine floods
Simulation of riverine flooding da
hybrid convolution approach
(Hochrainer-Stigler et al. 2014a
builds on results of the LISFLO
the Climate Cost project (Feyen
Watkiss 2011; Rojas et al. 3.2.1 Implementation of the impact scenario 2013)
Adam Cost project (Kundzewic
2010; Lugeri et al. 2010) Table 1 Summary of impacts and (bio-)physical impact models
Impact field
Impacts
(Bi
qua
Agriculture
Crop productivity of main crops (grain maize,
winter wheat, winter rape, soybean,
temporary grassland) and grassland due to
changes in temperature and precipitation
Re
Forestry
Biomass productivity in commercial
production forests due to changed
precipitation and temperature, bark beetle
disturbances on productivity of
commercial forests and protection
functionality of protection forests
Es
Catastrophe
Management
Building damages due to riverine floods
Sim (Bio)physical impact model used for
quantifying impact Regression analysis (Mitter et al. 2015b)
based on simulations with the biophysical
process model EPIC (Izaurralde et al. 2006) and the farm optimization model
PASMA (Schmid 2004) Regression analysis (Mitter et al. 2015b)
based on simulations with the biophysical
process model EPIC (Izaurralde et al. 2006) and the farm optimization model
PASMA (Schmid 2004) Estimation of productivity changes with
forestry revenue model PICUS 3G
(Schörghuber et al. 2010) and of damages
from spruce bark beetles with FISCEN
scenario model (Seidl et al. 2009, 2011);
impact of bark beetle disturbances on pro-
tection functionality based on expert guess
(Lexer et al. 2015) Simulation of riverine flooding damages in a
hybrid convolution approach
(Hochrainer-Stigler et al. 2014a) (which
builds on results of the LISFLOOD model,
the Climate Cost project (Feyen and
Watkiss 2011; Rojas et al. 2013) and on the
Adam Cost project (Kundzewicz et al. 2010; Lugeri et al. 2010) Simulation of riverine flooding damages in a
hybrid convolution approach
(Hochrainer-Stigler et al. 2014a) (which
builds on results of the LISFLOOD model,
the Climate Cost project (Feyen and
Watkiss 2011; Rojas et al. 2013) and on the
Adam Cost project (Kundzewicz et al. 2010; Lugeri et al 2010) Watkiss 2011; Rojas et al. 2013) and on the
Adam Cost project (Kundzewicz et al. 2010; Lugeri et al. 2010) 3.2.2 Implementation of the adaptation scenario The Adaptation scenario builds on the Impact scenario but additionally incorporates direct costs
and benefits (i.e., avoided impacts) of public adaptation measures in the three impact fields under
consideration. Adaptation costs are divided into operating costs (e.g., labor costs, contracting to
spatial planning bureaus, maintenance costs for public infrastructure) and investment costs. Changes in sectoral operating costs are modeled as shifts within the production cost structures
while holding unit costs constant (but having a different composition of costs). Changes in
government consumption patterns and levels (which are also a part of operating costs) are
implemented as additional consumption, financed via cuts in transfers to the private household
(and hence reduced private consumption). Accumulation effects of annual investment changes
are accounted for, resulting in a changed capital stock in 2050 with associated changed annual
capital costs (depreciation). Note that the deployed CGE model is not dynamic but comparative
static. The development of the capital stock is therefore no explicit part of the model but
accounted for when developing the adaptation pathway. Changes in investment are financed
via changed savings and thus corresponding changes in private consumption. Table 3 shows how
the indicative adaptation pathway (Fig. 2) translates into annual changes of sectoral costs as well
as changes in government consumption (annualized for 2050, relative to the Impact scenario). For details on the calculations, see Appendix A.2 (Table 2). The benefit of adaptation is avoided damage. The respective assumptions on the effective-
ness of different measures (i.e., by how much damages can be reduced) are summarized in
Table 3. Following the literature and expert estimates, we assume that agricultural crop yields
can be increased by 10% and damages in Forestry can be reduced by 30–40%. For the
Catastrophe Management impact field (flood protection), we use benefit-cost ratios to quantify
effectiveness. For the Adaptation scenario, we use mean values for a central simulation run, Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1333 but also provide results for a bandwidth (lower and upper bound) to address uncertainty. For
details, see Appendix A.3. i
ll
h
l
i
h Ad
i
i
h
di
f
bli
i
Table 2 Translation of the adaptation path into annual cost vectors for modeled sectors (agriculture, forestry,
water) and agents in 2050 (in million €)
Agriculture
Forestry
Water
Government
consumption
Aggregate
investment
Labor
+ 25
+ 69
+ 48
Capital (incl. land)
+ 126
+ 32
Research and development
+ 60
Civil engineering and planning
+ 165
Machinery
+ 213
+ 102
Construction (Investment)
+ 39
−45
−6
Total
+ 239
+ 336
+ 93
+ 226
−6 le 2 Translation of the adaptation path into annual cost vectors for modeled sectors (agriculture, forestry,
er) and agents in 2050 (in million €) but also provide results for a bandwidth (lower and upper bound) to address uncertainty. For
details, see Appendix A.3. Finally, note that also in the Adaptation scenario, we assume that expenditures of public service
provision are maintained at the Baseline level by adjusting transfers to households. In addition,
however, we allow for increased consumption for adaptation measures (as indicated in Fig. 1). 4.1 Economy-wide effects of public adaptation In 2050, climate change-induced annual GDP losses in the Impact scenario are −0.15%
relative to the Baseline (see Appendix A.4 for the detailed results). In the mean Adaptation In 2050, climate change induced annual GDP losses in the Impact scenario are
0.15%
relative to the Baseline (see Appendix A.4 for the detailed results). In the mean Adaptation
Table 3 Description, assumption, and sources of effectiveness of adaptation measures in the impact fields CATM
(Catastrophe Management), AGRI (Agriculture), and FORE (Forestry)
Impact
field
Adaptation measure/purpose
[lower] mean [upper]
value for analysis
Description of unit
of measurement
Source
CATM
Soft flood protection measures
[1.5] 9.2 [10]
Benefit/cost-ratio
(Kuik et al. 2016)
Green flood protection measures
[1.2] 1.8 [4.3]
Lower bound: 10th
percentile
Gray flood protection measures
[1] 3.9 [7.7]
Upper bound: 90th
percentile
AGRI
Change in agricultural crop
management practices
[5%] 10% [15%]
Yield increase
potential in
response to
adaptation
measures
(Mitter et al. 2015a;
Schönhart
et al. 2016)
FORE
Educational measures against bark
beetles; more intensive tending of
forests; better infrastructure in
forests
[20%] 32% [45%]
Damage reduction of
damages to
protective forest
(Kolström
et al. 2011;
Lexer 2017)
Educational measures against bark
beetles; more intensive tending of
forests; better infrastructure in
forests
[35%] 40% [45%]
Damage reduction of
damages to
commercial
timberland
Genetics: Better usage of
autochthonous species to increase
resilience of forests
[0%] 0.03% [0.03%]
Reduction in timber
growth loss Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1334 scenario, these losses can be reduced by public adaptation to only −0.06%. To put it
differently, adaptation leads to a net benefit of + 0.09%-points of annual GDP compared to
not adapting; however, there is still a residual loss when compared to the Baseline scenario
with no climate change. This benefit from adaptation is illustrated in Fig. 3 (black diamonds). The reasons for this GDP effect are twofold: First, there are positive effects from adaptation-
specific productivity gains (Agriculture) as well as positive employment effects. Second, there
are reductions of direct and indirect climate change impacts from the Forestry and Catastrophe
Management impact fields. The welfare loss, measured as Hicksian equivalent variation, in the Impact scenario is −
0.48% (again, see Appendix A.4 for the detailed results), which in the Adaptation scenario can
be reduced to −0.27%. Public adaptation therefore creates a welfare benefit of + 0.2%-points
(Fig. 3). 4.1 Economy-wide effects of public adaptation Note that the size of the welfare effect is larger than the GDP effect in the Impact and
the Adaptation scenarios. This is because some consumption expenditure is Bforced^ to shift
from generic (welfare enhancing) consumption to the renewal of destroyed assets. This shift
however only restores the original state (e.g., prior to an extreme event) and is thus reducing
other consumption possibilities (i.e., welfare). In contrast, the GDP effect is neutral to this re-
distribution within the consumption structure. Figure 3 furthermore shows how the total benefit of public adaptation on GDP, welfare, and
unemployment is distributed across impact fields. We find that the positive effect of adaptation
on GDP arises from Agriculture (due to productivity gains) and Forestry (especially due to the -0.1%
0.0%
+0.1%
+0.2%
+0.3%
+0.4%
GDP
Welfare
Unemployment
Agriculture
Forestry
Catastrophe
Management
Total effect of
adaptation
Fig. 3 Effects of adaptation on GDP, welfare, and unemployment for 2050 (Adaptation scenario relative to
Impact scenario, given in percentage point differences), distinguished by impact field and in total. Error bars
indicate different assumptions on effectiveness of adaptation measures to reduce climate change impacts -0.1%
0.0%
+0.1%
+0.2%
+0.3%
+0.4%
GDP
Welfare
Unemployment
Agriculture
Forestry
Catastrophe
Management
Total effect of
adaptation Total effect of
adaptation Unemployment Fig. 3 Effects of adaptation on GDP, welfare, and unemployment for 2050 (Adaptation scenario relative to
Impact scenario, given in percentage point differences), distinguished by impact field and in total. Error bars
indicate different assumptions on effectiveness of adaptation measures to reduce climate change impacts Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1335 reduction of damages to protection forests). For Catastrophe Management, the shift from a
capital-intensive to a more labor-intensive structure results in only a marginal GDP effect from
adaptation, as the effects offset each other. Substantial positive effects on welfare emerge from
reduced damage costs in Catastrophe Management and the associated reduction in forced
consumption. A positive contribution of adaptation to welfare also arises from Agriculture, due
to productivity gains and the moderating effect on food prices. In Forestry, we find positive
welfare effects primarily caused by the reduced damage to protection forests and thus more
public finances available to increase transfers to households. We also see that unemployment is
reduced by adaptation in all impact fields, which is driven by the focus on green and soft
adaptation as well as by higher expenditures for labor-intensive government consumption. 2 Note that the effect of adaptation in Catastrophe Management to transfers would be negative when performed in
isolation (since there is no large positive effect from these measures on employment and GDP), as expensive
structural measures need to be financed by cuts in transfers. However, the overall positive contribution of
adaptation in Forestry and Agriculture more than compensates for these transfer cuts. 4.2 Effects of public adaptation on government budgets Beginning with a description of results on the revenue side of the budget, the effect of
adaptation on the main tax revenue categories is shown in Fig. 4a, which compares the
Adaptation and Impact scenario in absolute terms (and with %-points differences on the
secondary axis). With adaptation, tax revenues increase for labor and production taxes
(due to higher economic activity and employment), but decline in value added tax, since
there is less forced consumption for restoring damaged assets and because the govern-
ment needs to finance its adaptation expenditure (which is taxed at lower rates than
private consumption). Figure 4b shows the expenditure side (again, Adaptation compared to the Impact
scenario). In the Adaptation scenario, relief payments to households can be reduced
substantially; thus, non-climate-related government consumption can be increased. How-
ever, direct public adaptation expenditures amount to additional € 219 million (0.1% of
total expenditure). Moreover, due to higher tax revenues and less payments for unem-
ployment benefits, transfers to households can be increased in the Adaptation scenario. Only when the effectiveness of adaptation is very low, transfers to households need to be
reduced to ensure stability of government consumption (service provision). Eventually,
the analysis allows for matching numerical assessment to the conceptual model from Fig. 1, which is presented in Figure A.1. Finally, Fig. 4 also shows the decomposition of the effects of adaptation across the three
impact fields. On the revenue side, we find that the relative contribution to higher labor taxes is
particularly strong for public adaptation in Forestry, as forest management is comparatively
labor-intensive. Revenues from value-added tax decline, however, as a result of adaptation in
Catastrophe Management, due to less forced consumption, leading to a slightly negative
contribution of adaptation in that regard. On the expenditure side, we find a significant
reduction in relief payments to private households from Catastrophe Management in the form
of structural flood protection. Adaptation expenditures increase particularly due to adaptation
in Catastrophe Management and Forestry, because both sectors are recipients of subsidies for,
e.g., ensuring the function of protection forests.2 2 Note that the effect of adaptation in Catastrophe Management to transfers would be negative when performed in
isolation (since there is no large positive effect from these measures on employment and GDP), as expensive
structural measures need to be financed by cuts in transfers. 4.1 Economy-wide effects of public adaptation Regarding the uncertainty of effectiveness of adaptation (see the error bars in Fig. 3), we find
that the results are robust in terms of the direction of the effects. 4.2 Effects of public adaptation on government budgets However, the overall positive contribution of
adaptation in Forestry and Agriculture more than compensates for these transfer cuts. Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1336 Discussion and conclusions
he first part of the paper we presented a modeling framework that allows for a more
-0.20%
-0.10%
0.00%
+0.10%
+0.20%
+0.30%
+0.40%
+0.50%
-100
-50
0
50
100
150
200
250
Production tax
Labor tax
Capital tax
Value added
tax
Other taxes
Mio. €2008
-2.50%
-2.00%
-1.50%
-1.00%
-0.50%
0.00%
+0.50%
+1.00%
+1.50%
+2.00%
+2.50%
-500
-400
-300
-200
-100
0
100
200
300
400
500
Non-climate
related
government
consumption
Additional
adaptation
expenditures
Relief
payments to
households
Transfers to
households
Unemployment
benefits
Mio. €2008
Agriculture
Forestry
Catastrophe Management
Total in Mio. €
%-points change (secondary axis)
(a) Revenue side
(b) Expenditure side
4 Decomposition of effects of public adaptation on tax revenue (a) and public expenditure (b) for the three
tation fields for 2050 (Adaptation scenario relative to Impact scenario). Diamonds show absolute total effect
riangles show percentage point differences between Adaptation and Impact scenarios (secondary axis). Error
indicate different assumptions on effectiveness of adaptation measures to reduce climate change impacts. that %-point changes are not shown for relief payments (−81%) and for additional adaptation expenditures
ch would be infinite when calculated since its value in the Baseline and Impact scenario would be zero)
g
p
g
g (
) -0.20%
-0.10%
0.00%
+0.10%
+0.20%
+0.30%
+0.40%
+0.50%
-100
-50
0
50
100
150
200
250
Production tax
Labor tax
Capital tax
Value added
tax
Other taxes
Mio. €2008
(a) Revenue side -2.50%
-2.00%
-1.50%
-1.00%
-0.50%
0.00%
+0.50%
+1.00%
+1.50%
+2.00%
+2.50%
-500
-400
-300
-200
-100
0
100
200
300
400
500
Non-climate
related
government
consumption
Additional
adaptation
expenditures
Relief
payments to
households
Transfers to
households
Unemployment
benefits
Mio. €2008
Agriculture
Forestry
Catastrophe Management
Total in Mio. €
%-points change (secondary axis)
(b) Expenditure side (b) Expenditure side Fig. 4 Decomposition of effects of public adaptation on tax revenue (a) and public expenditure (b) for the three
adaptation fields for 2050 (Adaptation scenario relative to Impact scenario). Diamonds show absolute total effect
and triangles show percentage point differences between Adaptation and Impact scenarios (secondary axis). Error
bars indicate different assumptions on effectiveness of adaptation measures to reduce climate change impacts. 4.2 Effects of public adaptation on government budgets Note that %-point changes are not shown for relief payments (−81%) and for additional adaptation expenditures
(which would be infinite when calculated since its value in the Baseline and Impact scenario would be zero) 5 Discussion and conclusions In the first part of the paper, we presented a modeling framework that allows for a more
detailed analysis of the budgetary effects of public adaptation to climate change, taking
account of economy-wide feedback effects. Until now, such analyses have not been available,
but are urgently needed for informed policy making, including the phasing and prioritization of Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1337 adaptation measures. The proposed framework can be applied to any country or region;
however, it involves considerable effort with respect to data preparation and stakeholder
consultation, as adaptation is often integrated in general public sector activities. adaptation measures. The proposed framework can be applied to any country or region;
however, it involves considerable effort with respect to data preparation and stakeholder
consultation, as adaptation is often integrated in general public sector activities. In the second part of the paper, we demonstrated the usefulness of the proposed framework
for the case of Austria. We show that public adaptation already involves considerable costs for
the federal budget (15% of the pre-selected budget is adaptation-relevant). These costs are
likely to rise in the future, due to ongoing climate change but also due to re-investment cycles
in public infrastructure. For requirements of robust government finances, these higher expen-
ditures on adaptation require that other public expenditures are reduced accordingly; however,
the extent depends on how tax revenues and social benefit payments are affected by adapta-
tion. To also capture these indirect effects of public adaptation, we investigated how public
adaptation in Agriculture, Forestry, and Catastrophe Management affects GDP, welfare, and
unemployment. The first key lesson learned is that due to positive effects on GDP, welfare and
employment, public adaptation can lead to a net benefit from a macroeconomic perspective. While the effectiveness of adaptation measures to reduce climate change impacts is still subject
to uncertainty, we find that the direction of our results is highly robust with regard to different
assumptions on the effectiveness of adaptation measures. When looking more closely at the government budget, we find that public adaptation has
the potential to increase government revenues (in total and for all categories except valued
added tax) because of higher economic activity as compared to a scenario with only the
impacts of climate change. 5 Discussion and conclusions On the expenditure side, public adaptation leads to a partial re-
distribution of government expenditures towards adaptation but also to a reduced need for
disaster relief payments. Moreover, reduced unemployment translates into less unemployment
payments. The effect on transfers tends to be positive but can be slightly negative when
adaptation measures’ effectiveness is very low. Overall, the second key lesson learned is that it
is possible that the positive indirect effects of public adaptation outweigh the direct public
costs of adaptation, with a resulting increase in the overall government budget, another insight
offered by the proposed framework. Regarding global adaptation strategy recommendations, our findings imply that adaptation
assessments should take a comprehensive perspective based on quantitative economic model-
ing, and include both the revenue and expenditure side as well as indirect effects. Computable
general equilibrium models are very useful in that respect. As we have shown, the macroeco-
nomic benefits of adaptation can outweigh the costs, because of indirect effects, e.g., on
economic activity in other sectors and employment. Such effects may turn cost-benefit
assessments in favor for adaptation measures with high direct costs, but strong positive indirect
effects. This is the case, e.g., for labor-intensive green adaptation measures. We also demonstrate the advantage of a close collaboration between academia and admin-
istration (especially from finance and treasury), in order to develop realistic and policy-
compatible adaptation pathways. Such a collaboration also raises awareness within the policy
domain and enhances the mainstreaming of climate change adaptation across the board. Regarding the generalizability of our results for other countries, we conclude the following. For countries with (i) similar budgetary structures as well as (ii) similar patterns of impacts (as
in Austria), the revealed mechanisms can lead to the same qualitative effects. Regarding the
first similarity—the budgetary structure—we know that across EU member states, these
structures are very homogenous (EUROSTAT 2018). Among OECD countries, the variations
are larger, yet the dominant revenue and expenditure categories are still the same (OECD
2018a, b). Similar impact patterns relate to high damages from flooding and forestry (loss of Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1338 ecosystem services) and moderate positive effects in Agriculture. As this is the case for many
developed regions and industrialized countries, the qualitative insights and mechanisms as
obtained from our case might also carry over to many other OECD and EU countries. References Agrawala S, Bosello F, Carraro C et al (2011a) Adapting to climate change: costs, benefits, and modelling
approaches. Int Rev Environ Resour Econ 5:245–284. https://doi.org/10.1561/101.00000043 Agrawala S, Bosello F, Carraro C et al (2011a) Adapting to climate change: costs, benefits, and modelling
approaches. Int Rev Environ Resour Econ 5:245–284. https://doi.org/10.1561/101.00000043 Agrawala S, Bosello F, Carraro C et al (2011b) Plan or react? Analysis of adaptation costs and benefits using
integrated assessment models. Clim Chang Econ 02:175–208. https://doi.org/10.1142/S2010007811000267 Agrawala S, Bosello F, Carraro C et al (2011b) Plan or react? Analysis of adaptation costs and benefits using
integrated assessment models. Clim Chang Econ 02:175–208. https://doi.org/10.1142/S2010007811000267 Armington PS (1969) A theory of demand for products distinguished by place of production (Une theorie de la
demande de produits differencies d’apres leur origine) (Una teoria de la demanda de productos
distinguiendolos segun el lugar de produccion). Staff Papers - International Monetary Fund 16:159. https://doi.org/10.2307/3866403 Armington PS (1969) A theory of demand for products distinguished by place of production (Une theorie de la
demande de produits differencies d’apres leur origine) (Una teoria de la demanda de productos
distinguiendolos segun el lugar de produccion). Staff Papers - International Monetary Fund 16:159. https://doi.org/10.2307/3866403 Bachner G (2017) Assessing the economy-wide effects of climate change adaptation options of land transport
systems in Austria. Reg Environ Chang 17:929–940. https://doi.org/10.1007/s10113-016-1089-x Bachner G (2017) Assessing the economy-wide effects of climate change adaptation options of land transport
systems in Austria. Reg Environ Chang 17:929–940. https://doi.org/10.1007/s10113-016-1089-x Bachner G, Bednar-Friedl B (2018) The effects of climate change impacts on public budgets and implication
fiscal counterbalancing instruments. Environ Model Assess. https://doi.org/10.1007/s10666-018-9617-3 Bachner G, Bednar-Friedl B (2018) The effects of climate change impacts on public budgets and implications of
fiscal counterbalancing instruments. Environ Model Assess. https://doi.org/10.1007/s10666-018-9617-3
h
d
i dl
b
i i
i
l
i
li
h Bachner G, Bednar-Friedl B, Nabernegg S, Steininger KW (2015) Macroeconomic evaluation of climate change
in Austria: a comparison across impact fields and total effects. In: Steininger KW, König M, Bednar-Friedl B
et al (eds) Economic evaluation of climate change impacts: development of a cross-sectoral framework and
results for Austria. Springer, Berlin, pp 415–440 Bachner G, Bednar-Friedl B, Nabernegg S, Steininger KW (2015) Macroeconomic evaluation of climate change
in Austria: a comparison across impact fields and total effects. 5 Discussion and conclusions As this paper is the first to analyze not only the direct but also the indirect effects of public
adaptation on government budgets, limitations and several directions for future research should
naturally be discussed. First, a better understanding of how the effectiveness of adaptation can
change over time is definitely needed. Second, while we develop an indicative and plausible
adaptation pathway together with professionals from relevant government departments, several
alternative adaptation pathways could be developed and compared with regard to their
macroeconomic and budgetary consequences. Third, it would be interesting to investigate in
more detail how different budgetary rules, such as flexible tax rates or additional foreign debt,
interact with public adaptation spending, an exercise which would require a more dynamic
modeling setting. Acknowledgements We thank Keith Williges for careful proof-reading. Funding information Open access funding provided by University of Graz. Funding for this research was
granted by the Austrian Climate and Energy Fund under its 6th Call of the Austrian Climate Research Program
(grant number B36862; project PACINAS). 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 repro-
duction 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. Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. References https://doi.org/10.1007/s10584-011-0340-1 Bosello F, Carraro C, De Cian E (2013) Adaptation can help mitigation: an integrated approach to post-2012
climate policy. Environ Dev Econ 18:270–290. https://doi.org/10.1017/S1355770X13000132 Bosello F, Carraro C, De Cian E (2013) Adaptation can help mitigation: an integrated approach to post-2012
climate policy. Environ Dev Econ 18:270–290. https://doi.org/10.1017/S1355770X13000132 p
y
p
g
Bräuer I, Umpfenbach K, Blobel D et al (2009) Klimawandel: Welche Belastungen entstehen
Tragfähigkeit der Öffentlichen Finanzen? Endbericht, Ecologic Institute, Berlin car J-C, Iglesias A, Feyen L et al (2011) Physical and economic consequences of climate change in Europe. P
N tl A
d S i U S A 108 2678 2683 htt
//d i
/10 1073/
1011612108 Ciscar J-C, Iglesias A, Feyen L et al (2011) Physical and economic consequences of climate change in E
Proc Natl Acad Sci U S A 108:2678–2683. https://doi.org/10.1073/pnas.1011612108 Ciscar J-C, Szabó L, van Regemorter D, Soria A (2012) The integration of PESETA sectoral economic impacts
into the GEM-E3 Europe model: methodology and results. Clim Chang 112:127–142. https://doi. org/10.1007/s10584-011-0343-y g
y
Bruin KC, Dellink RB, Tol RSJ (2009) AD-DICE: an implementation of adaptation in the DICE model. Clim
Chang 95:63–81. https://doi.org/10.1007/s10584-008-9535-5 Dellink R, Lanzi E, Chateau J (2017) The sectoral and regional economic consequences of climate change to
2060. Environ Resour Econ. https://doi.org/10.1007/s10640-017-0197-5 Eakin HC, Patt A (2011) Are adaptation studies effective, and what can enhance their practical impact? WIREs
Climate Change 2:141–153 European Commission (2016) Tracking climate expenditures. The common methodology for tracking and
monitoring climate expenditure under the European Structural and Investment Funds (2014–2020). Brussels, Belgium g
European Union (2008) Consolidated Version of the Treaty on the Functioning of the European Union EUROSTAT (2018) Government finance statistics - Statistics Explained. https://ec
explained/index.php/Government_finance_statistics. Accessed 26 Nov 2018 Feyen L, Watkiss P (2011). The impacts and economic costs of river floods in Europe and the costs and bene
of adaptation Fisher-Vanden K, Sue Wing I, Lanzi E, Popp D (2013) Modeling climate change feedbacks and adaptation
responses: recent approaches and shortcomings. Clim Chang 117:481–495. https://doi.org/10.1007/s10584-
012-0644-9 Gilmore EA, St.Clair T (2018) Budgeting for climate change: obstacles and opportunities at the US state le
Clim Pol 18:729–741. https://doi.org/10.1080/14693062.2017.1366891 Haasnoot M, Kwakkel JH, Walker WE, ter Maat J (2013) Dynamic adaptive policy pathways: a method for
crafting robust decisions for a deeply uncertain world. Glob Environ Chang 23:485–498. https://doi. References In: Steininger KW, König M, Bednar-Friedl B
et al (eds) Economic evaluation of climate change impacts: development of a cross-sectoral framework and
results for Austria. Springer, Berlin, pp 415–440 Bednar-Friedl B, Knittel N, Leitner M, Bachner G (2017) The cost of climate change adaptation for the Austrian
Federal Budget. PACINAS Working Paper #04. http://anpassung.ccca.at/pacinas/en/ergebnisse/. Accessed 1
Jun 2018 Bednar-Friedl B, Knittel N, Leitner M, Bachner G (2017) The cost of climate change adaptation for the Austrian
Federal Budget. PACINAS Working Paper #04. http://anpassung.ccca.at/pacinas/en/ergebnisse/. Accessed 1
Jun 2018 Bednar-Friedl B, Knittel N, Leitner M, Bachner G (2017) The cost of climate change adaptation for the Austrian
Federal Budget. PACINAS Working Paper #04. http://anpassung.ccca.at/pacinas/en/ergebnisse/. Accessed 1
Jun 2018 Bierbaum R, Smith JB, Lee A et al (2013) A comprehensive review of climate adaptation in the United States:
more than before, but less than needed. Mitig Adapt Strateg Glob Chang 18:361–406. https://doi. org/10.1007/s11027-012-9423-1 Bierbaum R, Smith JB, Lee A et al (2013) A comprehensive review of climate adaptation in the United States:
more than before, but less than needed. Mitig Adapt Strateg Glob Chang 18:361–406. https://doi. org/10.1007/s11027-012-9423-1 1339 Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 Biesbroek GR, Swart RJ, Carter TR et al (2010) Europe adapts to climate change: comparing national adaptation
t t
i
Gl b E
i
Ch
20 440 450 htt
//d i
/10 1016/j l
h 2010 03 005 strategies. Glob Environ Chang 20:440–450. https://doi.org/10.1016/j.gloenvcha.2010.03.005 BMF (2015) Strategiebericht zum Bundesfinanzrahmengesetz 2016-2019 gemäß § 14 BHG 2013. Bundesministerium für Finanzen, Vienna BMF (2015) Strategiebericht zum Bundesfinanzrahmengesetz 2016-2019 gemäß § 14 BHG 2013. Bundesministerium für Finanzen, Vienna BMF (2015) Strategiebericht zum Bundesfinanzrahmengesetz 2016-2019 gemäß § 14 BHG 2013. Bundesministerium für Finanzen, Vienna ,
Bosello F, De Cian E (2014) Climate change, sea level rise, and coastal disasters. A review of modeling practices. Energy Econ 46:593–605. https://doi.org/10.1016/j.eneco.2013.09.002 Bosello F, De Cian E (2014) Climate change, sea level rise, and coastal disasters. A review of modeling practices. Energy Econ 46:593–605. https://doi.org/10.1016/j.eneco.2013.09.002 gy
p
g
j
Bosello F, Nicholls RJ, Richards J et al (2012) Economic impacts of climate change in Europe: sea-level r
Clim Chang 112:63–81. https://doi.org/10.1007/s10584-011-0340-1 gy
p
g
j
Bosello F, Nicholls RJ, Richards J et al (2012) Economic impacts of climate change in Europe: sea-level rise. Clim Chang 112:63–81. 264. https://doi.org/10.1038/nclimate2863 264. https://doi.org/10.1038/nclimate2863 Lexer MJ (2017) Interview on climate change adaptation measures in Austria’s forests. Conducted during the
project PACINAS p j
Lexer MJ, Jandl R, Nabernegg S, Bednar-Friedl B (2015) Forestry. In: Steininger KW, König M, Bednar-Friedl B
et al (eds) Economic evaluation of climate change impacts. Development of a cross-sectoral framework and
results for Austria. Springer International Publishing, Cham, pp 147–167 Lis EM, Nickel C (2010) The impact of extreme weather events on budget balances. Int Tax Public Financ
378–399. https://doi.org/10.1007/s10797-010-9144-x Lugeri N, Kundzewicz ZW, Genovese E et al (2010) River flood risk and adaptation in Europe-assessment of the
present status. Mitig Adapt Strateg Glob Chang 15:621–639. https://doi.org/10.1007/s11027-009-9211-8 Margulis S, Narain U (2010) The costs to developing countries of adapting to climate change: new methods and
estimates - the global report of the economics of adaptation to climate change study. The World Bank McDonald J (2011) The role of law in adapting to climate change: the role of law in adapting to climate change. Wiley Interdiscip Rev Clim Chang 2:283–295. https://doi.org/10.1002/wcc.96 y
p
g
p
g
Mees HLP, Driessen PPJ, Runhaar HAC (2012) Exploring the scope of public and private responsibilities for
climate adaptation. J Environ Policy Plan 14:305–330. https://doi.org/10.1080/1523908X.2012.707407 ecky M, Raddatz C (2015) Fiscal responses after catastrophes and the enabling role of financial development. ld
k
h
//d i
/
/ b /lh Melecky M, Raddatz C (2015) Fiscal responses after catastrophes and the enabli
World Bank Econ Rev 29:129–149. https://doi.org/10.1093/wber/lht041 Mitter H, Heumesser C, Schmid E (2015a) Spatial modeling of robust crop production portfolios to assess
agricultural vulnerability and adaptation to climate change. Land Use Policy 46:75–90. https://doi. org/10.1016/j.landusepol.2015.01.010 g
j
p
Mitter H, Schönhart M, Meyer I et al (2015b) Agriculture. In: Steininger KW, König M, Bednar-Friedl B et al
(eds) Economic evaluation of climate change impacts. Development of a cross-sectoral framework and
results for Austria. Springer International Publishing, Cham, pp 123–146 Moss RH, Edmonds JA, Hibbard KA et al (2010) The next generation of scenarios for climate change research
and assessment. Nature 463:747–756. https://doi.org/10.1038/nature08823 Nakićenović N, Swart R (eds) (2000) Special report on emissions scenarios: a special report of Working Group
III of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge O’Neill BC, Kriegler E, Riahi K et al (2014) A new scenario framework for climate change research: the conc
of shared socioeconomic pathways. Clim Chang 122:387–400. References org/10.1016/j.gloenvcha.2012.12.006 Hallegatte S, Hourcade J-C, Dumas P (2007) Why economic dynamics matter in assessing climate change
damages: illustration on extreme events. Ecol Econ 62:330–340. https://doi.org/10.1016/j. ecolecon.2006.06.006 Hochrainer-Stigler S, Lugeri N, Radziejewski M (2014a) Up-scaling of impact dependent loss distributions: a
hybrid convolution approach for flood risk in Europe. Nat Hazards 70:1437–1451. https://doi.org/10.1007
/s11069-013-0885-6 Hochrainer-Stigler S, Mechler R, Pflug G, Williges K (2014b) Funding public adaptation to climate-related
disasters. Estimates for a global fund. Glob Environ Chang 25:87–96. https://doi.org/10.1016/j. gloenvcha.2014.01.011 g
Izaurralde RC, Williams JR, McGill WB et al (2006) Simulating soil C dynamics with EPIC: model description
and testing against long-term data. Ecol Model 192:362–384. https://doi.org/10.1016/j. ecolmodel.2005.07.010 Jones B, Keen M, Strand J (2013) Fiscal implications of climate change. Int Tax Public Financ 20:29–
https://doi.org/10.1007/s10797-012-9214-3 p
g
Knutti R, Sedláček J (2013) Robustness and uncertainties in the new CMIP5 climate model projections. Nat C
Chang 3:369–373. https://doi.org/10.1038/nclimate1716 g
p
g
Kolström M, Lindner M, Vilén T et al (2011) Reviewing the science and implementation of climate change
adaptation measures in European forestry. Forests 2:961–982. https://doi.org/10.3390/f2040961 p
p
y
p
g
Kuik O, Scussolini P, Mechler R, et al (2016) Assessing the economic case for adaptation to extreme events at
different scales 1340 Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 Kundzewicz ZW, Lugeri N, Dankers R et al (2010) Assessing river flood risk and adaptation in Europe-review of
projections for the future. Mitig Adapt Strateg Glob Chang 15:641–656. https://doi.org/10.1007/s11027-
010-9213-6 Leppänen S, Solanko L, Kosonen R (2015) The impact of climate change on regional government expenditures:
evidence from Russia. Environ Resour Econ. https://doi.org/10.1007/s10640-015-9977-y Leppänen S, Solanko L, Kosonen R (2015) The impact of climate change on regional government ex
evidence from Russia. Environ Resour Econ. https://doi.org/10.1007/s10640-015-9977-y Lesnikowski A, Ford J, Biesbroek R et al (2015) National-level progress on adaptation. Nat Clim Chang 6:26
264. https://doi.org/10.1038/nclimate2863 Lesnikowski A, Ford J, Biesbroek R et al (2015) National-level progress on adaptation. Nat Clim Cha
264 htt
//d i
/10 1038/
li
t 2863 264. https://doi.org/10.1038/nclimate2863 https://doi.org/10.1007/s10584-013-090 O’Neill BC, Kriegler E, Riahi K et al (2014) A new scenario framework for climate change research: the concept
of shared socioeconomic pathways. Clim Chang 122:387–400. https://doi.org/10.1007/s10584-013-0905-2
OECD (2015) The economic consequences of climate change. The economic consequences of climate change. OECD Publishing, Paris. https://doi.org/10.1787/9789264235410-en OECD (2015) The economic consequences of climate change. The economic consequences of climate change. OECD Publishing, Paris. https://doi.org/10.1787/9789264235410-en g
p
g
OECD (2018a) General government revenue (indicator). Dataset. doi.org/10.1787/b68b04ae-en. Accessed 26
Nov 2018 OECD (2018b) Central government spending (indicator). Dataset. doi.org/10.1787/83a23f1b-en. Accessed 26
Nov 2018 OECD Development Assistance Committee (2016) OECD DAC Rio Markers for Climate – Handbook. https://www.oecd.org/dac/environment-development/Revised%20climate%20marker%20handbook_
FINAL.pdf. Accessed 06 June 2018 Osberghaus D, Reif C (2010) Total costs and budgetary effects of adaptation to climate change: an assessment for
the European Union. ZEW-Centre for European Economic Research Discussion Paper Osberghaus D, Dannenberg A, Mennel T, Sturm B (2010) The role of the government in adaptation to climate
change. Environ Plan C Gov Policy 28:834–850. https://doi.org/10.1068/c09179j Ouattara B, Strobl E (2013) The fiscal implications o
https://doi.org/10.1016/j.ecolecon.2012.10.002 Ouattara B, Strobl E (2013) The fiscal implications of hurricane strikes in the Caribbean. Ecol Econ 85:105–115. https://doi.org/10.1016/j.ecolecon.2012.10.002 Rojas R, Feyen L, Watkiss P (2013) Climate change and river floods in the European Union: socio-economic
consequences and the costs and benefits of adaptation. Glob Environ Chang 23:1737–1751. https://doi. org/10.1016/j.gloenvcha.2013.08.006 Schinko T, Mechler R, Hochrainer-Stigler S (2016) A methodological framework to operationalize climate risk
management: managing sovereign climate-related extreme event risk in Austria. Mitig Adapt Strateg Glob
Chang. https://doi.org/10.1007/s11027-016-9713-0 Schmid E (2004) The positive agricultural sector model Austria - PASMA. University of Agriculture and Life
Sciences, Vienna Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1341 Schönhart M, Schauppenlehner T, Kuttner M et al (2016) Climate change impacts on farm production, landscape
appearance, and the environment: policy scenario results from an integrated field-farm-landscape model in
Austria. Agric Syst 145:39–50. https://doi.org/10.1016/j.agsy.2016.02.008 Schönhart M, Schauppenlehner T, Kuttner M et al (2016) Climate change impacts on farm production, landscape
appearance, and the environment: policy scenario results from an integrated field-farm-landscape model in
Austria. Agric Syst 145:39–50. https://doi.org/10.1016/j.agsy.2016.02.008 g
y
p
g
j g y
Schörghuber S, Seidl R, Rammer W, et al (2010) KlimAdapt - Ableitung von prioritären Maßnahmen zur
Adaption des Energiesystems an den Klimawandel - Arbeitspaket 3: Biomasse Bereitstellung. 264. https://doi.org/10.1038/nclimate2863 University of
Agriculture and Life Sciences, Vienna g
y
p
g
j g y
Schörghuber S, Seidl R, Rammer W, et al (2010) KlimAdapt - Ableitung von prioritären Maßnahmen zur
Adaption des Energiesystems an den Klimawandel - Arbeitspaket 3: Biomasse Bereitstellung. University of
Agriculture and Life Sciences, Vienna g
Seidl R, Schelhaas M-J, Lindner M, Lexer MJ (2009) Modelling bark beetle disturbances in a large scale forest
scenario model to assess climate change impacts and evaluate adaptive management strategies. Reg Environ
Chang 9:101–119. https://doi.org/10.1007/s10113-008-0068-2 Seidl R, Schelhaas M-J, Lindner M, Lexer MJ (2009) Modelling bark beetle disturbances in a large scale forest
scenario model to assess climate change impacts and evaluate adaptive management strategies. Reg Environ
Chang 9:101–119. https://doi.org/10.1007/s10113-008-0068-2 Seidl R, Schelhaas M-J, Lexer MJ (2011) Unraveling the drivers of intensifying forest disturbance regimes in
Europe: drivers of forest disturbance intensification. Glob Chang Biol 17:2842–2852. https://doi.org/10.1111
/j.1365-2486.2011.02452.x Seidl R, Schelhaas M-J, Lexer MJ (2011) Unraveling the drivers of intensifying forest disturbance regimes in
Europe: drivers of forest disturbance intensification. Glob Chang Biol 17:2842–2852. https://doi.org/10.1111
/j.1365-2486.2011.02452.x p
/j.1365-2486.2011.02452.x j
Siegmeier J, Mattauch L, Franks M et al (2018) The fiscal benefits of stringent climate change mitigation: an
overview. Clim Pol 18:352–367. https://doi.org/10.1080/14693062.2017.1400943 Siegmeier J, Mattauch L, Franks M et al (2018) The fiscal benefits of stringent climate change mitigation:
overview. Clim Pol 18:352–367. https://doi.org/10.1080/14693062.2017.1400943 Steininger KW, König M, Bednar-Friedl B et al (eds) (2015) Economic evaluation of climate change impacts. Steininger KW, König M, Bednar-Friedl B et al (eds) (2015) Economic evaluation of climate change impa
Development of a Cross-Sectoral Framework and Results for Austria. Springer, Berlin Steininger KW, König M, Bednar-Friedl B et al (eds) (2015) Economic evaluation of climate change impac
Development of a Cross-Sectoral Framework and Results for Austria Springer Berlin Steininger KW, Bednar-Friedl B, Formayer H, König M (2016) Consistent economic cross-sectoral climate
change impact scenario analysis: method and application to Austria. Clim Serv 1:39–52. https://doi. org/10.1016/j.cliser.2016.02.003 Steininger KW, Bednar-Friedl B, Formayer H, König M (2016) Consistent economic cross-sectoral climate
change impact scenario analysis: method and application to Austria. Clim Serv 1:39–52. https://doi. org/10.1016/j.cliser.2016.02.003 g
j
Sussman F, Krishnan N, Maher K et al (2014) Climate change adaptation cost in the US: what do we know? Clim
Pol 14:242–282. https://doi.org/10.1080/14693062.2013.777604 Sussman F, Krishnan N, Maher K et al (2014) Climate change adaptation cost in the US: what do we know? Clim
Pol 14:242–282. https://doi.org/10.1080/14693062.2013.777604 p
g
Tol RSJ (2005) Adaptation and mitigation: trade-offs in substance and methods. Environ Sci Pol 8:572–578. https://doi.org/10.1016/j.envsci.2005.06.011 Tol RSJ (2005) Adaptation and mitigation: trade-offs in substance and methods. Environ Sci Pol 8:572–578. https://doi.org/10.1016/j.envsci.2005.06.011 Tol RSJ (2005) Adaptation and mitigation: trade
https://doi.org/10.1016/j.envsci.2005.06.011 Urwin K, Jordan A (2008) Does public policy support or undermine climate change adaptation? Exploring policy
interplay across different scales of governance. Glob Environ Chang 18:180–191. https://doi.org/10.1016/j. gloenvcha.2007.08.002 Urwin K, Jordan A (2008) Does public policy support or undermine climate change adaptation? Exploring policy
interplay across different scales of governance. Glob Environ Chang 18:180–191. https://doi.org/10.1016/j. gloenvcha.2007.08.002 g
Watkiss P, Hunt A, Blyth W, Dyszynski J (2015) The use of new economic decision support tools for adaptation
assessment: a review of methods and applications, towards guidance on applicability. Clim Chang 132:401–
416. https://doi.org/10.1007/s10584-014-1250-9 Watkiss P, Hunt A, Blyth W, Dyszynski J (2015) The use of new economic decision support tools for adaptation
assessment: a review of methods and applications, towards guidance on applicability. Clim Chang 132:401–
416. https://doi.org/10.1007/s10584-014-1250-9
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https://openalex.org/W4236504910
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https://zenodo.org/records/1631369/files/article.pdf
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German
| null |
Krankenpflege
|
Deutsche medizinische Wochenschrift/Deutsche Medizinische Wochenschrift
| 1,900
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public-domain
| 429
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Krankenpflege. Die oft anerkannte Thatsache, dass Berlin sich über die Grenzen
Deutschlands hinaus durch eine ungewöhnlich grosse Zahl vorzüglicher
Privatkliniken auszeichnet, hat durch die am 11. d. M. eröffnete
Privat-Frauenklinik von Dr. K. Ab e 1 (IPotsdamerstrasse 92) eine neue
Bestätigung erfahren. Die nicht nur zweckmässig eingerichtete, sondern
auch architektonisch schöne Anstalt kann vollen Anspruch auf den Bei-
fall der Fachgenossen wie der sie aufsuchenden Patienten erheben. Die ganz aus Stein und Eisen erbaute Klinik verfügt über eine erfreu-
liche Fülle von Licht und Luft; beide, besonders für Kranke so überaus
wohlthätige Factoren werden namentlich von den an der Hinterfront der
Anstalt gelegenen Gärten der benachbarten Häuser in einer ungewöhn-
lichen Weise begünstigt. Die Vertheilung der Räumlichkeiten ist die in
neuerer Zeit übliche: Im obersten (IV.) Stock die Operationsräume, im
Souterrain bezw. Parterre die Wirthschaftsräume, im Hochparterre
Sprech- und Wartezimmer, Bureau, Zimmer der Oberin, in den übrigen
Stockwerken die Krankenzimmer, Laboratorium etc. Zu den Räumen
der mit der Anstalt verbundenen Poliklinik führt zweckmässiger Weise
ein Nebeneingang, so dass die ambulanten Patienten die eigentliche
Klinik nicht zu betreten brauchen, ebenso sind der Poliklinik besondere
Wartezimmer, Closets u. s. w. zugewiesen. Die Verbindung zwischen den
einzelnen Stockwerken wird durch einen elektrischen Aufzug, construirt
von Stiegler, hergestellt. Der durchweg vorhandene Cementfussboden
ist überall mit Linoleum bedeckt; hierdurch, sowie durch die an den
meisten Zimmern vorhandenen Doppelthüren wird möglichst jedes störende
Geräusch von den Patienten ferngehalten. Die Heizung findet durch
centrale Warmwasserleitung, die Beleuchtung theils durch Gasglühlicht,
theils
elektrisch durch Nernstlampen statt. Im Winter kann den
Zimmern vorgewärmte Luft zugeführt werden, wodurch gleichzeitig
ventilirt wird. An Luftraum und innerer Ausstattung bieten die
Krankenräume - insbesondere diejenigen für die TTT. Klasse - das
äusserste dar, was von einer Privatklinik mit Recht verlangt werden
kann. Der Operationsraum entspricht modernen Anforderungen der
Antiseptik. Bemerkenswerth ist, dass in der Anstalt nicht nur Nan-
heimer, sondern auch Moor-Bäder angewandt werden können. Vor-
trefflich
ausgestattet ist das zu mikroskopischen,
chemischen und
bacteriologischen Untersuchungen eingerichtete,
mit Arbeitsplätzen
für Curse versehene Laboratorium. Die Anstalt umfasst zur Zeit
28 Betten, kann aber auf 45 Betten gebracht worden. Dio Pensions-
preise stellen sich täglich für Kassenkranke auf 2,50 Mark, sonst für
die III. Klasse auf 3,50 Mark, für die II. auf 6-7,50 Mark, für die
I. auf 10 Mark und mehr. Die von der ärztlichen Leitung völlig ge-
trennte Verwaltung ruht in den Händen der von der Martin'schen
Frauenklinik her vortheilhaft bekannten Oberin Frau Horn. DEUTSCBIF MIEDiCIIflSCIIIE WOC}TEINSCHPJFP. DEUTSCBIF MIEDiCIIflSCIIIE WOC}TEINSCHPJFP. No. 46 No. 46 748 Druck von G. Bern8tein in J3erlin. Krankenpflege. J. S. J. S. Druck von G. Bern8tein in J3erlin.
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https://bmcimmunol.biomedcentral.com/counter/pdf/10.1186/1471-2172-10-6
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English
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Chemokines are secreted by monocytes following OK-432 (lyophilized Streptococcus pyogenes) stimulation
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BMC immunology
| 2,009
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cc-by
| 10,569
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BioMed Central BioMed Central This article is available from: http://www.biomedcentral.com/1471-2172/10/6 This article is available from: http://www.biomedcentral.com/1471-2172/10/6 © 2009 Olsnes 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. Abstract Background: OK-432, penicillin-killed Streptococcus pyogenes, is used in treating lymphangiomas
and carcinomas. We have studied in vitro the role of mononuclear phagocytes (MNPs), including
purified monocytes (MOs), in the immune response to OK-432. MIP-1α/β and MCP-1 secretions
were assessed in whole blood (WB), peripheral blood mononuclear cells (PBMCs) and purified
MOs, after in vitro stimulation with OK-432 with or without adherence for 24 hours. Results: OK-432 stimulated MNPs to secrete MCP-1 and MIP-1α/β in healthy individuals and in
head and neck squamous cell carcinoma (HNSCC) patients, except for OK-432 stimulation of WB
giving a minimal MIP-1α/β response. Upon culture on low-attachment wells, a spontaneous
chemokine secretion was observed, with an unchanged secretion following OK-432 stimulation. Inhibition of Syk kinase and/or PI-3 kinase did not significantly change the chemokine response to
OK-432, except for MIP-1α production being increased upon Syk inhibitor addition and an
increased MCP-1 response upon addition of both inhibitors. Adhesion may possibly involve β1 and/
or β3 integrins, not β2, whereas β1–3 integrins may act as co-stimulatory receptors for OK-432. Based on direct blockage of CD36 or CD18 by antibodies, MCP-1 production may be mediated by
CD18 while MIP-1β and MCP-1 production may occur upon binding to CD36. Conclusion: Adherent human MOs produce MCP-1 and MIP-1α/β upon stimulation with OK-432. CD36 modulates MIP-1β and MCP-1 response. Thus, to some extent OK-432 acts as a substance
whereby only MOs adhered to surfaces secrete MCP-1 and MIP-1α/β, in part explaining why OK-
432 is suited as a biological response modifying drug. Open
Research article
Chemokines are secreted by monocytes following OK-432
(lyophilized Streptococcus pyogenes) stimulation
Carla Olsnes*1, Helen Stavang1,2, Karl Brokstad3, Jan Olofsson1,2 and
Hans J Aarstad1,2 Open Access Address: 1Department of Surgical Sciences, Faculty of Medicine, University of Bergen, Jonas Lies vei 66, 5021, Bergen, Norway, 2Department of
Head and Neck Surgery, Haukeland University Hospital, 5021, Bergen, Norway and 3Broegelmann Research Laboratory, Gades Institute,
University of Bergen, 5021, Bergen, Norway Email: Carla Olsnes* - carla.olsnes@ore.uib.no; Helen Stavang - helen.stavang@ore.uib.no; Karl Brokstad - karl.brokstad@gades.uib.no;
Jan Olofsson - jan.olofsson@ore.uib.no; Hans J Aarstad - hans.jorgen.aarstad@helse-bergen.no
* Corresponding author Email: Carla Olsnes* - carla.olsnes@ore.uib.no; Helen Stavang - helen.stavang@ore.uib.no; Karl Brokstad - karl.b
Jan Olofsson - jan.olofsson@ore.uib.no; Hans J Aarstad - hans.jorgen.aarstad@helse-bergen.no arla.olsnes@ore.uib.no; Helen Stavang - helen.stavang@ore.uib.no; Karl Brokstad - karl.brokstad@gades.uib.no;
on@ore.uib.no; Hans J Aarstad - hans.jorgen.aarstad@helse-bergen.no * Corresponding author Received: 16 December 2008
Accepted: 28 January 2009 Received: 16 December 2008
Accepted: 28 January 2009 Published: 28 January 2009 Published: 28 January 2009 BMC Immunology 2009, 10:6
doi:10.1186/1471-2172-10-6 BMC Immunology 2009, 10:6
doi:10.1186/1471-2172-10-6 Page 1 of 11
(page number not for citation purposes) http://www.biomedcentral.com/1471-2172/10/6 http://www.biomedcentral.com/1471-2172/10/6 BMC Immunology 2009, 10:6 cancer [1,2] and lymphangiomas [3]. Killed bacterial tox-
ins [4] along with bacillus Calmette-Guerin (BCG) [5], β-
glucan [6], interferons [7] and monoclonal antibodies [8]
are examples of BRMs used in cancer treatment. from tonsillitis, impetigo, necrotizing fasciitis, and scarlet
fever to lethal toxic shock syndrome [13]. Thus, studying
the interactions of OK-432 with the immune system may
shed light into the biology of these diseases as well. One major MNP function is secretion of chemokines,
such as monocyte chemoattractant protein (MCP) -1 and
macrophage inflammatory protein (MIP) -1α/β [14]. Chemokines have been shown to be secreted in response
to OK-432 stimulation [15] and have the main effect of
attracting new leucocytes to the site from where they are
secreted [16]. It should be of interest, to determine the
modality of chemokine secretion from MOs and Mϕs
upon OK-432 stimulation. We have therefore found it
pertinent to address this issue in the present investigation. Japan has a long standing tradition in using penicillin-
killed lyophilized Streptococcus pyogenes, denominated
OK-432 or picibanil, as a biological response modifier
(BRM) for treatment of cancer [1,2]. Sakamoto et al. [1]
published in 2001 a meta-analysis showing a 20% 5-year
survival improvement with immunochemotherapy, com-
pared to chemotherapy alone, following OK-432 treat-
ment in patients diagnosed with non-small-cell lung
cancer. This meta-analysis was based on 1,520 patients
enrolled in 11 randomized clinical phase III trials. Fur-
thermore, Oba et al [2] published in 2007 a meta-analysis
including 8009 gastric carcinoma patients from 8 rand-
omized clinical phase III trials and concluded that com-
pared to control conditions, addition of OK-432
treatment increased survival. There are also reports sug-
gesting that patients with other cancers, such as head and
neck squamous cell carcinoma (HNSCC) [9], may benefit
from OK-432 treatment. OK-432 has also been used as a
maturation factor for DCs cells as part of vaccination ther-
apy of cancer patients [10]. Priming provided by adherence, presumably associated
with MNP differentiation from MO to Mϕ, contributes to
interleukin secretion following OK-432 MNP stimulation
[17]. Therefore, it is of interest to study to what extent the
MNP chemokine response to OK-432 depends on adher-
ence. Molecularly, we have found it relevant to investigate
the role of β-integrin receptors, as these receptors partici-
pate during differentiation of MNP from MO to Mϕ. http://www.biomedcentral.com/1471-2172/10/6 Fur-
thermore, these receptors may be functionally blocked by
addition of piceatannol (Syk kinase inhibitor) and/or
LY294002 (phosphoinositide (PI)-3 kinase inhibitor)
[18-22]. OK-432 may stimulate TLR receptors. In addition
to OK-432, we have studied chemokine secretion upon
stimulation with the known TLR2 agonist LTA and TLR4
agonist LPS. Moreover, we explored the role of the TLR2
co-receptor CD36 [23] in OK-432 stimulation. Lymphangiomas are benign neoplasias of lymphatic ori-
gin, often congenital, that may extend around vital struc-
tures [11]. Surgical removal of lymphangiomas has been
the standard treatment, but may be technically difficult. Injection of OK-432 into cystic lymphangioma lesions
may lead to shrinkage and subsequent cure as first
reported by Ogita [3]. Today, injection with OK-432 is
established as a major treatment option for lymphangi-
omas [11], and is an important example of the use of
BRMs in medicine. We have determined that in vitro purified adherent MNPs
may secrete chemokines MCP-1, MIP-1α and MIP-1β fol-
lowing OK-432 stimulation. MOs deprived of adherence
did not respond to OK-432 stimulation as assessed by
chemokine secretion. Inhibition of Syk and PI-3 kinase
did not block the stimulatory effect of OK-432. MIP-1α/β
production in WB and MOs was higher upon stimulation
with LTA or LPS while OK-432 stimulation gave a higher
MCP-1 secretion in WB and MOs in comparison with LTA
or LPS. CD36, and to some extent CD18, participate in the
OK-432-modulated MNP chemokine response. OK-432 presumably exerts its effect by activating the
immune system to secrete toxic substances, which in turn
eliminate tumor cells [12]. It is, however, relatively little
known about the mechanisms of action of OK-432. The
principal cells responding to the drug, the engaged recep-
tors or signal transduction pathways, are to a surprisingly
large extent unknown. This should be an important area
of study. The efficiency of OK-432 treatment needs to be
improved in order to make OK-432 a better cancer treat-
ment drug. Additional drugs, which could improve OK-
432's response, as well as studying to what extent other
(cancer) drugs potentially interfere with the receptors and
signal transduction pathways driving the OK-432
response, should therefore be determined as to optimize
and improve treatment. Background as monocytes (MOs) in blood and become, e.g., tissue
macrophages (Mϕs) or dendritic cells (DCs) upon
extravasation into tissues. The innate immune system is
presumably involved when biological response modifiers
(BRMs) are utilized in the treatment of diseases such as g
The innate immune system detects and eliminates invad-
ing foreign material through non-specific defense mecha-
nisms elicited by, e.g., mononuclear phagocytes (MNP). MNPs originate as monoblasts in the bone marrow, reside Page 1 of 11
(page number not for citation purposes) Page 1 of 11
(page number not for citation purposes) http://www.biomedcentral.com/1471-2172/10/6 BMC Immunology 2009, 10:6 In vitro chemokine production in unstimulated WB, PBMCs
or MOs
Figure 1
In vitro chemokine production in unstimulated WB,
PBMCs or MOs. Human WB, PBMCs or MOs were plated
in parallel on regular (R) or low attachment (L) plates and left
unstimulated for 24 h (A). Supernatants were then collected
and analyzed using Multiplex (MIP) or ELISA (MCP-1). WB (n
= 8); PBMC (MIP-1α/β: n = 7; MCP-1: n = 4); MO (n = 6). In itro chemok
or MOs
Figure 1 chemokine response profile as healthy controls (Fig. 2). With respect to MCP-1, close correlation between protein
levels and mRNA levels was observed in three healthy
donors (Fig. 3A) as well as two HNSCC patients, tested on
two separate days (Fig. 3C) suggesting a pre-transcrip-
tional regulation upon in vitro OK-432 stimulation of
MOs. Chemokine production following OK-432 versus LTA or
LPS stimulation In separate experiments,
MOs, stimulated with piceatannol (120 μM) (B) or wortman-
nin (100 nM) (D) for 30 min followed by stimulation with
OK-432 for an additional 24 h, were analyzed by ELISA or
real-time PCR with respect to MCP-1 production and gene
expression, respectively. A, B, and D: n = 3; C: n = 2. OK-432 augments MCP-1 gene expression in MOs from
healthy donors and HNSCC patients and correlates with
MCP-1 protein levels upon inhibition with piceatannol and
wortmannin
Figure 3
OK-432 augments MCP-1 gene expression in MOs
from healthy donors and HNSCC patients and corre-
lates with MCP-1 protein levels upon inhibition with
piceatannol and wortmannin. MCP-1 gene expression
was assessed in MOs stimulated with OK-432 for 24 h from
three randomly selected healthy donors (A) or two HNSCC
patients on two separate days (C). In separate experiments,
MOs, stimulated with piceatannol (120 μM) (B) or wortman-
nin (100 nM) (D) for 30 min followed by stimulation with
OK-432 for an additional 24 h, were analyzed by ELISA or
real-time PCR with respect to MCP-1 production and gene
expression, respectively. A, B, and D: n = 3; C: n = 2. MIP-1α/β upon stimulating PBMCs versus MOs (Fig. 2). Stimulated WB gave a lower MIP-1α secretion compared
to PBMCs or MOs (Fig. 2). The effect of OK-432 was also
tested with MOs from HNSCC patients giving the same OK-432 stimulates release of chemokines in WB, PBMCs,
and isolated human MOs from healthy donors and HNSCC
patients
Figure 2
OK-432 stimulates release of chemokines in WB,
PBMCs, and isolated human MOs from healthy
donors and HNSCC patients. Human WB, PBMCs or
MOs were stimulated with media or OK-432 (0.01 KE/ml)
for 24 h (A). MOs isolated from four HNSCC patients were
stimulated for 24 h with media or OK-432 (0.01 KE/ml) (B). Supernatants were then collected and analyzed using Multi-
plex (MIP) or ELISA (MCP-1). A: WB (n = 8); PBMC (n = 12);
MO (n = 27); B: HNSCC (n = 4). l
f
MO f OK-432 augments MCP-1 gene expression in MOs from
healthy donors and HNSCC patients and correlates with
MCP-1 protein levels upon inhibition with piceatannol and
wortmannin
Figure 3
OK-432 augments MCP-1 gene expression in MOs
from healthy donors and HNSCC patients and corre-
lates with MCP-1 protein levels upon inhibition with
piceatannol and wortmannin. Chemokine production following OK-432 versus LTA or
LPS stimulation Tested were WB (Fig. 4) and MOs (Fig. 5) stimulated in
parallel with OK-432 or LTA (TLR2 ligand) or LPS (TLR4
ligand). In WB stimulated with OK-432 or LTA, the
response was primarily seen with MCP-1, and only to
some extent with MIP-1α/β, whereas the LPS-stimulated
response was determined by MIP-1α/β secretion. In puri-
fied MOs, OK-432 and LTA stimulated secretion of MCP-
1. LPS inhibited MCP-1 production. Thus, it appears that
OK-432 stimulation is more akin to LTA than to LPS stim-
ulation. In vitro che
or MOs
Figure 1 p
,
g
In vitro chemokine production in unstimulated WB,
PBMCs or MOs. Human WB, PBMCs or MOs were plated
in parallel on regular (R) or low attachment (L) plates and left
unstimulated for 24 h (A). Supernatants were then collected
and analyzed using Multiplex (MIP) or ELISA (MCP-1). WB (n
= 8); PBMC (MIP-1α/β: n = 7; MCP-1: n = 4); MO (n = 6). MIP-1α/β upon stimulating PBMCs versus MOs (Fig. 2). Stimulated WB gave a lower MIP-1α secretion compared
to PBMCs or MOs (Fig. 2). The effect of OK-432 was also
tested with MOs from HNSCC patients giving the same
= 8); PBMC (MIP-1α/β: n = 7; MCP-1: n = 4); MO (n = 6). OK-432 stimulates release of chemokines in WB, PBMCs,
and isolated human MOs from healthy donors and HNSCC
patients
Figure 2
OK-432 stimulates release of chemokines in WB,
PBMCs, and isolated human MOs from healthy
donors and HNSCC patients. Human WB, PBMCs or
MOs were stimulated with media or OK-432 (0.01 KE/ml)
for 24 h (A). MOs isolated from four HNSCC patients were
stimulated for 24 h with media or OK-432 (0.01 KE/ml) (B). Supernatants were then collected and analyzed using Multi-
plex (MIP) or ELISA (MCP-1). A: WB (n = 8); PBMC (n = 12);
MO (n = 27); B: HNSCC (n = 4). OK-432 augments MCP-1 gene expression in MOs from
healthy donors and HNSCC patients and correlates with
MCP-1 protein levels upon inhibition with piceatannol and
wortmannin
Figure 3
OK-432 augments MCP-1 gene expression in MOs
from healthy donors and HNSCC patients and corre-
lates with MCP-1 protein levels upon inhibition with
piceatannol and wortmannin. MCP-1 gene expression
was assessed in MOs stimulated with OK-432 for 24 h from
three randomly selected healthy donors (A) or two HNSCC
patients on two separate days (C). Results In vitro chemokine production in unstimulated cultures
Upon adherence, only MCP-1 was to some extent secreted
(Fig. 1). Upon low adherence conditions, MCP-1 and
MIP-1α/β were secreted at all studied conditions (Fig. 1). Chemokine production following OK-432 stimulation
At all tested regular growth conditions, OK-432 stimu-
lated the release of MIP-1α/β, and MCP-1 (all p < 0.05)
(Fig. 2). We determined a similar secretion for MCP-1 and Furthermore,S. pyogenes is an important pathogen causing
human disease. The diseases caused by S. pyogenes range Page 2 of 11
(page number not for citation purposes) http://www.biomedcentral.com/1471-2172/10/6 Chemokine
stimulation
Figure 4 Chemokine production follows OK-432, LTA or LPS
in vitro stimulation of WB. Human WB was stimulated in
parallel with OK-432 (0.01 KE/ml), LTA (0.5 μg/ml), LPS (1
μg/ml), or left unstimulated for 24 h. Supernatants were then
collected and analyzed using Multiplex (MIP) or ELISA (MCP-
1). Control, OK-432, LPS: n = 8; LTA: n = 4. Chemokine production following OK-432 versus LTA or
LPS stimulation MCP-1 gene expression
was assessed in MOs stimulated with OK-432 for 24 h from
three randomly selected healthy donors (A) or two HNSCC
patients on two separate days (C). In separate experiments,
MOs, stimulated with piceatannol (120 μM) (B) or wortman-
nin (100 nM) (D) for 30 min followed by stimulation with
OK-432 for an additional 24 h, were analyzed by ELISA or
real-time PCR with respect to MCP-1 production and gene
expression, respectively. A, B, and D: n = 3; C: n = 2. OK-432 st
and isolate
patients
Figure 2 OK 432 stimulates release of chemokines in WB, PBMCs,
and isolated human MOs from healthy donors and HNSCC
patients
Figure 2
OK-432 stimulates release of chemokines in WB,
PBMCs, and isolated human MOs from healthy
donors and HNSCC patients. Human WB, PBMCs or
MOs were stimulated with media or OK-432 (0.01 KE/ml)
for 24 h (A). MOs isolated from four HNSCC patients were
stimulated for 24 h with media or OK-432 (0.01 KE/ml) (B). Supernatants were then collected and analyzed using Multi-
plex (MIP) or ELISA (MCP-1). A: WB (n = 8); PBMC (n = 12);
MO (n = 27); B: HNSCC (n = 4). Page 3 of 11
(page number not for citation purposes) BMC Immunology 2009, 10:6 http://www.biomedcentral.com/1471-2172/10/6 Chemokine production follows OK-432, LTA or LPS in vitro
stimulation of WB
Figure 4
Chemokine production follows OK-432, LTA or LPS
in vitro stimulation of WB. Human WB was stimulated in
parallel with OK-432 (0.01 KE/ml), LTA (0.5 μg/ml), LPS (1
μg/ml), or left unstimulated for 24 h. Supernatants were then
collected and analyzed using Multiplex (MIP) or ELISA (MCP-
1). Control, OK-432, LPS: n = 8; LTA: n = 4. more, MIP-1β response was reduced by 74% (19 ± 3 vs 5
± 2 ng/ml; p < 0.02), and the MCP-1 response by 94% (54
± 9 vs 3.1 ± 1.7 ng/ml; p < 0.02). In MOs, MCP-1 (19.8 ±
6.4 vs -0.2 ± 0.2 ng/ml; p < 0.02) response was completely
blocked, while MIP-1α was reduced by 92% 17.9 ± 10.7
vs 1.5 ± 1.1 ng/ml; p < 0.05) and MIP-1β by 98% 12.1 ±
3.8 vs 0.2 ± 0.6 ng/ml; p < 0.03). Thus, it appears that
chemokine responses following OK-432-stimulation are
almost absent if the cells are minimally allowed to adhere
to a surface. Stimulation of MOs with OK-432 is independent of Syk
and PI-3 kinase In order to determine whether Syk kinase activation was
necessary for the observed chemokine production in OK-
432-stimulated MOs, the Syk kinase inhibitor piceatannol
(120 μM, (Fig. 3B), 30 μM (not shown), 1 μM (Fig. 8) or
0.5 μM (not shown), or diluent control, was added for 30
minutes to adherent MOs prior to addition of OK-432 for
24 hours. Western blot (Wb) analysis (Fig. 9) shows that Chemokine production following OK-432 stimulation of
PBMC, MO-depleted PBMCs or MOs Chemokine secretion was determined from PBMCs, MOs
separated by adherence and MO-depleted PBMCs (Fig. 7). MO-depleted PBMCs (PB) and MOs secreted 18% and
36% of the PBMC-derived MCP-1, respectively (Figure 7). Thus, MOs are the major source for MCP-1 production. In
contrast, PBMCs stimulated with LPS gave a negative
MCP-1 production since stimulation with LPS gives lower
MCP-1 secretions compared to media controls. Chemokine production following OK-432 stimulation of
WB, PBMCs and MOs cultured on regular or low-
attachment culture wells Chemokine secretion was compared in WB and MNPs cul-
tured on regular (R) or low-attachment (L) wells (Fig. 6). In WB, the MCP-1 response was reduced by 90% at the L
compared to the R condition (48 ± 4 vs 7 ± 27 ng/ml; p <
0.02). In PBMCs, MIP-1α response was abolished at the L
condition 24 ± 9.7 vs -1 ± 5.5 ng/ml; p < 0.04). Further- Page 4 of 11
0.02). In PBMCs, MIP-1α response was abolished at the L
condition 24 ± 9.7 vs -1 ± 5.5 ng/ml; p < 0.04). Further-
OK-432, LTA or LPS in vitro stimulation of MOs
Figure 5
OK-432, LTA or LPS in vitro stimulation of MOs. MOs were stimulated in parallel with OK-432 (0.01 KE/ml),
LTA (0.5 μg/ml), LPS (1 μg/ml), or left unstimulated for 24 h. Supernatants were then collected and analyzed using Multi-
plex (MIP) or ELISA (MCP-1). MIP-1α (Control, OK-432,
LPS: n = 9; LTA: n = 7).MIP-1β (Control, OK-432, LPS: n =
10; LTA: n = 8); MCP-1 (Control, OK-432: n = 19; LTA: n =
9; LPS: n = 16). OK-432 stimulation of adherent or suspended WB, PBMCs,
and MOs
Figure 6
OK-432 stimulation of adherent or suspended WB,
PBMCs, and MOs. WB, PBMCs or MOs were stimulated
with OK-432 (0.01 KE/ml) or left unstimulated on regular (R)
or low-attachment (L) plates for 24 h. Supernatants were
analyzed using Multiplex (MIP) or ELISA (MCP-1). WB: n = 8;
PBMC: n = 8; MO: n = 6. OK-432 stimulation of adherent or suspended WB, PBMCs,
and MOs
Figure 6
OK-432 stimulation of adherent or suspended WB,
PBMCs, and MOs. WB, PBMCs or MOs were stimulated
with OK-432 (0.01 KE/ml) or left unstimulated on regular (R)
or low-attachment (L) plates for 24 h. Supernatants were
analyzed using Multiplex (MIP) or ELISA (MCP-1). WB: n = 8;
PBMC: n = 8; MO: n = 6. OK-432 stimulation of adherent or suspended WB, PBMCs,
and MOs
Figure 6
OK-432 stimulation of adherent or suspended WB,
PBMCs, and MOs. WB, PBMCs or MOs were stimulated
with OK-432 (0.01 KE/ml) or left unstimulated on regular (R)
or low-attachment (L) plates for 24 h. Supernatants were
analyzed using Multiplex (MIP) or ELISA (MCP-1). WB: n = 8;
PBMC: n = 8; MO: n = 6. Syk and PI-
lation of M
Figure 8 With respect to mRNA, MCP-1 levels are also
reduced when exposed for 30 min to 120 μM piceatannol
followed by 24-hour incubation with OK-432 (Fig. 3B, bot-
tom). Thus, piceatannol, at high, likely unspecific, concen-
trations (120 μM) lowers MCP-1 secretion while at low,
specific, concentrations (0.5, 1 or 30 μM), has no effect on
the tested chemokines, despite exhibiting efficacy in lower-
ing Syk kinase phosphorylation. Syk kinase becomes phosphorylated following adherence
of MOs and is further phosphorylated following OK-432
stimulation but this phosphorylation is substantially
reduced in MOs exposed to piceatannol (30 μM) for 30
min before stimulation with OK-432 for an additional 15
min. Addition of piceatannol had no effect on chemokine
production, as measured by MCP-1, in human MOs unless
it was used at high, unspecific concentrations (120 μM)
(Fig. 3B, top). With respect to mRNA, MCP-1 levels are also
reduced when exposed for 30 min to 120 μM piceatannol
followed by 24-hour incubation with OK-432 (Fig. 3B, bot-
tom). Thus, piceatannol, at high, likely unspecific, concen-
trations (120 μM) lowers MCP-1 secretion while at low,
specific, concentrations (0.5, 1 or 30 μM), has no effect on
the tested chemokines, despite exhibiting efficacy in lower-
ing Syk kinase phosphorylation. did not significantly reduce MIP-1α, MIP-1β, or MCP-1
production in OK-432-stimulated MOs. Furthermore, a
combination of piceatannol (Pic) (30 μM), and
LY294002 (LY) (50 μM), or diluent control (ethanol,
EtOH), was used throughout the entire MO isolation pro-
cedure as well as during incubation with media (Fig. 8C)
or OK-432 for an additional 24 hours (Fig. 8D). Incuba-
tion with media blocked spontaneous in vitro secretion of
all tested chemokines, but no significant effect was
observed in the ability of MOs to produce MIP-1α, and
MIP-1β upon OK-432 stimulation. On the contrary, MCP-
1 production appears to be augmented following OK-432
stimulation, pointing to the existence of an additional
MCP-1 pathway, dependent on Syk and PI-3 kinase,
throughout the isolation and adherence protocol. The role of PI-3 kinase in OK-432's ability to stimulate
MOs was tested where LY294002 (Fig. 8) or Wortmannin
(Fig. 3D) was added for 30 min to freshly isolated MOs
followed by incubation with OK-432 for 24 hours. West-
ern blot analysis (Fig. 9) shows that PI-3 kinase is not
phosphorylated following adherence of MOs but
becomes phosphorylated following OK-432 stimulation. Chemokine production following OK-432 stimulation of
WB, PBMCs and MOs cultured on regular or low-
attachment culture wells OK-432, LTA or LPS in vitro stimulation of MOs
Figure 5
OK-432, LTA or LPS in vitro stimulation of MOs. MOs were stimulated in parallel with OK-432 (0.01 KE/ml),
LTA (0.5 μg/ml), LPS (1 μg/ml), or left unstimulated for 24 h. Supernatants were then collected and analyzed using Multi-
plex (MIP) or ELISA (MCP-1). MIP-1α (Control, OK-432,
LPS: n = 9; LTA: n = 7).MIP-1β (Control, OK-432, LPS: n =
10; LTA: n = 8); MCP-1 (Control, OK-432: n = 19; LTA: n =
9; LPS: n = 16). OK-432, LTA or LPS in vitro stimulation of MOs
Figure 5
OK-432, LTA or LPS in vitro stimulation of MOs. MOs were stimulated in parallel with OK-432 (0.01 KE/ml),
LTA (0.5 μg/ml), LPS (1 μg/ml), or left unstimulated for 24 h. Supernatants were then collected and analyzed using Multi-
plex (MIP) or ELISA (MCP-1). MIP-1α (Control, OK-432,
LPS: n = 9; LTA: n = 7).MIP-1β (Control, OK-432, LPS: n =
10; LTA: n = 8); MCP-1 (Control, OK-432: n = 19; LTA: n =
9; LPS: n = 16). ,
g
OK-432, LTA or LPS in vitro stimulation of MOs. MOs were stimulated in parallel with OK-432 (0.01 KE/ml),
LTA (0.5 μg/ml), LPS (1 μg/ml), or left unstimulated for 24 h. Supernatants were then collected and analyzed using Multi-
plex (MIP) or ELISA (MCP-1). MIP-1α (Control, OK-432,
LPS: n = 9; LTA: n = 7).MIP-1β (Control, OK-432, LPS: n =
10; LTA: n = 8); MCP-1 (Control, OK-432: n = 19; LTA: n =
9; LPS: n = 16). OK-432 stimulation of adherent or suspended WB, PBMCs,
and MOs
Figure 6
OK-432 stimulation of adherent or suspended WB,
PBMCs, and MOs. WB, PBMCs or MOs were stimulated
with OK-432 (0.01 KE/ml) or left unstimulated on regular (R)
or low-attachment (L) plates for 24 h. Supernatants were
analyzed using Multiplex (MIP) or ELISA (MCP-1). WB: n = 8;
PBMC: n = 8; MO: n = 6. OK-432 stimulation of adherent or suspended WB, PBMCs,
and MOs
Figure 6
OK-432 stimulation of adherent or suspended WB,
PBMCs, and MOs. WB, PBMCs or MOs were stimulated
with OK-432 (0.01 KE/ml) or left unstimulated on regular (R)
or low-attachment (L) plates for 24 h. Supernatants were
analyzed using Multiplex (MIP) or ELISA (MCP-1). WB: n = 8;
PBMC: n = 8; MO: n = 6. Syk and PI-
lation of M
Figure 8 This OK-432-dependent PI-3 kinase phosphorylation is
decreased when MOs are exposed to LY294002 for 30 min
before stimulation with OK-432 for 15 min. Addition of
LY294002 (Fig. 8B) or Wortmannin (results not shown) Syk and PI-
lation of M
Figure 8 Syk and PI-
lation of M
Figure 8 Syk and PI 3 kinase independent signaling for OK 432 stimu
lation of MOs
Figure 8
Syk and PI-3 kinase-independent signaling for OK-
432 stimulation of MOs. Piceatannol (1 μM) or diluent
control was added to MO cultures for 30 min prior to addi-
tion of OK-432 for 24 h (A). LY294002 (50 μM) was added
to MOs for 30 min prior to addition of OK-432 for 24 h (B). In separate experiments, a combination of LY294002 (50
μM) and piceatannol (30 μM) was added to whole blood and
throughout the monocyte isolation procedure (C) and fol-
lowed by stimulation with OK-432 or left unstimulated for
24 h (D). Supernatants were analyzed using Multiplex (MIP)
or ELISA (MCP-1). A: n = 4; B: n = 5; C: n = 4; D: n = 4. Syk and PI 3 kinase independent signaling for OK 432 stimu
lation of MOs
Figure 8
Syk and PI-3 kinase-independent signaling for OK-
432 stimulation of MOs. Piceatannol (1 μM) or diluent
control was added to MO cultures for 30 min prior to addi-
tion of OK-432 for 24 h (A). LY294002 (50 μM) was added
to MOs for 30 min prior to addition of OK-432 for 24 h (B). In separate experiments, a combination of LY294002 (50
μM) and piceatannol (30 μM) was added to whole blood and
throughout the monocyte isolation procedure (C) and fol-
lowed by stimulation with OK-432 or left unstimulated for
24 h (D). Supernatants were analyzed using Multiplex (MIP)
or ELISA (MCP-1). A: n = 4; B: n = 5; C: n = 4; D: n = 4. p
pg
MO-depleted PBMCs secrete less chemokines than
PBMCs upon OK-432 stimulation. PBMCs, MOs, or PBs
(MO-depleted PBMCs) were left unstimulated or stimulated
in parallel with OK-432 (0.01 KE/ml) or LPS (1 μg/ml) for 24
h. Supernatants were analyzed using Multiplex (MIP) or ELISA
(MCP-1). n = 2. Syk kinase becomes phosphorylated following adherence
of MOs and is further phosphorylated following OK-432
stimulation but this phosphorylation is substantially
reduced in MOs exposed to piceatannol (30 μM) for 30
min before stimulation with OK-432 for an additional 15
min. Addition of piceatannol had no effect on chemokine
production, as measured by MCP-1, in human MOs unless
it was used at high, unspecific concentrations (120 μM)
(Fig. 3B, top). Chemokine production following OK-432 stimulation of
WB, PBMCs and MOs cultured on regular or low-
attachment culture wells Page 4 of 11
(page number not for citation purposes) BMC Immunology 2009, 10:6 http://www.biomedcentral.com/1471-2172/10/6 Syk and PI-3 kinase-independent signaling for OK-432 stimu-
lation of MOs
Figure 8
Syk and PI-3 kinase-independent signaling for OK-
432 stimulation of MOs. Piceatannol (1 μM) or diluent
control was added to MO cultures for 30 min prior to addi-
tion of OK-432 for 24 h (A). LY294002 (50 μM) was added
to MOs for 30 min prior to addition of OK-432 for 24 h (B). In separate experiments, a combination of LY294002 (50
μM) and piceatannol (30 μM) was added to whole blood and
throughout the monocyte isolation procedure (C) and fol-
lowed by stimulation with OK-432 or left unstimulated for
24 h (D). Supernatants were analyzed using Multiplex (MIP)
or ELISA (MCP-1). A: n = 4; B: n = 5; C: n = 4; D: n = 4. MO-depleted PBMCs secrete less chemokines than PBMCs
upon OK-432 stimulation
Figure 7
MO-depleted PBMCs secrete less chemokines than
PBMCs upon OK-432 stimulation. PBMCs, MOs, or PBs
(MO-depleted PBMCs) were left unstimulated or stimulated
in parallel with OK-432 (0.01 KE/ml) or LPS (1 μg/ml) for 24
h. Supernatants were analyzed using Multiplex (MIP) or ELISA
(MCP-1). n = 2. Role of CD36 on stimulation of MOs with OK-432 f
f
Anti-CD36 antibody or isotype control was added to wells
followed by addition of PBMCs, incubated for 40 min,
and OK-432 was added for an additional 24 hours (Fig. 10B). CD36 addition decreased MIP-1β and MCP-1 (both
p = 0.028) but not MIP-1α secretion. Thus, in MOs, OK-
432 elicits both MIP-1β and MCP-1 production in part via
CD36 acting as a co-receptor. All studied chemokines were secreted without particular
stimulation factors at low adherence conditions,
whereas MIP-1α/β secretion was completely inhibited by
adherence. At low adherence conditions, the secretion
response of MIP-1α/β and MCP-1 from PBMCs and MOs
was abolished upon OK-432 stimulation. This might
indicate that MNPs undergoing adherence fundamen-
tally alter their chemokine secretion responses and sup-
ports that MO to Mϕ differentiation interferes with the
regulation of chemokine secretion. This is also sup-
ported by our observations of MIP-1α-derived OK-432
stimulated secretion being lower in WB compared to
PBMCs and MOs. Diapedesis depends on, e.g., integrin
receptor engagement co-triggering differentiation of
MOs to Mϕs [18]. Such stimulation may be provided
when MOs are isolated by in vitro adherence. Syk phos-
phorylation is a necessary part of β1–3 integrin-generated
activation [19-21], whereas PI-3 kinase phosphorylation
is required in β2 integrin-generated activation [20]. We
have presently shown that adherence of MOs stimulated
phosphorylation of Syk but not PI-3 kinase, as deter-
mined by Western blots. Thus, it appears that possibly
both β1 and αvβ3 integrins, but not β2 integrin, play a role
in mediating MO adhesion. Based on Western blots,
binding of OK-432 to MOs elicited a higher Syk phos-
phorylation compared to unstimulated MOs, possibly
suggesting an adherence-mediated priming effect. PI-3 Page 6 of 11
(page number not for citation purposes) Role of β2-integrin (CD18) during stimulation of MOs with
OK-432 Treating MOs with a neutralizing F(ab) – anti-CD18 anti-
body (Fig. 10A) prior to OK-432 stimulation, the secre-
tion of MCP-1 (statistically nonsignificant), but not the
other tested chemokines, decreased. Thus, the β2 integrin
(CD18) may possibly be involved in MCP-1 secretion fol-
lowing OK-432 activation of human isolated MOs. Page 5 of 11
(page number not for citation purposes) Page 5 of 11
(page number not for citation purposes) http://www.biomedcentral.com/1471-2172/10/6 BMC Immunology 2009, 10:6 OK-432 promotes Syk or PI-3 kinase phosphorylation in
MOs
Figure 9
OK-432 promotes Syk or PI-3 kinase phosphorylation
in MOs. MOs, isolated by the monocyte negative isolation
kit, were incubated either with piceatannol, Pic. (30 μM) or
diluent control (A) or LY294002, LY (50 μM) (B) and either
left unstimulated or further stimulated with OK-432 for 15
min. Blots shown are representative of two separate experi-
ments performed with pooled MOs obtained from two dif-
ferent blood donors. Role of CD18 and CD36 in OK-432 activation of MOs
Figure 10
Role of CD18 and CD36 in OK-432 activation of MOs. MOs were incubated either with isotype control or anti-
CD18/F(ab) (12.5 μg/ml) for 24 h (A). PBMCs were incu-
bated either with isotype control or anti-CD36 (2.5 μg/ml)
(B) prior to incubation with OK-432 for 24 h. Supernatants
were analyzed for shown chemokines by Multiplex (MIP) or
ELISA (MCP-1). A: n = 3; B: n = 6. CD18: MCP-1 (NS). chemokine response in purified MOs from HNSCC
OK-432 promotes Syk or PI-3 kinase phosphorylation in
MOs
Figure 9
OK-432 promotes Syk or PI-3 kinase phosphorylation
in MOs. MOs, isolated by the monocyte negative isolation
kit, were incubated either with piceatannol, Pic. (30 μM) or
diluent control (A) or LY294002, LY (50 μM) (B) and either
left unstimulated or further stimulated with OK-432 for 15
min. Blots shown are representative of two separate experi-
ments performed with pooled MOs obtained from two dif-
ferent blood donors. Role of CD18 and CD36 in OK-432 activation of MOs
Figure 10
Role of CD18 and CD36 in OK-432 activation of MOs. MOs were incubated either with isotype control or anti-
CD18/F(ab) (12.5 μg/ml) for 24 h (A). PBMCs were incu-
bated either with isotype control or anti-CD36 (2.5 μg/ml)
(B) prior to incubation with OK-432 for 24 h. Supernatants
were analyzed for shown chemokines by Multiplex (MIP) or
ELISA (MCP-1). A: n = 3; B: n = 6. Role of β2-integrin (CD18) during stimulation of MOs with
OK-432 CD18: MCP-1 (NS). Role of CD18 and CD36 in OK-432 activation of MOs
Figure 10
Role of CD18 and CD36 in OK-432 activation of MOs. MOs were incubated either with isotype control or anti-
CD18/F(ab) (12.5 μg/ml) for 24 h (A). PBMCs were incu-
bated either with isotype control or anti-CD36 (2.5 μg/ml)
(B) prior to incubation with OK-432 for 24 h. Supernatants
were analyzed for shown chemokines by Multiplex (MIP) or
ELISA (MCP-1). A: n = 3; B: n = 6. CD18: MCP-1 (NS). OK-432 pr
MOs
Figure 9 OK-432 promotes Syk or PI-3 kinase phosphorylation
in MOs. MOs, isolated by the monocyte negative isolation
kit, were incubated either with piceatannol, Pic. (30 μM) or
diluent control (A) or LY294002, LY (50 μM) (B) and either
left unstimulated or further stimulated with OK-432 for 15
min. Blots shown are representative of two separate experi-
ments performed with pooled MOs obtained from two dif-
ferent blood donors. chemokine response in purified MOs from HNSCC
patients. Discussion We have studied in vitro OK-432-stimulated mononuclear
MNPs chemokine secretion. Purified MOs, PBMCs or WB
secreted MCP-1, MIP-1α, and MIP-1β following OK-432
stimulation at a similar rate, except for a substantially
lower MIP-1α secretion from WB. This is in line with other
investigators, e.g., showing that S. pyogenes directly acti-
vates Mϕs to secrete chemokines [24]. Cytokine mRNA
quantification of MCP-1 transcripts showed that OK-432
stimulation was followed by cytokine mRNA synthesis
within hours. There was a correlation between measured
cytokine mRNA levels and cytokine-secreted levels sug-
gesting that OK-432 mainly exerts its effects at the pre-
transcriptional level. We furthermore separated by adher-
ence most of the MOs from a sample of PBMCs. Upon
OK-432 stimulation both these cell fractions secreted
MCP-1 at about twice the level of the MO-depleted frac-
tion. These findings support that MNPs are the main
source for MCP-1 within PBMCs. OK-432 also elicited a Page 6 of 11
(page number not for citation purposes) Page 6 of 11
(page number not for citation purposes) http://www.biomedcentral.com/1471-2172/10/6 BMC Immunology 2009, 10:6 kinase phosphorylation occurred only upon stimulation
with OK-432. We have shown that addition of either one
of these inhibitors to OK-432-stimulated MOs did not
change chemokine secretion. On the contrary, with addi-
tion of both of these inhibitors, MCP-1 secretion was
increased, pointing to the existence of other possible sig-
naling pathways. In addition, presence of both inhibi-
tors throughout the isolation and separation of
monocytes blocked the unstimulated secretion of chem-
okines supporting that β integrins are important for
chemokine secretion during the adhesion process. Our
observed Syk phosphorylation upon OK-432 binding
pointed towards an integrin dependence and resonated
with Cuzzola et al [22], who have shown that the MO
interleukin response to Group A Streptococci (GAS) was
dependent on the β2 integrin. We explored whether this
applied to the OK-432-mediated activation of MOs by
adding a blocking anti-CD18 antibody. MCP-1 produc-
tion appears to be partially inhibited by the anti-CD18
antibody. Thus, the β2 integrin may play a role in medi-
ating the effects of OK-432 on MOs. Nevertheless, other
receptors may phosphorylate either Syk or PI-3 kinase or
both and their nature has yet to be established. upon OK-432 stimulation after initiation of MO differen-
tiation [17]. The present report thus adds more support to
the notion that OK-432 is a target-seeking substance. Discussion Only
when MNPs are stimulated by, e.g., tumor cells or intercel-
lular substances [29], one observes a chemokine response. The present results argue in favor of chemokine secretion
being an important effector function of MNPs during OK-
432 stimulation. This is important as to designing future
BRM treatments with OK-432. Platinum-based drugs, e.g.,
negatively interfere more with monocyte than lymphocyte
function [31] and should thus be avoided in combination
with OK-432 treatment. MIP-1α/β and MCP-1 secretion is induced by stimulation
of MOs with OK-432. These chemokines are ligands of the
receptors CXCR1, CCR1, CCR2 and CCR5, present on pol-
ymorphonuclear granulocytes, basophiles, granulocytes,
MOs, Mϕs, DCs, NK cells, T, and B cells [32]. Thus, the
shown chemokine secretion after OK-432 stimulation of
MOs may co-explain the systemic effects of OK-432 on the
immune system. Similar findings were made by Veckman
et al [24] upon stimulating Mϕs with pathogenic live bac-
teria (serotype T1M1 (IH32030)) isolated from a child
with bacteremia. In contrast, we have stimulated mono-
cytes with a heat- and penicillin G-killed, low-virulence
SU strain of S. pyogenes and found very low levels of
RANTES (CCL5). Therefore, it is plausible that virulence
status/degree, strain, and preparation of bacteria influence
chemokine production and MOs may have a different
chemokine profile compared to Mϕs. This becomes espe-
cially interesting when the emphasis is on OK-432 being
a lyophilized, killed S. pyogenes. S. pyogenes is an impor-
tant pathogen causing human disease. The diseases
caused by S. pyogenes range from tonsillitis, impetigo, and
scarlet fever to lethal toxic shock syndrome [13]. The
shown mechanisms of MNP activation by OK-432 thus
shed light on these diseases as well. OK-432 probably stimulates MOs via TLR2 [15] and/or
TLR4 [25]. In order to study the role of these receptors,
MOs were stimulated in parallel with OK-432, and the
known TLR2 or TLR4 agonists, LTA and LPS, respectively. Results showed that MOs respond to OK-432 in a similar
fashion to LTA stimulation, thus, arguing for primary
TLR2-dependent stimulation of MOs by OK-432. A potential candidate for co-stimulation of MOs is
CD36. This receptor has been found to function as a co-
receptor for TLR2 receptor [26]. We determined a
reduced MIP-1β and MCP-1, but an unchanged MIP-1α
response upon employing blocking anti-CD36 antibod-
ies. Thus, CD36 may function as an important receptor
in the OK-432 stimulation of MOs with respect to chem-
okine secretion. Conclusion
h
f We have found that MCP-1 and MIP-1α/β production by
OK-432-stimulated human purified adherent MOs occurs
in most healthy individuals. MIP-1α secretion is mostly
found following differentiation stimuli of MOs to become
Mϕs. Furthermore, MOs stimulated with OK-432 respond
with chemokine secretion dependent on adherent growth
conditions. OK-432 may rely on β2 integrin stimulation. CD36 modulates the MIP-1β and MCP-1 response to OK-
432. To some extent OK-432 may act as a target-seeking
substance whereby only MOs adhered, e.g., to a target,
secrete substantial amounts of chemokines in part
explaining why OK-432 is suited as a BRM drug. Other co-stimulating receptors than the presently shown
ones may be important for chemokine production following
OK-432 stimulation of MNPs. Examples include CD62L (L-
selectin receptor), a mediator of MOs rolling on and interact-
ing with endothelial cells [27] and CD162 (P-selectin glyco-
protein ligand-1), a promoter in the interaction between
MOs and platelets [28]. Future studies are needed to estab-
lish their roles in OK-432 stimulation in MOs. The shown MNP stage-dependence for MIP-1α secretion
and adherence-dependent chemokine response to OK-
432 supports the hypothesis that chemokine secretion is
less favoured when MOs are stimulated by OK-432 in pb
compared to extravasal stimulation. This is in line with
our previous observation that TNF-α is primarily secreted Page 7 of 11
(page number not for citation purposes) Experimental conditions In different experimental series, MOs (1 × 105 cells/well)
were cultured either with piceatannol (0.5, 1, 30 or 120
μM – final concentration), LY294002 (50 μM – final con-
centration), wortmannin (100 nM – final concentration)
or anti-CD36 (2.5 μg/ml – final concentration) or anti-
mouse IgG1 isotype control (2.5 μg/ml – final concentra-
tion) or pepsin-degraded (Fab) anti-CD18 (12.5 μg/ml –
final concentration) or anti-rat IgG2B isotype control (12.5
μg/ml – final concentration) for 30 min before OK-432
(0.01 KE/ml – final concentration) was added and after 24
hours the supernatants were collected and stored at -80°C
for further analysis. Whole blood, PBMC, and monocyte preparation
Whole blood was diluted 1:1 with Ultraculture (Lonza,
Basel, Switzerland), allocated to a 24-well plate (Nunc A/
S, Roskilde, Denmark) and upon experiment comple-
tion, the supernatant was collected after the whole
blood/Ultraculture mixture was centrifuged. PBMCs
were separated by density gradient centrifugation with
Lymphoprep® (Nycomed, Oslo, Norway) as density gra-
dient medium in LeukoSep tubes (Greiner-Bio One
GmbH, Frickenhausen, Germany). MOs were, unless Reagents and antibodies OK-432 (Picibanil) (Chugai Pharmaceutical Co. Ltd.,
Tokyo, Japan) was dissolved in sterile water as a stock Page 7 of 11
(page number not for citation purposes) http://www.biomedcentral.com/1471-2172/10/6 BMC Immunology 2009, 10:6 otherwise stated, isolated from blood by gradient centrif-
ugation followed by adherence to plastic. The PBMC
yield of 7 ml blood (6 × 105 cells/well) was allocated to
a 24-well plate (Nunc) with RPMI 1640 (Lonza) supple-
mented with amphotericin B (2.5 μg/ml) and glucose
(2% v/v) (both Sigma), HEPES (1% v/v), L-glutamine
(1% v/v), penicillin (100 IU/ml), streptomycin (100 μg/
ml), sodium bicarbonate (2.7% v/v), sodium pyruvate
(1% v/v)(all from Lonza) and 20% AB serum to a total
volume of 0.5 ml/well. After 40 minutes pre-incubation,
the adherent MOs were purified by washing, and then
cultured in complete RPMI 1640 (Lonza)/20% AB serum
with 0.5 ml/well. The method yields in general cultures
which contain at least 75% MOs, as determined by non-
specific esterase stain (NSES), and more than 90% viable
cells, as tested by tryphan blue stain. In some experi-
ments, MOs were also isolated using the negative isola-
tion kit (Dynal Biotech, Oslo, Norway) according to the
manufacturer's protocol. Briefly, PBMCs, isolated by gra-
dient centrifugation using Lymphoprep-containing Leu-
koSep tubes, were resuspended in PBS/0.1% bovine
serum albumin (BSA) (Sigma) and exposed to an equal
mixture of blocking reagent and antibody mix for 10 min
at 4°C. After 10 min, PBMCs were washed, added fresh
PBS/0.1% BSA, added to washed beads, and incubated
for 15 min with gentle tilting and rotation at 4°C. After
incubation, resuspended rosettes were pipetted and
placed in the Dynal MPC magnet for 5 min. The super-
natant was collected and placed in the Dynal MPC mag-
net for an additional 2 min. The collected MO-pure
supernatant (95% purity – NSES) was washed, cells
counted, and added to a 24-well plate. solution with a concentration of 1 KE (Klinische Einheit)/
ml = 0.1 mg/ml. LTA from S. pyogenes and LPS from E.coli
026:B6 (Sigma, St.Louis, MO, USA) were dissolved in PBS
as a stock solution of 5 mg/ml (LTA) and 25 μg/ml (LPS)
and used at a final concentration of 0.5 μg/ml and 1 μg/
ml, respectively. Reagents and antibodies F(ab)
(degraded by pepsin (Sigma)), azide-free, blocking rat
anti-human CD18 (Chemicon International, Temecula,
CA, USA) was dissolved in sterile PBS as a 100 μg/ml stock
solution. Anti-rat IgG2B isotype control (R&D Systems
Europe Ltd.) was dissolved in sterile PBS as a 500 μg/ml
stock solution. Patients and controls Patients admitted to surgery for head and neck squamous
cell carcinoma (HNSCC) gave written consent before par-
ticipating in the study. All patients were males. Patient 1
was 60 years and had tonsillar carcinoma (T3N0M0G1). Patient 2 had carcinoma of the tongue (T2N2M0G2). Patient 3 had carcinoma of the basal tongue (T2N2M0G2). Patient 4 had laryngeal carcinoma (T4N1M0). All patients
donated blood approximately 2 weeks post-operatively as
is appropriate in the event of starting treatment with OK-
432. Whole blood from controls was obtained from ran-
domly selected, healthy donors at Haukeland University
Hospital. As selection was done randomly, age and sex of
donors were not taken into consideration, i.e. not
recorded. The study was approved by the Regional Com-
mittee for Medical Ethics. Reagents and antibodies The Syk inhibitor piceatannol (Calbio-
chem, La Jolla, CA, USA) was dissolved in ethanol as a
stock solution with a concentration of 2 mg/ml (8000
μM) and used at a final concentration of 0.5, 1, 30 or 120
μM [18,19,33]. The PI-3 kinase inhibitor, LY294002
(Sigma) was dissolved in sterile water at a concentration
of 5 mg/ml (14.5 mM) and used at a final concentration
of 50 μM. The PI-3 kinase inhibitor, wortmannin (Sigma)
was dissolved in DMSO at a concentration of 1 mg/ml
(2.3 mM) and used at a final concentration of 100 nM. Azide-free, blocking mouse anti-human CD36 (HyCult,
Uden, The Netherlands) came as a 100 μg/ml stock solu-
tion and anti-mouse rat IgG1 isotype control (R&D Sys-
tems Europe Ltd., Abingdon, Great Britain) was dissolved
in sterile PBS as a 250 μg/ml stock solution. F(ab)
(degraded by pepsin (Sigma)), azide-free, blocking rat
anti-human CD18 (Chemicon International, Temecula,
CA, USA) was dissolved in sterile PBS as a 100 μg/ml stock
solution. Anti-rat IgG2B isotype control (R&D Systems
Europe Ltd.) was dissolved in sterile PBS as a 500 μg/ml
stock solution. solution with a concentration of 1 KE (Klinische Einheit)/
ml = 0.1 mg/ml. LTA from S. pyogenes and LPS from E.coli
026:B6 (Sigma, St.Louis, MO, USA) were dissolved in PBS
as a stock solution of 5 mg/ml (LTA) and 25 μg/ml (LPS)
and used at a final concentration of 0.5 μg/ml and 1 μg/
ml, respectively. The Syk inhibitor piceatannol (Calbio-
chem, La Jolla, CA, USA) was dissolved in ethanol as a
stock solution with a concentration of 2 mg/ml (8000
μM) and used at a final concentration of 0.5, 1, 30 or 120
μM [18,19,33]. The PI-3 kinase inhibitor, LY294002
(Sigma) was dissolved in sterile water at a concentration
of 5 mg/ml (14.5 mM) and used at a final concentration
of 50 μM. The PI-3 kinase inhibitor, wortmannin (Sigma)
was dissolved in DMSO at a concentration of 1 mg/ml
(2.3 mM) and used at a final concentration of 100 nM. Azide-free, blocking mouse anti-human CD36 (HyCult,
Uden, The Netherlands) came as a 100 μg/ml stock solu-
tion and anti-mouse rat IgG1 isotype control (R&D Sys-
tems Europe Ltd., Abingdon, Great Britain) was dissolved
in sterile PBS as a 250 μg/ml stock solution. Reverse transcription TaqMan RT reagents (Applied Biosystems, Foster City, CA,
USA) were used to reverse transcribe total RNA from OK-
432-stimulated MOs obtained from normal blood
donors. Final concentration of the RT reaction was 1 ×
TaqMan RT buffer, 5.5 mM MgCl 2, 2 mM dNTP mixture
(500 μM of each dNTP), 2.5 μM random hexamers, 0.4
units/μl RNase inhibitor, 2.5 units/μl Multiscribe RT, 5–
25 ng RNA, and DEPC water to a total volume of 50 μl. The RT thermal cycling was performed on a Mastercycler
gradient thermal cycler (Eppendorf, Hamburg, Germany)
under the following conditions: primer incubation for 10
min at 25°C, reverse transcription for 30 minutes at 48°C,
and inactivation of reverse transcriptase for 5 minutes at
95°C. Real-time quantitative RT-PCR analysis Real-time quantitative RT-PCR analysis
The ABI PRISM 7700 Sequence Detection System
(Applied Biosystems) with 96-well plates was used to per-
form real-time quantitative RT-PCR. Reactions were car-
ried out in a total volume of 25 μl containing 12.5 μl of 2
× TaqMan Universal PCR Master Mix (Applied Biosys-
tems), 300 nM (0.25 μl) forward primer, 300 nM (0.25
μl) reverse primer, 200 nM (1 μl) TaqMan probe, 8 μl
water, and 3 μl cDNA template. Thermocycler conditions
were as follows: incubation for 2 minutes at 50°C, fol-
lowed by incubation for 10 minutes at 95°C and 40 cycles
of two-step (denaturing for 15 seconds at 95°C followed
by annealing/extension for 1 minute at 60°C) PCR. As a
reference gene, CD68 was used to normalize the gene Western blot analysis y
MOs from 7 ml blood were isolated by gradient centrifu-
gation in Lymphoprep-containing LeukoSep tubes, fol-
lowed by the monocyte negative isolation kit (Dynal
Biotech), allowed to adhere for 40 minutes, after which
piceatannol or LY294002 or diluent control was added for
30 min, followed by incubation with OK-432 for 15 min. Upon experiment completion, cells were washed once
with cold PBS, added lysis buffer (50 mM Tris-HCl, pH
7.4, 1% NP40, 0.25% sodium deoxycholate, 150 mM
NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM sodium
orthovanadate, 1 mM NaF, and 1× complete proteinase
inhibitor (Roche, Nutley, NJ, USA)) and scraped gently
using a cell scraper. The cell lysate was clarified by centrif-
ugation at 16,000 g for one minute at 4°C. Protein levels
were measured using the Bio-Rad BCA Protein Assay (Bio-
Rad, Hercules, CA, USA). Samples were added 1× loading
buffer and the lysate was boiled for 5 minutes. Samples (6
μg/lane) and a Precision Plus Protein Kaleidoscope stand-
ard (Bio-Rad) were electrophoresed on a 10% SDS poly-
acrylamide gel for 60 minutes at 200 V and transferred to
nitrocellulose membranes (Bio-Rad) in electroblotting
buffer (25 mM Tris base, 0.2 M glycine, and 20% metha-
nol) for 60 minutes at 100 V. Membrane was blocked with
5% BSA in TBS-Tween for one hour at RT with gentle tilt-
ing. The blots were exposed either to a polyclonal anti-
body against phosphorylated Syk or PI3 kinase p85/p55
or total Syk or total PI3 kinase p85 (all: Cell Signaling,
Beverly, MA, USA) (all: 1:1000 in 5% BSA in TBS-Tween)
overnight at 4°C with gentle shaking. After washing, an
HRP-linked goat anti-rabbit IgG (H + L) antibody (Bio-
Rad) (1:2000) was added for one hour at room tempera-
ture with gentle tilting. Proteins were detected using lumi-
nol/enhancer/hydrogen peroxide – based ECL and
visualized on the Molecular Imager ChemiDoc XRS Sys-
tem (Bio-Rad). http://www.biomedcentral.com/1471-2172/10/6 http://www.biomedcentral.com/1471-2172/10/6 http://www.biomedcentral.com/1471-2172/10/6 expression of MCP-1. To generate a standard curve for rel-
ative gene expression determination, cDNA synthesized
from pooled total RNA of MOs obtained from healthy
blood donors was used. The standard curves generated for
the cDNA analyzed had slopes with a mean of -3.3. (buffer RLT containing beta-mercaptoethanol (Sigma)),
homogenized by vortexing for 30 s, and the homogenate
was applied to an Rneasy spin column after addition of
ethanol in order to bind total RNA to the membrane. Contaminants were removed by washing with buffers
RW1 and RPE, and total RNA was eluted using water. In
addition, total RNA was isolated from MOs of normal
blood donors and subsequently pooled for use in generat-
ing the standard curve. Primer and probe design Primer and probes for the TaqMan assays were designed
using the Primer Express software (Applied Biosystems). Intron-spanning primers were used to prevent amplifica-
tion of genomic DNA. The candidate genes were MCP-1
[GenBank: NM_002982] (70 bp-amplicon); primers: (F)
5'-CTCTCGCCTCCAGCATGAA-3', (R) 5'-GGAATGAAG-
GTGGCTGCTATGA-3',
probe:
5'-CCGCCCTTCTGT-
GCCTGCTGC-3'and the housekeeping gene used was
CD68 [GeneBank: BC015557] (67 bp-amplicon); prim-
ers: (F) 5'-CCCCACGCAGCAAAGTG-3', (R) 5'-CCAG-
GGGTGCTTGGAGATCT-3, probe: 5'-TCTCGGCTCAGAA
TGCATCCCTTCG-3'). Designed primers and probes were
purchased from MedProbe (Oslo, Norway). Page 9 of 11
(page number not for citation purposes) Total RNA extraction In some experiments, total RNA was isolated immediately
after harvesting MOs (24 hours post-stimulation), using
the RNeasy Minikit's (Qiagen, Hilden, Germany) protocol
for isolation of total RNA from animal cells grown in
monolayer, as recommended by the manufacturer. Briefly, adherent MOs were added 350 μl lysis buffer Page 8 of 11
(page number not for citation purposes) Page 8 of 11
(page number not for citation purposes) BMC Immunology 2009, 10:6 Luminex fluorescent bead-based technology (Luminex
Corporation Austin, TX, USA). Luminex fluorescent bead-based technology (Luminex
Corporation Austin, TX, USA). Luminex fluorescent bead-based technology (Luminex
Corporation Austin, TX, USA). Statistical analyses The statistical program package Statistical Package for the
Social Sciences (Ver. 16 SPSS, Chicago, Ill., USA) was
used. Chemokine levels and expression were compared by
the Wilcoxon signed-rank test (two-tailed). Differences
were considered significant at p < 0.05. 8. Knecht R, Peters S, Solbach C, Baghi M, Gstottner W, Hambek M:
EGFR antibody-supplemented TPE-chemotherapy. Preclini-
cal investigations to a novel approach for head and neck can-
cer induction treatment. Anticancer Res 2003, 23:4789-95. 9. 9. Kumazawa H, Yamashita T, Tachikawa T, Minamino M, Nakata Y:
Local injection of OK-432/fibrinogen gel into head and neck
carcinomas. Eur J Cancer 1994, 30A:1741-4. 10. Naito K, Ueda Y, Itoh T, Fuji N, Shimizu K, Yano Y, Yamamoto Y,
Imura K, Kohara J, Iwamoto A, et al.: Mature dendritic cells gen-
erated from patient-derived peripheral blood monocytes in
one-step culture using streptococcal preparation OK-432
exert an enhanced antigen-presenting capacity. Int J Oncol
2006, 28:1481-9. References 1. Sakamoto J, Teramukai S, Watanabe Y, Hayata Y, Okayasu T, Naka-
zato H, Ohashi Y: Meta-analysis of Adjuvant Immunochemo-
therapy Using OK-432 in Patients With Resected Non-
Small-Cell Lung Cancer. J Immunother 2001, 24:250-256. 1. Sakamoto J, Teramukai S, Watanabe Y, Hayata Y, Okayasu T, Naka-
zato H, Ohashi Y: Meta-analysis of Adjuvant Immunochemo-
therapy Using OK-432 in Patients With Resected Non-
Small-Cell Lung Cancer. J Immunother 2001, 24:250-256. g
J
2. Oba K, Teramukai S, Kobayashi M, Matsui T, Kodera Y, Sakamoto J:
Efficacy of adjuvant immunochemotherapy with polysaccha-
ride K for patients with curative resections of gastric cancer. Cancer Immunol Immunother 2006, 56(6):905-911. ( )
3. Ogita S, Tsuto T, Deguchi E, Tokiwa K, Nagashima M, Iwai N: OK-
432 therapy for unresectable lymphangiomas in children. J
Pediatr Surg 1991, 26:263-8. g
4. Okamoto M, Ohe G, Oshikawa T, Nishikawa H, Furuichi S, Yoshida
H, Matsuno T, Saito M, Sato M: Induction of Th1-type cytokines
by lipoteichoic acid-related preparation isolated from OK-
432, a penicillin-killed streptococcal agent. Immunopharmacol-
ogy 2000, 49:363-76. gy
,
5. Baran J, Baj-Krzyworzeka M, Weglarczyk K, Ruggiero I, Zembala M:
Modulation of monocyte-tumour cell interactions by Myco-
bacterium vaccae. Cancer Immunol Immunother 2004, 53:1127-34. 6. Ooi VE, Liu F: Immunomodulation and anti-cancer activity of
polysaccharide-protein complexes. Curr Med Chem 2000,
7:715-29. gy
5. Baran J, Baj-Krzyworzeka M, Weglarczyk K, Ruggiero I, Zembala M:
Modulation of monocyte-tumour cell interactions by Myco-
bacterium vaccae. Cancer Immunol Immunother 2004, 53:1127-34. 6. Ooi VE, Liu F: Immunomodulation and anti-cancer activity of
polysaccharide-protein complexes. Curr Med Chem 2000,
7:715-29. 7. Moschos SJ, Edington HD, Lan SR, Rao UN, Jukic D, Shipe-Spotloe J,
Kirkwood JM: Neoadjuvant treatment of regional stage IIIB
melanoma with high-dose interferon alfa-2b induces objec-
tive tumor regression in association with modulation of
tumor infiltrating host cellular immune responses. J Clin Oncol
2006, 24:3164-71. Acknowledgements This work has been supported by the Norwegian Cancer Society and the
Strategic Research Program at Helse Bergen, Norway. We thank Dagny
Ann Sandnes for expert ELISA analysis, Petra Vogelsang for assistance with
the Western blot/Molecular Imager ChemiDoc XRS System, Broegelmann
Research Laboratory for providing lab and instruments for Western blot
analysis and The Research Core Facility (FFS) at Haukeland University Hos-
pital for general lab and instrument access. Competing interests
h
h
d
l
h
h The authors declare that they have no competing interests. The authors declare that they have no competing interests. py
13. Cleary PP: Streptococcus moves inward. Nat Med 2006,
12:384-6. 14. Van Ginderachter JA, Movahedi K, Hassanzadeh Ghassabeh G, Meer-
schaut S, Beschin A, Raes G, De Baetselier P: Classical and alterna-
tive activation of mononuclear phagocytes: picking the best
of both worlds for tumor promotion. Immunobiology 2006,
211:487-501. Abbreviations BRM: biological response modifier; HNSCC: head and
neck squamous cell carcinoma; MIP: macrophage inflam-
matory protein; MCP: monocyte chemoattractant protein;
pb: peripheral blood; PI: phosphoinositide; pSyk: phos-
phoSyk; TLR: Toll-like receptor. 11. Knipping S, Goetze G, Neumann K, Bloching M: Sclerotherapy of
cervical cysts with Picibanil (OK-432). Eur Arch Otorhinolaryngol
2006, 264:423-7. 12. Ryoma Y, Moriya Y, Okamoto M, Kanaya I, Saito M, Sato M: Biolog-
ical effect of OK-432 (picibanil) and possible application to
dendritic cell therapy. Anticancer Res 2004, 24:3295-301. MCP-1 protein analysis The contents of MCP-1 in supernatants were determined
by enzyme-linked immunosorbant assay kit (ELISA) man-
ufactured by R&D Systems (R&D Systems). All procedures
were performed according to the specifications of the
manufacturer. In short, 96-well microtiter plates (Costar
Corning, NY, USA) were coated overnight at room tem-
perature with monoclonal mouse anti-human MCP-1
capture antibodies. Diluted samples and recombinant
human MCP-1 standard were added and incubated for 2
h at room temperature followed by addition of bioti-
nylated polyclonal goat anti-human MCP-1. The plates
were incubated for 20 minutes at room temperature with
streptavidin-conjugated horseradish peroxidase. Tetra-
methyl-benzidine (TMB) (Sigma) and H2O2 were used as
substrate. Absorbency values were measured at 450 nm
using Softmax Pro version 4.0 on an Emax Precision
microtiter plate reader (Molecular Devices, Sunnyvale,
CA, USA). The lower detection level was 15.62 pg/ml. Chemokine levels obtained with media controls (after 24
h) were substracted from those obtained with OK-432-,
LTA-, LPS-stimulation. All media controls tested directly
after monocyte purification had nondetectable chemok-
ine levels. Multiplex cytokine analysis Chemokines in supernatants were detected using the
Luminex immunobead technology. A 25-plex or 5-plex
chemokine kit (all from Invitrogen/Biosource, Carlsbad,
CA, USA), were used to analyze cytokine levels in super-
natants from successfully completed experiments. In
short, antibody-coupled beads were incubated with target
analyte after which they were incubated with biotinylated
detection antibody before finally being incubated with
streptavidin-phycoerythrin. Standard sensitivity was 10
pg/ml (MIP-1α, MIP-1β). Samples were then read by the
Bio-Plex array reader (Invitrogen/Biosource), using Page 9 of 11
(page number not for citation purposes) Page 9 of 11
(page number not for citation purposes) BMC Immunology 2009, 10:6 http://www.biomedcentral.com/1471-2172/10/6 http://www.biomedcentral.com/1471-2172/10/6 Authors' contributions CO performed the Western blot analysis, Multiplex analy-
sis, all the experiments and data analysis. HS did the RT-
PCR experiments and analysis. KB operated the Luminex
machine. JO provided OK-432. CO and HJA were respon-
sible for the concept and design of the experiments and
primarily writing the manuscript. All authors have read
and approved the manuscript. 15. Graveline R, Segura M, Radzioch D, Gottschalk M: TLR2-depend-
ent recognition of Streptococcus suis is modulated by the
presence of capsular polysaccharide which modifies macro-
phage responsiveness. Int Immunol 2007, 19:375-89. p
g
p
16. Moser B, Wolf M, Walz A, Loetscher P: Chemokines: multiple
levels of leukocyte migration control. Trends Immunol 2004,
25:75-8. Page 10 of 11
(page number not for citation purposes) Page 10 of 11
(page number not for citation purposes) BMC Immunology 2009, 10:6 http://www.biomedcentral.com/1471-2172/10/6 http://www.biomedcentral.com/1471-2172/10/6 17. Olsnes C, Stavang H, Olofsson J, Aarstad HJ: TNF-alpha is
secreted by monocytes in transit to become macrophages,
but not by peripheral blood monocytes, following OK-432
(lyophilized S. pyogenes) stimulation. Scand J Immunol 2007,
66:684-93. 18. Schenkel AR, Mamdouh Z, Muller WA: Locomotion of monocytes
on endothelium is a critical step during extravasation. Nat
Immunol 2004, 5:393-400. 19. Vines CM, Potter JW, Xu Y, Geahlen RL, Costello PS, Tybulewicz VL,
Lowell CA, Chang PW, Gresham HD, Willman CL: Inhibition of
beta 2 integrin receptor and Syk kinase signaling in mono-
cytes by the Src family kinase Fgr. Immunity 2001, 15:507-19. 20
G
G
C
2 g
y
20. Reyes-Reyes M, Mora N, Gonzalez G, Rosales C: beta1 and beta2
integrins activate different signalling pathways in mono-
cytes. Biochem J 2002, 363:273-80. y
J
21. Butler B, Williams MP, Blystone SD: Ligand-dependent activation
of integrin alpha vbeta 3. J Biol Chem 2003, 278:5264-70. 22. Cuzzola M, Mancuso G, Beninati C, Biondo C, Genovese F, Tomasello
F, Flo TH, Espevik T, Teti G: Beta 2 integrins are involved in
cytokine responses to whole Gram-positive bacteria. J Immu-
nol 2000, 164:5871-6. 23. Collot-Teixeira S, Martin J, McDermott-Roe C, Poston R, McGregor
JL: CD36 and macrophages in atherosclerosis. Cardiovasc Res
2007, 75:468-77. 24. Veckman V, Miettinen M, Matikainen S, Lande R, Giacomini E, Coccia
EM, Julkunen I: Lactobacilli and streptococci induce inflamma-
tory chemokine production in human macrophages that
stimulates Th1 cell chemotaxis. J Leukoc Biol 2003, 74:395-402. 25. Authors' contributions Okamoto M, Oshikawa T, Tano T, Ahmed SU, Kan S, Sasai A, Akashi
S, Miyake K, Moriya Y, Ryoma Y, et al.: Mechanism of anticancer
host response induced by OK-432, a streptococcal prepara-
tion, mediated by phagocytosis and Toll-like receptor 4 sign-
aling. J Immunother 2006, 29:78-86. g
J
26. Triantafilou M, Gamper FG, Haston RM, Mouratis MA, Morath S, Har-
tung T, Triantafilou K: Membrane sorting of toll-like receptor
(TLR)-2/6 and TLR2/1 heterodimers at the cell surface
determines heterotypic associations with CD36 and intracel-
lular targeting. J Biol Chem 2006, 281:31002-11. g
g
J
27. Tacke F, Randolph GJ: Migratory fate and differentiation of
blood monocyte subsets. Immunobiology 2006, 211:609-18. 28. Ma YQ, Plow EF, Geng JG: P-selectin binding to P-selectin glyc-
oprotein ligand-1 induces an intermediate state of
alphaMbeta2 activation and acts cooperatively with extra-
cellular stimuli to support maximal adhesion of human neu-
trophils. Blood 2004, 104:2549-56. p
29. Coussens LM, Werb Z: Inflammation and cancer. Nature 2002,
420:860-7. 30. Otto F, Schmid P, Mackensen A, Wehr U, Seiz A, Braun M, Galanos
C, Mertelsmann R, Engelhardt R: Phase II trial of intravenous
endotoxin in patients with colorectal and non-small cell lung
cancer. Eur J Cancer 1996, 32A:1712-8. J
31. Briegert M, Kaina B: Human monocytes, but not dendritic cells
derived from them, are defective in base excision repair and
hypersensitive to methylating agents. Cancer Res 2007,
67:26-31. 32. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M: The
chemokine system in diverse forms of macrophage activa-
tion and polarization. Trends Immunol 2004, 25:677-8. p
33. Huang ZY, Barreda DR, Worth RG, Indik ZK, Kim MK, Chien P, Sch-
reiber AD: Differential kinase requirements in human and
mouse Fc-gamma receptor phagocytosis and endocytosis. J
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LXVI.—<i>On the special protection of appendages in process of regeneration after artificial mutilation among insects</i>
|
Annals & magazine of natural history
| 1,900
|
public-domain
| 2,111
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LXVI.—On the
special protection of
appendages in process
of regeneration after
artificial mutilation
among insects
Edmond Bordage
Published online: 22 Sep 2009. LXVI.—On the
special protection of
appendages in process
of regeneration after
artificial mutilation
among insects
Edmond Bordage
Published online: 22 Sep 2009. To cite this article: Edmond Bordage (1900) LXVI.—On the special
protection of appendages in process of regeneration after artificial
mutilation among insects , Annals and Magazine of Natural History: Series
7, 5:30, 501-503, DOI: 10.1080/00222930008678323 This article was downloaded by: [York University Libraries]
On: 31 December 2014, At: 13:47
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number:
1072954 Registered office: Mortimer House, 37-41 Mortimer
Street, London W1T 3JH, UK PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of
all the information (the “Content”) contained in the publications
on our platform. However, Taylor & Francis, our agents, and our
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to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication
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in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private
study purposes. Any substantial or systematic reproduction,
redistribution, reselling, loan, sub-licensing, systematic supply,
or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions ity Libraries] at 13:47 31 December 2014 ork University Libraries] at 13:47 31 December 2014 Special Protection of Appeltdages among [nsects. 50I LXVI.--On the Special Protection o( Appendages 52 Process
of Refeneration a])er Artificial Mutilation amon.q Insects. By EDMOND BORDAGE *. WHE:N a limb is removed owing to self-mutilation from the
body of' an Arthropod, such as a crab or a Phasmid for in-
stanc% regeneration starts from tho very surface of the
section so produced. The same thing, however, rarely
happens when, on the other hand, regeneration follows the
artificial severance of a limb. * Translated from ' Comptes Rendus,' exxix. (1899) pp: 501-504, by
Wilfred Mark Webb, F.L,~. From a separate impressmn communi-
cated by the Author.
t I1. H. Brindley, ' On certain Character.~ of Reproduced Appendages
in Arthropoda,' 1898. * Translated from ' Comptes Rendus,' exxix. (1899) pp: 501-504, by
Wilfred Mark Webb, F.L,~. From a separate impressmn communi-
cated by the Author.
1
i dl
' O
i
Ch
f
d
d A
d y
t I1. H. Brindley, ' On certain Character.~ of Reproduced Appendages
in Arthropoda,' 1898. PLEASE SCROLL DOWN FOR ARTICLE Among Mantidm, B]attidmt,
and the Orthoptera saltatoria, for exampl% artificial cuts are
followed by contraction and by more or less marked displace-
ment of the divided muscles, which are retracted within the
chitinous covering of the limb, so that, if regeneration follow~
the part in process of growth remains entirely hidden until
the next moult. The chitinous ease therefore in this instance
plays the part of a protector. York University Libraries] at 13:47 31 December 2014 p
y
p
p
It may even happen among the Mantid~e~ which possess the
power ot" self mutilation developed in a high degree, or among
the Blattidee, where it is much less marked, that contraction
of the muscles may be produced in the interior of the tro-
chanter, or even inside the joint, if developed, that forms the
haunch (coxa)~ after self-mutilation along the trochantero-
femoral suture. In this ease, if there be regeneration the
part in process of growth will remain hidden until the next
moult. Most often, however, among the Manti&e, and more rarely
in the Blatti&e~ this contraction does not take place after
self mutilation. In this case one can very soon see whether there will be
regenerat~ion without its being necessary to wait for the next
moult. It is true that one cannot perceive the various parts
which make up the rudiment in process of growth, for it is
coiled upon itself~ and, what is more~ covered over by the
non-ehitinized cuticle, which protects it after the manner of
a pocket. This cuticle lacks transparency on account of its
brownish coloration ; but the very slightly marked projection
which this protective pouch forms at the end of the trochani~er
shows nevertheless that the work of regeneration is going on. g
g
g
In the Phasmidm the rudiment in process of growth destined 502 Special Protection of Appemlages among Insects. to replace a limb detached by self-mutilation sometimes shows
i~se]f~ but in an indistinct manner, coiled up under the pro-
tective cuticle, which has some degree of transparency in
certain species. p
So far as the Mantidse and Blattid~e are concerned, I
noticed that the amount of withdrawal of the muscles divided
by self-mutilation within the chitinous covering was propof
tional to the violence of the efforts made by the insects in
getting rid of the limb. When self-mutilation took place
easily the contraction was practically nil. York University Libraries] at 13:47 31 December 2014 y
p
y
It now remains for us to examine the particular case pre-
sented by the Phasmida~. y
When artificial separation takes place in the region com-
prising the femur and the upper two thirds of the tibia, cou-
traction of the severed muscles is most marked. When~ again,
similar cuts are made in the upper part of the region formed
by the lower third of the tibia, contraction is still produced,
and as it is exactly there that the power of regeneration begins
to show itself, the part in process of growth remains hidden
until the following moult. Then in proportion as the cuts
approach the tarsus~ contraction becomes less and less evident,
until in the neighbourhood of the tibio-tarsal articulation the
divided muscles practically remain m position ; so that the
part in process of regeneration may be visible before a moult
takes place. The result is the same when the cuts are made
on any of the first three joints of the tarsus. y
j
On examining the internal structure of the limb we find
that it is precisely in the spot under discussion--upon the
lower portion of the tibia and the first joints of the tarsus--
that the muscles are inserted which move the tarsus as a
whole and its various parts. Cuts made in this region pass
through the surfaces ot the chitinous covering to which these
muscles are attached and t¥om which they run towards the
joints below which they have to move. Under these circum-
stances one can understand why the contraction will be slight
or not produced at all. It is only possible to produce it when
the cut is more or less remo~e from the upper attachment-
surface of the divided muscles: which is not the case. In
other insects there is complete withdrawal of the cut muscles
when t},e section passes through the tarsal region. It is
evident that these particular features point to differences in
the number and position of the attachment-surfaces of the
muscles in question, differences which are revealed upon
careful dissection. It follows, then, that among the Phasmidm par~s in process
of regeneration in the region we are considering are more or _New Species of Prionocalus,]'~'om Ecuador and Peru. 503 less apparent from the outset of their formation. * In the most perfectly regenerated limb~ there is a tetramerous tarsus.
I have nevertheless obtained after cuts made upon the third joint of the
t 2. A pentamerous tarsus as perfect as the normal one.
3. A bent and monstrous tarsus of six incompletely separated joints.
But these are rare exceptions to the rule.
Tetramery after regeneration
has been determined anaong the Phasmi&e not only in the four genera I
quoted in my previous papers~ but also in Anehiale, Acanthoderus, Lopa-
phus, .Diapheromera~ and probably in Cyphocrania, Diura~ and .Bacteria
me.vicana, the total nmnber of species of Orthoptera cursoria in which
tetramery has been determined being thus twenty-five tarsus :--
1 A pentamerous tarsus with incompletely separated joints 1. A pentamerous tarsus with incompletely separated
2 A pentamerous tarsus as perfect as the normal one York University Libraries] at 13:47 31 December 2014 It is interesting to compare the slowness of the growth of
parts in process of regeneration after artificial removal~ as
well as that--much less marked nevertheless--of limbs in-
tended to replace those removed by self-mutilation, among
Phasmida~ with the marvellous rapidity of such growth which
has been iound among the Mantidze and Blattid~e. Whilst
among the latter limbs regenerated after self- or artificial
mutilation may begin to be of use to the insect immediately
after the next moult, in the Phasmi&e limbs in course of
regeneration cannot become useful until after the second or
third moult. I have noted that the same holds good for
the Orthoptera saltatoria. Downloaded by [York University Libraries] at 13:47 31 Downloaded by [York University Libr LXVII.~New Species of the Coleopterous Geuus Prionocalus
from Ecuador and ff eru. By CIIAS. O. WATI~I~HOUSE
V P E S THE British Museum has recently received a few specimens
of Longicorns of the genus Prionocalus. One I refer with a
slight doubt to P. cacicus~ Whit% but in the type the tubercle 1. A pentamerous tarsus with incompletely separated
2 A pentamerous tarsus as perfect as the normal one 2. A pentamerous tarsus as perfect as the normal one. 3. A bent and monstrous tarsus of six incompletely separated joints. But these are rare exceptions to the rule. Tetramery after regeneration
has been determined anaong the Phasmi&e not only in the four genera I
quoted in my previous papers~ but also in Anehiale, Acanthoderus, Lopa-
phus, .Diapheromera~ and probably in Cyphocrania, Diura~ and .Bacteria
me.vicana, the total nmnber of species of Orthoptera cursoria in which
tetramery has been determined being thus twenty five 2. A pentamerous tarsus as perfect as the normal one. 3. A bent and monstrous tarsus of six incompletely separated joints. But these are rare exceptions to the rule. Tetramery after regeneration
has been determined anaong the Phasmi&e not only in the four genera I
quoted in my previous papers~ but also in Anehiale, Acanthoderus, Lopa-
phus, .Diapheromera~ and probably in Cyphocrania, Diura~ and .Bacteria
me.vicana, the total nmnber of species of Orthoptera cursoria in which
tetramery has been determined being thus twenty five 2. A pentamerous tarsus as perfect as the normal one. 3. A bent and monstrous tarsus of six incompletely separated joints. But these are rare exceptions to the rule. York University Libraries] at 13:47 31 December 2014 As I have
already said, bowever~ growth proceeds with the greatest
slowness; it fbllows therefore that during the time which
elapses before the next moult the part in process of growth
barely forms a minute projection from 1 to 2 millim, in length. It is covered by a thin protective cuticle of a brown eolour~
moulding itself exactly upon the rudiment of the limb~ which
up to the present shows no separation into joints. It is only
after the next moult that the limb, beginning to be of appre-
ciable lengtl b will show any distinct traces of division into
tarsal joints. The growth is so slow that it is only after two
or even three moults that the mutilated limb is completed arid
becomes serviceable to the insect *. York University Libraries] at 13:47 31 December 2014 Tetramery after regeneration
has been determined anaong the Phasmi&e not only in the four genera I
quoted in my previous papers~ but also in Anehiale, Acanthoderus, Lopa-
phus, .Diapheromera~ and probably in Cyphocrania, Diura~ and .Bacteria
me.vicana, the total nmnber of species of Orthoptera cursoria in which
tetramery has been determined being thus twenty-five
|
https://openalex.org/W4239226752
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https://journals.plos.org/ploscompbiol/article/file?id=10.1371/journal.pcbi.1003875&type=printable
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English
| null |
Correction: Structure-Based Druggability Assessment of the Mammalian Structural Proteome with Inclusion of Light Protein Flexibility
|
PLOS computational biology/PLoS computational biology
| 2,014
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cc-by
| 323
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Correction
Correction: Structure-Based Druggability Assessment of
the Mammalian Structural Proteome with Inclusion of
Light Protein Flexibility Correction
Correction: Structure-Based Druggability Assessment of
the Mammalian Structural Proteome with Inclusion of
Light Protein Flexibility Correction A number of figure legends are associated with the wrong
figures in the published article. g
p
The legend of Figure 6 should be associated with Figure 4. The legend of Figure 6 should be associated with Figure 4. The legend of Figure 4 should be associated with Figure 5. The legend of Figure 5 should be associated with Figure 6. The legend of Figure 5 should be associated with Figure 6. Reference 1. Loving KA, Lin A, Cheng AC (2014) Structure-Based Druggability Assessment of
the Mammalian Structural Proteome with Inclusion of Light Protein Flexibility. PLoS Comput Biol 10(7): e1003741. doi:10.1371/journal.pcbi.1003741 Citation: The PLOS Computational Biology Staff (2014) Correction: Structure-
Based Druggability Assessment of the Mammalian Structural Proteome with
Inclusion of Light Protein Flexibility. PLoS Comput Biol 10(9): e1003875. doi:10. 1371/journal.pcbi.1003875 1. Loving KA, Lin A, Cheng AC (2014) Structure-Based Druggability Assessment of
the Mammalian Structural Proteome with Inclusion of Light Protein Flexibility.
PLoS Comput Biol 10(7): e1003741. doi:10.1371/journal.pcbi.1003741 PLOS Computational Biology | www.ploscompbiol.org Correction: Structure-Based Druggability Assessment of
the Mammalian Structural Proteome with Inclusion of
Light Protein Flexibility The PLOS Computational Biology Staff The PLOS Computational Biology Staff The PLOS Computational Biology Staff A number of figure legends are associated with the wrong
figures in the published article. PLOS Computational Biology | www.ploscompbiol.org Published September 9, 2014 Published September 9, 2014 Copyright: 2014 The PLOS Computational Biology Staff. 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. PLOS Computational Biology | www.ploscompbiol.org 1 September 2014 | Volume 10 | Issue 9 | e1003875 September 2014 | Volume 10 | Issue 9 | e1003875
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English
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Review on Stochastic Approach to Inflation
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Universe
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cc-by
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universe universe universe Review Review Diego Cruces 1,2 Diego Cruces 1,2 1
Institut de Ciencies del Cosmos (ICCUB), Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain;
dcruces@ub.edu; Tel.: +34-93-402-0817 1
Institut de Ciencies del Cosmos (ICCUB), Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain;
dcruces@ub.edu; Tel.: +34-93-402-0817 2
Departament de Física Quàntica i Astrofísica, Faculty of Physics, Universitat de Barcelona, Martí i Franquès 1,
08028 Barcelona, Spain 2
Departament de Física Quàntica i Astrofísica, Faculty of Physics, Universitat de Barcelona, Martí i Franquès 1,
08028 Barcelona, Spain 2
Departament de Física Quàntica i Astrofísica, Faculty of Physics, Universitat de Barcelona, Martí i Franquès 1,
08028 Barcelona, Spain Abstract: We present a review on the state-of-the-art of the mathematical framework known as
stochastic inflation, paying special attention to its derivation, and giving references for the readers
interested in results coming from the application of the stochastic framework to different inflationary
scenarios, especially to those of interest for primordial black hole formation. During the derivation of
the stochastic formalism, we will emphasise two aspects in particular: the difference between the
separate universe approach and the true long wavelength limit of scalar inhomogeneities and the
generically non-Markovian nature of the noises that appear in the stochastic equations. Keywords: stochastic inflation; primordial black holes; early universe; inflation 1. Introduction Although cosmological inflation was first introduced as a possible solution to the hot
Big Bang model problems [1,2], the study of vacuum fluctuations during this accelerated
expansion of the universe also gives an explanation to the anisotropies observed in the
Cosmic Microwave Background (CMB). The idea is that the different scales of fluctuations
leave the observable universe during inflation, long after it, they reenter the observable
universe at different times, being the scales reentering the cosmological horizon (horizon
from now on) during the time of recombination the responsible for the CMB anisotropies. In fact, the almost gaussianity and scale invariance of the vacuum quantum fluctuations
predicted by Slow Roll (SR) inflation has been remarkably well confirmed by the recent
Plank satellite mission [3,4]. Citation: Cruces, D. Review on
Stochastic Approach to Inflation. Universe 2022, 8, 334. https://
doi.org/10.3390/universe8060334
Academic Editor: Yi-Fu Cai
Received: 28 March 2022
Accepted: 15 June 2022
Published: 17 June 2022 Citation: Cruces, D. Review on
Stochastic Approach to Inflation. Universe 2022, 8, 334. https://
doi.org/10.3390/universe8060334
Academic Editor: Yi-Fu Cai
Received: 28 March 2022
Accepted: 15 June 2022
Published: 17 June 2022 universe universe Citation: Cruces, D. Review on
Stochastic Approach to Inflation. Universe 2022, 8, 334. https://
doi.org/10.3390/universe8060334 It is however true that, although inflation provides a causal mechanism to generate
CMB anisotropies, the CMB is only accessible to a restricted range of scales, constraining
the inflationary phase only during a limited time interval. In order to be accessible to
smaller scales of inflation, that reenter the horizon before the recombination epoch, we
must then seek for any other hint that help us to extend the time interval of inflation that
we can constraint. Primordial Black Holes (PBHs) is one of those hints [5]. In addition
to the fact that PBHs can probe the missing scales of inflation, they represent natural
candidates not only for dark matter (DM) [6], but also as the seed of supermassive BHs at
the center of massive galaxies [7] and even as the progenitors of some events that radiate
the gravitational waves detected by the LIGO/VIRGO collaboration [8]. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. g
y
PBHs are expected to form if the amplitude of density perturbations from inflation is
big enough such that when they reenter the horizon they will collapse into a Black Hole
(BH). This is not the case for CMB scales, for which the SR approximation holds and where
the amplitude of inhomogeneities is too small to form PBHs; however, for smaller scales,
this amplitude is less constrained and it could grow until PBHs are possible to form, in this
case, SR is not a good approximation and other inflationary regimes arise such as Ultra
Slow Roll (USR) and Constant Roll (CR). Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). Since the over-densities responsible for the formation of PBHs must be large enough,
they will be found at the tail of the probability distribution of density perturbations, which https://www.mdpi.com/journal/universe Universe 2022, 8, 334. https://doi.org/10.3390/universe8060334 Universe 2022, 8, 334 2 of 39 makes them exponentially rare [9]. At the same time, a large over-density at small scales
can modify the large-scale dynamics of the universe in a non-perturbative way. Thus, in
order to predict the abundance of PBHs, a precise statistical knowledge of inflationary
perturbations is highly desirable. The hope of the stochastic approach to inflation is that it incorporates quantum cor-
rections to the inflationary dynamics in a non-perturbative way [10]. In this approach,
wavelengths that are well outside the horizon are approximated in powers of spatial gradi-
ents rather than on amplitudes (as in linear theory). At the same time though, those modes
are influenced by the quantum sector by receiving quantum-kicks from stochastic forces
generated by the perturbative sub-horizon modes. The success of the stochastic formalism
resides in the fact that it allows to reduce a quantum problem into a statistical one and it
has been widely used in the literature [10–68]. Since this manuscript is mainly devoted to stochastic inflation, we will briefly review
the long history behind it, which will also help us to understand why the paper is organized
as it is. Soon after Starobisnky first proposed the idea of treating the short-wavelength part
of the field as a white noise which is included as a source term in the equation of motion
for the classical (long-wavelength) field in [10], stochastic inflation immediately gained
huge popularity [11–24], manly because of the possibility of solving the Fokker-Planck
equation that govern the evolution of the probability distribution of the amplitude of
the perturbations of the scalar field during inflation in an exact way for some specific
form of the potential, or even for generic potentials with the help of the first time passage
analysis [69] as firstly done in [24]. In the references above, the heuristic derivation of
Starobinsky, which was only valid in single-field slow-roll inflationary models, was ex-
tended beyond SR and multifield inflation. At the same time, Morikawa et al. put the stochastic formalism on a more firm
ground [25,26], where the heuristic derivation of the equations of motion of stochastic
inflation done by previous authors was refined. More concretely, they were able to derive
the same stochastic equations integrating out the short-wavelength part of the scalar
field in the path integral. Although this derivation lead to the same equations of motion
as the heuristic argument followed by previous authors, it is very useful in order to
understand what are the approximations that lead to the stochastic equations first presented
by Starobinsky and its consequences. y
y
q
The most important of these approximations is probably to compute the variance of the
noises in a pure de-Sitter background. This approach has been vastly used in mort applica-
tions
of
the
stochastic
formalism
until
today. Examples
of
this
can
be
found in [27,29–33,35,37–39,50,54,56], but more importantly, with the aid of this approach,
the
well-known
δN
formalism
[70–73]
was
extended
to
an
stochastic
δN
formalism [36,41,46,47,53,61,62,66] which allow us to compute the probability distribu-
tion of the curvature perturbation and hence to connect the results of stochastic inflation
with PBH formation as done for example in [52,58,59,63,64,67]. p
However, computing the noises in a pure de-Sitter background has important con-
sequences, in fact, it was checked in [35,37,40,45,47–49] that, under this approximation,
stochastic formalism recovers the standard result of Quantum Field Theory (QFT) for
test scalar fields on a fixed inflationary background, which give us a hint that thos ap-
proach does not correctly take into account the backreaction of the scalar field in the
metric. As pointed out in [74], this method misses the coupling between the long- and
short-wavelength sectors; more concretely, the background in which the noises should be
computed is not exactly de-Sitter, but it is both slow-roll and stochastically corrected. This
means that the results obtained with a stochastic formalism that computes the noises in a
pure de-Sitter background should not be taken seriously if we are looking for deviations
from de-Sitter shuch as in the spectral index. This important limitation of the stochastic
formalism as presented by Starobinsky (which will be further studied in Section 6.1.1), led
to some authors to present alternative ways of solving the equations of motion of stochastic Universe 2022, 8, 334 3 of 39 3 of 39 inflation beyond the usual Fokker-Plank equation with de-Sitter noises, some examples are
the recursive stategy presented in [42–44] or numerical codes as in [67]. inflation beyond the usual Fokker-Plank equation with de-Sitter noises, some examples are
the recursive stategy presented in [42–44] or numerical codes as in [67]. Another important simplification that is usually performed in the stochastic framework
is to split long- and short-wavelengths modes via a sharp window function (a step function). This choice leads to white noises which are very easy to handle both analytically and
numerically. However, it was noted in [28] that this choice of window function leads to
some problems in the asymptotic of the noise correlator at large spatial distances. This is
why some works with smooth window functions that lead to coloured noises have also
been proposed [34,68]. p
p
If we now go back to the historical controversies of stochastic inflation, we can find the
issue of the time variable in which this formalism must be formulated. Already in one of
the first works by Starobinsky [24], it was pointed out that the number of e-folds, (defined
as N = R
Hdt, where t is the cosmic time and H is the Hubble parameter) seem to be a
very appropriate time variable. This choice was later justified by connecting stochastic
inflation with results form QFT in curved space times [35,37,47]; however, as also pointed
out by these references, the choice of N as time variable is only a consequence of writing
the stochastic equations in terms only of the scalar field. In fact, one can check that the
only way to write all the scalar degrees of freedom in terms solely of the scalar field is to
use the uniform-N gauge in the separate universe approach [60], which justifies the use
of N as time variable since, by definition, in this gauge it is unperturbed. It was not until
recently [65] when the stochastic formalism was formulated in another gauge, making it
clear that the choice of N as time variable is only a consequence of the gauge choice. Finally, it was lately noticed that the stochastic formalism of inflation was leading to
some inconsistencies at next-to-leading order in SR parameters even at linear level. For
example, it was checked in [55] that the linearization of the stochastic equations do not
exactly reproduce the well-known equation of motion of the scalar linear perturbations in
the long-wavelength limit (the Mukhanov-Sasaki equation, see Section 4.1). Furthermore,
in [57] it was demonstrated that, during an ultra-slow-roll (USR) inflationary regime, some
noises that appear in the stochastic equations of motion were incompatible with the rest of
the system. The origin of these inconsistencies can be traced back to the use of the separate
universe approach in the construction of the stochastic formalism and they can be solved
by including the momentum constraint in the separate universe approach [65]. This review will give a detailed derivation of the stochastic formalism to inflation
rather than giving a list of results obtained within this formalism. During the derivation
we will emphasise all controversial aspects that have been indicated in the above summary
of the long history of the stochastic formalism, from the difference between the separate
universe approach and the true long-wavelength limit of scalar inhomogeneities to the
construction of the noises using an exact de-Sitter background. g
g
The review is organized as follows: after presenting the basic inflationary concepts
with the aid of the homogeneous picture of inflation in Section 2, we will start study-
ing inflationary inhomogeneities, focusing on its long-wavelength behaviour. In order
to do so we will present different approximations to the exact Arnowitt-Deser-Misner
(ADM) equations of Section 3. These approximations are linear perturbation theory
(in Section 4), gradient expansion (in Section 5) and the stochastic formalism (in Section 6),
which combines the two approximation schemes presented before. Finally, in Section 7 we
will give some conclusions. 2. Homogeneous Inflationary Scenarios and Slow-Roll Parameters As we have already mentioned, PBHs represent natural candidates for dark matter
(DM) (latest constraints on this idea can be found in [75]). However, one possibility to
statistically generate enough PBHs for this to hold one needs, at least, a power spectrum
of primordial curvature perturbations several order of magnitudes larger than the one
observed in the cosmic microwave background (CMB). Note that this is not the only
possibility; one could statistically generate enough PBHs with a large non-gaussianity in Universe 2022, 8, 334 4 of 39 the probability distribution of fluctuations even without enhancing the power spectrum. This scenario is discussed in [76]. It is known that a period of Slow-Roll (SR), of which the predictions of the CMB are
based upon, cannot lead to the appropriate power spectrum necessary to generate enough
PBHs to match the DM abundance [77] ( For the non-linear relation between the inflationary
power spectrum and PBHS abundance, under the assumption of gaussianity, the interested
reader can see [78]). Thus, one necessarily needs an inflationary epoch evolving beyond SR. A possibility is the introduction of an inflection point in the inflationary potential [79]. This
leads the inflaton to undergo a so-called Ultra-Slow-Roll (USR) phase of inflation [80,81]. Taking into account that the statistics of PBHs from non-gaussian fluctuations has yet to be
fully developed. The single field USR option with standard kinetic term seems then to be
the best [76]. Such a system is described by the Einstein-Hilbert action with a minimally
coupled scalar field i.e.,: S = 1
2
Z p−g
h
M2
PLR −(∇φ)2 −2V(φ)
i
,
(1) (1) whose homogeneous solution is an universe described by a Friedman-Lemaitre-Robertson-
Walker (FLRW) metric: ds2 = −dt2 + a(t)2d⃗x · d⃗x , ds2 = −dt2 + a(t)2d⃗x · d⃗x ,
(2) (2) where a(t) represents the scale factor. where a(t) represents the scale factor. The equation of motion of the scalar field in the universe described by (2) has the
following equation of motion: ¨φb + 3Hb ˙φb + Vφb
φb
= 0 ,
(3) (3) where Vφb ≡∂V(φb)
∂φb , Hb ≡˙a
a is the Hubble parameter, and a dot denotes a derivative with
respect to the cosmic time t. Finally, the super-script “b” stands for “background”, the
meaning of which will be clarified later on. 2. Homogeneous Inflationary Scenarios and Slow-Roll Parameters (9) (9) As we will show later on, an exponential decrease of ϵ1 makes the power spectrum of
curvature perturbation increase. •
Both SR and USR are, at least approximately, sub-cases of Constant-Roll (CR). Here
¨φb
Hb ˙φb = κ where κ is a constant. SR is realized when κ = 0 while USR when κ = −3. We will not analyse further this generic case. •
Both SR and USR are, at least approximately, sub-cases of Constant-Roll (CR). Here
¨φb
Hb ˙φb = κ where κ is a constant. SR is realized when κ = 0 while USR when κ = −3. We will not analyse further this generic case. It is important to remark that, given a potential related to PBH formation, the SR
and USR phases alternate. Thus, the equations of motion (6) and (8) will always be an
approximation of the system. 2. Homogeneous Inflationary Scenarios and Slow-Roll Parameters The Friedmann equation is The Friedmann equation is
Hb2
=
1
3M2
PL
˙φb2
2
+ V
φb
. (4) (4) The Slow-Roll (SR) parameters ϵi define the rate of change of the Hubble parameter: ϵ1 ≡−
˙Hb
Hb2 =
˙φb2
2H2M2
PL
≪1 ;
ϵi+1 ≡
˙ϵi
Hϵi
with
i ≥1,
(5) (5) where, to write the final expressions, we have used the Friedmann equation and the
equation of motion of the field. q
We can now define different inflationary regimes depending on the values of the ϵis: •
Slow-Roll (SR): The field is slowly rolling down a potential with an almost constant
velocity, which makes the acceleration negligible. In this case the equation of motion
(3) is approximately
•
Slow-Roll (SR): The field is slowly rolling down a potential with an almost constant
velocity, which makes the acceleration negligible. In this case the equation of motion
(3) is approximately
b
b
b 3Hb ˙φb + Vφ
φb
≃0 . (6) (6) The SR parameters are much smaller than one (ϵi ≪1) and can be written in terms of
the potential as ϵSR
1
≃
1
2M2
PL
Vφb
V
!2
;
ϵSR
2
≃
2
M2
PL
Vφbφb
V
! −4ϵSR
1
. (7) (7) Universe 2022, 8, 334 5 of 39 •
Ultra-Slow-Roll (USR): The field is moving along an exactly flat potential
Vφ = 0
,
which makes the acceleration relevant. In this case the equation of motion (3) is •
Ultra-Slow-Roll (USR): The field is moving along an exactly flat potential
Vφ = 0
,
which makes the acceleration relevant. In this case the equation of motion (3) is ¨φb + 3Hb ˙φb = 0 . (8) (8) From (8) one can infer that the velocity of the field (and hence ϵ1) exponentially
decreases, which makes some ϵi ∼O(1). More precisely: From (8) one can infer that the velocity of the field (and hence ϵ1) exponentially
decreases, which makes some ϵi ∼O(1). More precisely: ϵUSR
i
= −6 + 2ϵUSR
1
when i even. ϵUSR
i
= 2ϵUSR
1
when i>1 and odd. (9) ϵUSR
i
= −6 + 2ϵUSR
1
when i even. ϵUSR
i
= 2ϵUSR
1
when i>1 and odd. (9) ϵUSR
i
= 2ϵUSR
1
when i>1 and odd. 3. Inhomogeneities during Inflation: The ADM Formalism Although it is very useful in order to understand the inflationary dynamics, the
homogeneous picture of inflation presented in Section 2 is not very realistic and some
inhomogeneities must be introduced in order to explain the universe we currently observe. Thus, we must define a general metric which is not restricted to homogeneity and isotropy. An option which is very useful in the context of inflation is to work in the so-called Arnowitt-
Deser-Misner (ADM) formalism [82]. This framework supposes that the four-dimensional
space-time is foliated into a family of three-dimensional hypersurfaces Σt, labeled by its
time coordinate t. In order to work in the ADM formalism, it is convenient to write the metric as: ds2 = gµνdxµdxν = −α2dt2 + γij(dxi + βidt)(dxj + βjdt) ,
(10) (10) and the action (1) presented in the previous section becomes: and the action (1) presented in the previous section becomes: γ
h
M2
PL
αR(3) + α
KijKij −K2
−2αV(φ) + α−1
˙φ −βi∂iφ −αγij∂iφ∂jφ
i
,
(11) (11) where we have introduced many new terms: where we have introduced many new terms: •
α is the lapse function, which measures the rate of flow of proper time with respect to
coordinate time t as one moves normally to Σt. •
α is the lapse function, which measures the rate of flow of proper time with respect to
coordinate time t as one moves normally to Σt. •
βi is the shift vector, which measures how much the local spatial coordinate system
shifts tangential to Σt, when moving from Σt to Σt+δt along the normal direction to Σt. •
γij represents the induced metric on the hypersurface Σt, that we we will decompose •
βi is the shift vector, which measures how much the local spatial coordinate system
shifts tangential to Σt, when moving from Σt to Σt+δt along the normal direction to Σt •
βi is the shift vector, which measures how much the local spatial coordinate system
shifts tangential to Σt, when moving from Σt to Σt+δt along the normal direction to Σt. •
βi is the shift vector, which measures how much the local spatial coordinate system
shifts tangential to Σt, when moving from Σt to Σt+δt along the normal direction to Σt. 3. Inhomogeneities during Inflation: The ADM Formalism •
γij represents the induced metric on the hypersurface Σt, that we we will decompose
as γij = a2e2ζ ˜γij with det ˜γij = 1 so that the scale factor a is explicitely present. (3)
(3) g
g
t+δt
g
•
γij represents the induced metric on the hypersurface Σt, that we we will decompose
as γij = a2e2ζ ˜γij with det ˜γij = 1 so that the scale factor a is explicitely present. p
y p
R(3)
ij
is the Ricci tensor of the spatial metric, and hence R(3) ≡γijR(3)
ij . •
R(3)
ij
is the Ricci tensor of the spatial metric, and hence R(3) ≡γijR(3)
ij . j
j
•
Finally, Kij is the extrinsic curvature (K ≡γijKij), which is defined as: j
j
•
Finally, Kij is the extrinsic curvature (K ≡γijKij), which is defined as: Kij ≡−∇inj = −1
2α
∂tγij −Diβj −Djβi
,
(12) (12) where ni ≡(−α, 0, 0, 0) is the unit vector normal to the spatial hypersurfaces and ∇µ
and Di are the covariant derivatives with respect to gµν and γij, respectively. Universe 2022, 8, 334 6 of 39 It is also convenient to decompose the extrinsic curvature into its trace and traceless part as: Kij = γij
3 K + a2e2ζ ˜Aij ,
(13) (13) where ˜γij ˜Aij = 0. Note that,in the homogeneous limit, i.e., when α = 1, βi = 0 and
γij = a2δij and hence the metric (10) reduces to (2), we can identify the extrinsic curvature
with the background Hubble parameter, namely K = −3 ˙a
a = −3Hb. We will then define a
more general inhomogeneous Hubble parameter as H ≡−K
3 . This makes sense because K
represents the expansion rate of the constant time hypersurfaces. In the ADM formalism, the lapse function and the shift vector act as Lagrange multi-
pliers for the action (11), and hence they generate two constraints: the Hamiltonian and the
momentum constraints, which are, respectively: R(3) −˜Aij ˜Aij + 2
3K2 =
2
M2
PL
E ,
(14)
Dj ˜Aij −2
3 DiK =
1
M2
PL
Ji ,
(15) (14) (15) where E ≡Tµνnµnν and Ji ≡Tµjnµγj
i and Tµν is, in our case, the stress-energy tensor of the
scalar field, i.e., Tµν = ∇µφ∇νφ −1
2 gµν(∇αφ∇αφ + 2V(φ)) . 3. Inhomogeneities during Inflation: The ADM Formalism (16) (16) The two remaining variables, γij and Kij are the dynamical ones and their evolution
equations are given by: •
For γij: •
For γij: (∂t −βk∂k)ζ + ˙a
a = −1
3(αK −∂kβk),
(17)
(∂t −βk∂k) ˜γij = −2α ˜Aij + ˜γik∂jβk + ˜γjk∂iβk −2
3 ˜γij∂kβk. (18) (17) (18) •
for Kij: (∂t −βk∂k)K = α
˜Aij ˜Aij + 1
3K2
−DkDkα + 4πGα(E + Sk
k),
(19) (19) (∂t −βk∂k) ˜Aij = e−2ζ
a2
α
R(3)
ij −γij
3 R(3)
−
DiDjα −γij
3 DkDkα
+ α(K ˜Aij −2 ˜Aik ˜Ak
j ) + ˜Aik∂jβk + ˜Ajk∂iβk −2
3
˜Aij∂kβk
−8πGαe−2ζ
a2
Sij −γij
3 Sk
k
,
(2 (20) where Sij = Tij and Sk
k = γklSlk. where Sij = Tij and Sk
k = γklSlk. where Sij = Tij and Sk
k = γklSlk. where Sij = Tij and Sk
k = γklSlk. Finally, and although it can be recovered using the ADM equations just presented, it is
also worthy to present here the Klein-Gordon equation for the evolution of the scalar field: Finally, and although it can be recovered using the ADM equations just presented, it is
also worthy to present here the Klein-Gordon equation for the evolution of the scalar field: 1
√−g∂µ
p−ggµν∂νφ
−Vφ = 0 . (21) (21) We will present more detail in the following section, but it is worth remarking here
that the homogeneous equations of Section 2 are straightforwardly recovered setting α = 1,
βi = 0 and γij = a2δij g
βi = 0 and γij = a2δij. g
βi = 0 and γij = a2δij. β
γ j
j
Since the ADM equations are equivalent to the Einstein equations but much easier to
implement numerically, they are very useful to study inhomogeneous space-times in an Universe 2022, 8, 334 7 of 39 7 of 39 exact way; however, the numerical methods capable of solving exactly these equations are
computationally expensive [83]. Fortunately there exist some very useful approximations
that can be done when studying the inflationary universe that, not only allow us to con-
siderably simplify the numerical way of solving the ADM equations, but they even admit
some analytical solutions as we will see in the following. 4. Linear Perturbation Theory Since, as explained in the introduction, the deviations from an exactly homogeneous
and isotropic FLRW universe that we observe are very small, it makes sense to solve the
system of ADM Equations (14)–(21) by expanding them around a FLRW background. If
this expansion is done up to first order, we call it linear perturbation theory. There are
many reviews on this topic [84–88]; however, since we are taking a slightly different point
of view from most of them, we will go through linear perturbation theory in some detail. With this in mind, the ADM metric (10) will be written as: gµν ≃gb
µν + δgµν ,
(22) (22) where gb
µν is the homogeneous and isotropic metric of Section 2 and δgµν represent the
perturbation. This implies that the lapse function, the shift vector and the spatial metric are
approximated as: α ≃1 + A ,
βj ≃aBj ,
e2ζ ≃1 + 2D ,
˜γij ≃δij −2Eij
(23) ,
˜γij ≃δij −2Eij
(23) (23) where Eij is traceless. The reason why Eij is traceless is because is the perturbation of ˜γij,
which has unit determinant. Precisely, any matrix with unit determinant can be written as: ˜γij = e−2Mij , where Mij is traceless. Note that the last two linearizations in (23) leads to here Mij is traceless. Note that the last two linearizations in (23) leads to γij ≃a2(1 + 2D)δij −2Eij
. (24) (24) Finally, the scalar field responsible for inflation must also be linearized, i.e., φ ≃φb + δφ . (25) (25) It can be easily shown that, of the linear variables introduced above, A, D and δφ
transform as scalars under rotations in the background space-time coordinates, Bi as a
3-vector and Eij as a 3D-tensor. This does not mean that the only scalar components are
A, D and δφ. In fact, we know from Euclidean 3D vector calculus that a vector can be
decomposed as: Bi = BS
i + BV
i
with
∂iBS
j −∂jBS
i = 0
and
∂iBV
i = 0 ,
(26) (26) and hence and hence BS
i = ∂iB ,
(27) BS
i = ∂iB , (27) where B is some scalar field. where B is some scalar field. 4. Linear Perturbation Theory Similarly, for a tensor field we have Similarly, for a tensor field we have Eij = ES
ij + EV
ij + ET
ij ,
(28) Eij = ES
ij + EV
ij + ET
ij , Eij = ES
ij + EV
ij + ET
ij , (28) Universe 2022, 8, 334 8 of 39 where where where ES
ij =
∂i∂j −1
3δij∇2
E ,
EV
ij = 1
2
∂jEi + ∂iEj
with
∂iEi = 0 ,
∂iET
ij = 0 ,
(29)
δijET
ij = 0 ,
(30) (29) (30) where E is again a scalar field. where E is again a scalar field. The procedure explained above allow us to decompose the perturbations into a scalar,
vector and tensor sector. These sectors have the characteristic of evolve independently one
from each other at linear order in perturbation theory, which make them easier to handle. During this review we will be mostly focused on scalar perturbations of the metric since
they are the ones that couple to the scalar field perturbation δφ. This allows us to write the
perturbed metric of (23) as: ds2 = −(1 + 2A)dt2 + 2a∂iBdxidt + a2
(1 + 2D)δij −2
∂i∂j −1
3δij∇2
E
dxidxj . (31) (31) Similarly to what we have just done with the metric, we can split each one of the ADM
equations presented in Section 3 into an homogeneous and a perturbed part as follows: Similarly to what we have just done with the metric, we can split each one of the ADM
equations presented in Section 3 into an homogeneous and a perturbed part as follows: •
Hamiltonian constraint (14). •
Hamiltonian constraint (14). 0 =R(3) −˜Aij ˜Aij + 2
3K2 −
2
M2
PL
E
≃
6
Hb2
−
2
M2
PL
˙φb2
2
+ V(φb)
+
"
−12Hb
HbA −˙D + 1
3
∇2
a B
−4∇2
a2
D + 1
3∇2E
−
2
M2
PL
˙φb ˙δφ −
˙φb2
A + Vφbδφ
#
. (32) (32) Note that in (32) the term inside the first square brackets corresponds to the back-
ground Hamiltonian constraint (4) of Section 2, being the term inside the second
square brackets the linearization of the Hamiltonian constraint. 4. Linear Perturbation Theory We will follow this
same notation for the rest of the ADM equations. •
Momentum constraint (15). 0 = Dj ˜Aij −2
3 DiK −
1
M2
PL
Ji
≃[0]
+
"
∂i
−2HbA + 2 ˙D + 2
3∇2 ˙E +
1
M2
PL
˙φbδφ
!#
. (33) (33) As it can be seen in (33), the momentum constraint do not have a contribution at
the background level, which is logical due to the presence of spatial derivatives,
which cannot appear in an exactly homogeneous and isotropic global background. However, it does have an homogeneous contribution in the perturbative sector due to
the presence of a total spatial derivative, this is of crucial importance for the rest of
the review. Universe 2022, 8, 334 9 of 39 •
Evolution equation for ζ (17). 0 = (∂t −βk∂k)ζ + ˙a
a = −1
3(αK −∂kβk)
≃
−Hb + ˙a
a
+
˙D −HbA −δH −1
3
∇2
a B
,
(34) (34) where we have used the identification K ≡−3H explained below Equation (13). From
(34) we can easily identify the perturbation of the Hubble parameter, i.e., H = Hb + δH = Hb +
−HbA + ˙D −1
3
∇2
a B
. (35) (35) •
Evolution equation for ˜Aij (18). •
Evolution equation for ˜Aij (18). •
Evolution equation for ˜Aij (18). 0 = (∂t −βk∂k) ˜γij = −2α ˜Aij + ˜γik∂jβk + ˜γjk∂iβk −2
3 ˜γij∂kβk
≃[0]
+
2 ˜Aij −2
∂i∂j −1
3δij∇2
B
a + ˙E
. (36) +
2 ˜Aij −2
∂i∂j −1
3δij∇2
B
a + ˙E
. (36) (36) •
Evolution equation for K (19). •
Evolution equation for K (19). •
Evolution equation for K (19). 0 = (∂t −βk∂k)K = α
˜Aij ˜Aij + 1
3K2
−DkDkα + 4πGα(E + Sk
k)
≃
"
−3 ˙Hb −3
Hb2
−
1
M2
PL
˙φb2
−V(φb)
#
+
6 ˙HbA + 3Hb ˙A −3 ¨D + ∇2
a
˙B + ∇2
a2 A + 6
Hb2
A −6Hb ˙D + Hb ∇2
a B
−
1
M2
PL
2 ˙φb ˙δφ −2
˙φb2
A −Vφbδφ
. (37) (37) •
Evolution equation for ˜Aij (20). •
Evolution equation for ˜Aij (20). •
Evolution equation for ˜Aij (20). 0 = (∂t −βk∂k) ˜Aij −e−2ζ
a2
α
R(3)
ij −γij
3 R(3)
−
DiDjα −γij
3 DkDkα
−α(K ˜Aij −2 ˜Aik ˜Ak
j ) −˜Aik∂jβk −˜Ajk∂iβk + 2
3
˜Aij∂kβk + 8πGαe−2ζ
a2
Sij −γij
3 Sk
k
[ ] 0 = (∂t −βk∂k) ˜Aij −e−2ζ
a2
α
R(3)
ij −γij
3 R(3)
−
DiDjα −γij
3 DkDkα
−α(K ˜Aij −2 ˜Aik ˜Ak
j ) −˜Aik∂jβk −˜Ajk∂iβk + 2
3
˜Aij∂kβk + 8πGαe−2ζ
a2
Sij −γij
3 Sk
k
≃[0]
+
"
∂i∂j −1
3δij∇2
˙B
a + 2 HbB
a
+ ¨E + 3Hb ˙E + A + D + 1
3∇2E
!#
. +
"
∂i∂j −1
3δij∇2
˙B
a + 2 HbB
a
+ ¨E + 3Hb ˙E + A + D + 1
3∇2E
!#
. (38) +
"
∂i∂j −1
3δij∇2
˙B
a + 2 HbB
a
+ ¨E + 3Hb ˙E + A + D + 1
3∇2E
!#
. (38) (38) •
Evolution equation for the scalar field (21). •
Evolution equation for the scalar field (21). 0 =
1
√−g∂µ
p−ggµν∂νφ
−Vφ
≃
h
¨φb + 3Hb ˙φb + Vφb(φb)
i
+
¨δφ + 3Hb ˙δφ + Vφbφbδφ −∇2
a2 δφ + 2Vφb A −˙φb
˙A −3 ˙D + ∇2
a B
. (39) (39) Universe 2022, 8, 334 10 of 39 10 of 39 Once we have seen how linear perturbation theory works and what are the linear
equations that describe small inhomogeneities during an inflationary epoch, it is important
to have a physical intuition about what a perturbation of the metric really means. By
definition, a perturbation is the difference between the value of a quantity in the real and
inhomogeneous space-time and its value on the idealized FLRW background. This seems
trivial; however, in order to make such a comparison, it is necessary to compute these
two values at the same space-time point. Since the quantities to compare live in different
space-times, we require a pointwise correspondence between them, which is given by a
coordinate system xµ such that the point Pb in the background space-time and the point
P in the perturbed space-time, which have the same coordinate values, correspond to
each other. •
Evolution equation for ˜Aij (20). The freedom in the choice among these coordinate systems is called the gauge choice. The way the different gauges are related in linear perturbation theory (for gauge trans-
formations beyond linear perturbation theory see for example [89]) is via an infinitesimal
gauge transformation of the coordinates: ˜xµ = xµ + δxµ
(40) (40) We can split the vector δxµ into its time an space components δxµ =
λ0, λi
, and
following the same idea as when we decomposed the perturbations in the metric, λi can
be written as λi = λi
⊥+ ∂iη, where λi
⊥is a 3-dimensional divergenless vector and η is a
scalar function. In terms of these functions, we can impose the gauge invariance of the
perturbed metric (31) to deduce the gauge transformation of each one of the scalar variables
in the metric: D →˜D = D + aHbλ0 + 1
3∇2η ,
A →˜A = A + aHbλ0 + a ˙λ0 ,
E →˜E = E −η ,
B →˜B = B + a ˙η −λ0 . (41) (41) B →˜B = B + a ˙η −λ0 . (41) Finally, the scalar field perturbation will transform as: Finally, the scalar field perturbation will transform as: Finally, the scalar field perturbation will transform as: δφ →˜δφ + a ˙φλ0 . (42) (42) From (41) and (42) we can clearly see that the freedom on the choice of the gauge allow
us to set two out of the five scalar perturbations to zero by choosing η and λ0 accordingly. This reduces the scalar degrees of freedom to three (which further reduces to two when
using the ADM equations for a single scalar field), which can be written in terms of gauge
invariant, and hence physical, variables: the two Bardeen potentials [90] Ψ ≡−D −1
3∇2E −aHb B + a ˙E
,
Φ ≡A + aHb(B + a ˙E) + a d
dt(B + a ˙E) ,
(43) (43) and the Mukhanov-Sasaki (MS) variable and the Mukhanov-Sasaki (MS) variable Q ≡δφ −
˙φb
Hb
D + 1
3∇2E
. (44) (44) During this review we will pay special attention to the MS variable. •
Evolution equation for ˜Aij (20). In fact, it can be
shown that, by rearranging the linearized ADM equations of (32)–(39), the MS variable Universe 2022, 8, 334 11 of 39 follows a simple equation of motion which, written in terms of the SR parameters defined
in Section 2, is: follows a simple equation of motion which, written in terms of the SR parameters defined
in Section 2, is: ¨Q + 3Hb ˙Q +
−∇2
a2 +
Hb2
−3
2ϵ2 + 1
2ϵ1ϵ2 −1
4ϵ2
2 −1
2ϵ2ϵ3
Q = 0 . (45) (45) In order to solve (45), we must take into account that subhorizon scales during inflation
are microscopic, and hence we must study the behavior of the inflationary scalar field using
quantum field theory (QFT). The way to proceed is similar to what we do when quantizing
the harmonic oscillator: ˆQ(x, t), which is now a quantum operator, can be expressed in
Fourier space as: ˆQ(x, t) =
Z
dk
(2π)3/2 ˆQk(t) ,
(46) (46) where where ˆQk(t) = e−ik·xQk(t)ak + e−ik·xQ∗
k(t)a†
k ,
(47) (47) and ak and a†
k are the usual creation and annihilation operators, that satisfy the usual
commutation relation: and ak and a†
k are the usual creation and annihilation operators, that satisfy the usual
commutation relation: [ak, a†
k’] = δ(3)(k −k’)
(48) (48) With this construction, the variable Qk of (47) is the solution of the MS Equation (45)
in Fourier space, i.e.,: ¨Qk + 3Hb ˙Qk +
k2
a2 +
Hb2
−3
2ϵ2 + 1
2ϵ1ϵ2 −1
4ϵ2
2 −1
2ϵ2ϵ3
Qk = 0
(49) (49) If we impose the Bunch-Davies vacuum [91] to be recovered at early time, we can
only write analitically a solution for (49) if a specific condition is satisfied. In order to see
what is this condition we will define the variable z as z = a
˙φb
Hb and the conformal time τ as
adτ = dt, with this, the analytical solution of (49) in terms of the conformal time only exists
if ν2 ≡1
4 + τ2 1
z
d2z
dτ2
(50) (50) is a constant. It is easy to check that this is the case generically; in fact we can integrate by
parts τ = R dt
a to get:
1 is a constant. •
Evolution equation for ˜Aij (20). It is easy to check that this is the case generically; in fact we can integrate by
parts τ = R dt
a to get:
1 τ ≃−1
aHb (1 + O(ϵ1)) . See for example Appendix E of [65] for the derivation. The equation above, together
with the fact that 1
z
d2z
dτ2 = a2
Hb2
2 −ϵ1 + 3
2ϵ2 −1
2ϵ1ϵ2 + 1
4ϵ2
2 + 1
2ϵ2ϵ3
, makes ν defined in (50) to be a constant generically up to O(ϵ1). Note that, since in SR, ϵi
is also a constant up to O(ϵ1), in this case we have that νSR is constant up to O
ϵSR
i
2
. The analytical solution in this case is: Qk = e
i
2 π(ν+ 1
2)
√π
2a
√
−τH(1)
ν (−kτ) ,
(51) (51) where H(1)
ν
is the Hankel function of first class. where H(1)
ν
is the Hankel function of first class. In order to finish this reminder of linear perturbation theory, let us introduce a very
useful quantity that characterizes the properties of the perturbations: the power spectrum. We will give a physical interpretation of the power spectrum later on and for the moment Universe 2022, 8, 334 12 of 39 12 of 39 we will focus only on its mathematical definition. The power spectrum of a generic quantity
X(x, t) is defined as the Fourier transform of the two-point correlation function, i.e.: ⟨0|X(x1, t)X(x2, t)|0⟩=
Z
dk
(2π)3 |Xk(t)|2 ≡
Z dk
k PX(k, t)sin(kr)
kr
,
(52) (52) where r = |x1 −x2| and k = |k|. From (52) it is clear that the power spectrum is defined as: PX(k, t) ≡
k3
2π2 |Xk|2 . (53) (53) Another interesting quantity derived from the power spectrum is the spectral index
X
1
hi h i d fi
d Another interesting quantity derived from the power spectrum is the spectral index
nX
s −1, which is defined as: Another interesting quantity derived from the power spectrum is the spectral index
nX
s −1, which is defined as: g q
nX
s −1, which is defined as: nX
s −1, which is defined as: nX
s −1 ≡d log PX
d log k . (54) (54) 4.1. Long Wavelength Limit of Linear Perturbation Theory As explained in the introduction, many quantities of interest such as the power spec-
trum of the scalar tilt are usually computed in the long wavelength limit, or superhorizon
scales. This makes sense because, as long as inflation is taking place, the exponential
expansion of the universe stretch the perturbations of the quantum fields from microscopic
to cosmological scales. In this way, the inhomogeneities that re-enter the horizon once
inflation has finished and hence the ones of interest today, were in its long-wavelength limit
when inflation ended. In this section we will take a close look to this limit and its physical
consequences. We will study the behaviour of perturbations with characteristic wavelength
L much larger that a local Hubble radius H(−1)
l
in such a way that if we consider L to be
infinitely large compared with H(−1)
l
, we can interpret the region inside H(−1)
l
as a local uni-
verse without perturbations, i.e., homogeneous and isotropic. In other words, if
L
H(−1)
l
→∞, or equivalently, if
k
alHl ≃kτl →0, then the region inside H(−1)
l
represents a local FLRW
universe. With this in mind, let us study the long wavelength limit of perturbations during
inflation. In order to do so we will take two different point of view: the first one is very
intuitive and we will call it the k →0 limit, the second one is slightly less intuitive but it is
very useful when dealing with non-linear perturbations, this is the so-called linear separate
universe approach of k = 0 case. Although they are very similar, the two point of view are
not exactly equivalent as we will see in the following. or equivalently, if
k
alHl ≃kτl →0, then the region inside H(−1)
l
represents a local FLRW
universe. With this in mind, let us study the long wavelength limit of perturbations during
inflation. In order to do so we will take two different point of view: the first one is very
intuitive and we will call it the k →0 limit, the second one is slightly less intuitive but it is
very useful when dealing with non-linear perturbations, this is the so-called linear separate
universe approach of k = 0 case. Although they are very similar, the two point of view are
not exactly equivalent as we will see in the following. 4.1.1. K →0 Limit 4.1. Long Wavelength Limit of Linear Perturbation Theory . Long Wavelength Limit of Linear Perturbation Theory 4.1.1. K →0 Limit Since each spatial derivative introduces a factor k in Fourier space, it would be logical
to think that in the k →0 limit of the ADM equations we must neglect any term that
contains a spatial derivative (This will be done in the next subsection). However, by doing
that, we would be neglecting terms that actually contribute in the k →0 limit. g
g
y
In the following we will enumerate the two cases in which neglecting a term with a
spatial derivative would be too naive: 1.
Non-local terms. 1. Non-local terms. As an example, let us imagine a re-scaling of the spatial coordinates xi →˜xi = (1 + δ)xi . (55) (55) It is easy to realize that if we rewrite δ in terms of the transformations parameters
of (41) we get:
2 It is easy to realize that if we rewrite δ in terms of the transformations parameters
of (41) we get:
2 ∇2η = δ . (56) (56) The fact that a transformation as the one described in (55) is perfectly allowed in a
FLRW universe together with (56) and the transformations rules of the perturbed Universe 2022, 8, 334 13 of 39 13 of 39 metric variables of (41) immediately tell us that variables like ∇2E cannot generically
be neglected in the long wavelength limit even if they contain a Laplacian. In order to
avoid this problem, we will only neglect terms that contain extra spatial derivatives,
by extra we mean that we will neglect a term like ∂iX if and only if it is compared
with X, but we will not neglect it if it appear alone. 2. Equations with overall spatial derivatives. Equations with overall spatial derivatives. A clear example is the momentum constraint of (33). Because it contains a total spatial
derivative, it gives non-trivial information even in the k →0 limit, namely: A clear example is the momentum constraint of (33). Because it contains a total spatial
derivative, it gives non-trivial information even in the k →0 limit, namely: HbA −˙D −1
3∇2 ˙E −
1
2M2
PL
˙φbδφ = 0 . (57) (57) The same happens with the evolution equation for ˜Aij (38). 4.1.2. K = 0 or Linear Separate Universe Approach 4.1.2. K = 0 or Linear Separate Universe Approach 4.1.2. K = 0 or Linear Separate Universe Approach The second point of view to solve linear perturbation theory in the long wavelength
limit is the linear separate universe approach [93–95], in this case we will forget for a
moment about the perturbed ADM equations and we will take advantage of the fact that,
in the long wavelength limit, each local patch represents an homogeneous and isotropic
universe such that its line element is: ds2
l = −dt2
l + al(tl)2δijdxi
ldxj
l ,
(63) (63) where, as before, the subscript l stands for local. As a consequence, the constraints and
equations of motion will be the same as the ones presented in Section 2, i.e., where, as before, the subscript l stands for local. As a consequence, the constraints and
equations of motion will be the same as the ones presented in Section 2, i.e., 3M2
PLH2
l = 1
2
d
dtl
φl
2
+ V(φl) ,
d
dtl
Hl + H2
l = −
1
3M2
PL
" d
dtl
φl
2
−V(φl)
#
,
d2φl
dt2
l
+ 3Hl
d
dtl
φl + Vφl(φl) = 0 . (64) (64) The first thing to realize here is that the momentum constraint is missing. This is
because in the separate universe approach we are not only assuming that each patch is
homogeneous and isotropic (we were also making this assumption in the k →0 limit
where the momentum constraint plays a role), but we are also assuming that each patch
evolve independently from each other. Since the momentum constraint gives information
about the interaction between the different FLRW patches due to the presence of a spatial
derivative, it makes sense that it does not appear in the linear separate universe approach. Before proceeding, let us clarify here that when we talk about the information about the
interaction between different patches encoded in the momentum constraint, which makes
these patches evolve in a not completely independent way, we do not claim that what
happens in one patch will affect the others, but rather that all the patches as an ensemble
must satisfy some conditions (given by the momentum constraint) that are absent if we
look to a single patch but that are important when comparing them. The same happens with the evolution equation for ˜Aij (38). Universe 2022, 8, 334 14 of 39 14 of 39 The same happens with the evolution equation for ˜Aij (38). Taking these two important point into account one can re-derive the equation of motion
for the MS variable Q in the k
→
0 limit. The result is, as expected, the
Equation (45) but without the Laplacian, i.e., ¨Q + 3Hb ˙Q +
Hb2
−3
2ϵ2 + 1
2ϵ1ϵ2 −1
4ϵ2
2 −1
2ϵ2ϵ3
Q = 0 . (58) (58) If we want to know the solution for Equation (58) we can simply take the kτ →0
limit of the solution (51) obtained before (we will do that later on); however, this would be
restricted to ν (defined in (50)) being a constant. In this section we will solve independently
the long wavelength equation of motion for Q without any assumption, i.e., valid at all
orders in slow-roll parameters. The price to pay is that, since the initial conditions are given
at sub-horizon scales and (58) is only valid at super-horizon scales, we will not specify any
initial condition here. It can be checked that (58) can be written as a total derivative as follows: 2M2
PLHb
a3 ˙φb
d
dt
"
a3 ˙φb
2M2
PL
˙Q
Hb −ϵ2
2 Q
#
= 0 . (59) (59) For convenience, we will define the comoving curvature perturbation as: Rc ≡−Hb
˙φb Q . (60) (60) It terms of Rc, (59) takes a very simple form: It terms of Rc, (59) takes a very simple form: a3ϵ1 ˙Rc = C1 ,
(61) (61) where C1 is a constant. Therefore, the solution of the MS equation in the long wavelength
limit (58) is Q =
˙φb
Hb Rc = C2
˙φb
Hb + C1
˙φb
Hb
Z
dt
a3ϵ1
,
(62) (62) where C2 is also a constant. Solution (62) is the well-known exact solution in the k →0 limit
for the single component scalar field case [85,92]. The term proportional to C1 is usually
known as decaying mode, name inherited from its SR behaviour where ˙φb is roughly
constant, since a ∼e−Hbt during inflation, we can clearly see that the term proportional to
C1 decays as e−3Hbt during SR. However, this is not the case beyond SR, for example in USR
we have ˙φb ∼e−3Hbt (see (8)) and hence ϵ1 ∼e−6Hbt, which makes the term proportional
to C1 be approximately constant whereas the one proportional to C2 decays, contrary to
what happens in SR. 4.1.2. K = 0 or Linear Separate Universe Approach Within this approximation, we can only see the effect of perturbations if we compare
different patches between them or with the global background. Knowing that the differ-
ences must be perturbative, we can follow our results from linear perturbation theory
and write: Hl ≃Hb + δH = Hb −HbA + ˙D −1
3
∇2
a B ,
dtl ≃(1 + A)dt ,
φl ≃φb + δφ . (65) (65) The term 1
3
∇2
a B can be set to zero without loss of generality, this is because since each
patch is independent from each other, we can always choose an orthogonal threading for all
of them, in which βi (and hence B) is zero. Another way of reasoning is that, as checked in
the k →0 limit of perturbation theory, a term with a Laplacian can only be important in the
long wavelength limit if it contains non-local information, but at the same time, non-local
terms would give some information about the interaction between local patches, which is
in contradiction with the absence of the momentum constraint (and hence with the separate
universe assumption) as explained above. If we also take into account that the scalar perturbation related with the traceless part
of the spatial metric (i.e., E) does not appear in (65) so neither in (64), we can set ∇2Bsep = ∇2Esep = 0 . (66) (66) Universe 2022, 8, 334 15 of 39 15 of 39 in the separate universe approach (as indicated with the superscript “sep”). Note that,
although (66) coincides with the Newtonian (or longitudinal) gauge, this is not a gauge
choice, but a consequence of the separate universe approach. q
p
pp
With this in mind, we can write the equations in (64) in terms of the global background
and perturbations over it as follows: −6
Hb2
Asep + 6Hb ˙Dsep =
1
M2
PL
˙φbδφsep −
˙φb2
Asep + Vφbδφsep
,
6 ˙HbAsep + 3Hb ˙Asep −3 ¨Dsep + 6
Hb2
Asep −6Hb ˙Dsep =
1
M2
PL
2 ˙φb ˙δφsep −
˙φb2
Asep −Vφbδφsep
,
¨δφsep + 3Hb ˙δφsep + Vφbφbδφsep = −2Vφb Asep + ˙φb ˙Asep −3 ˙Dsep
. 4.1.2. K = 0 or Linear Separate Universe Approach (67) (67) As one can see from (67), the linear separate universe approach does not use the
whole system of ADM equations presented in Section 4, it only uses the long-wavelength
version of: (a) the Hamiltonian constraint (32), (b) the evolution equation for the trace of the
extrinsic curvature (37) and (c) the equation of motion of the scalar field (39). All of them
setting both ∇2Bsep and ∇2Esep to zero accordingly with the separate universe assumption. In order to see what are the consequences of this reduction in the number of equations
and variables when comparing with the k →0 limit of Section 4.1.1, we can follow a similar
procedure to what we did when defining the MS variable and write down a “separate-
universe” gauge invariant variable [96,97], meaning that it is gauge invariant under time
reparametrizations. Qsep ≡δφsep −
˙φb
Hb Dsep . (68) (68) Similarly to what we did with the MS Equation (45), we can rearrange the equations
of the linear separate universe approach (67) and write a single equation of motion for Qsep,
the result, in terms of the SR parameters, is: ϵ1ϵ2
3(3 −ϵ1)
˙Qsep +
Hb2
−3
2ϵ2 + 1
2ϵ1ϵ2 −1
4ϵ2
2 −1
2ϵ2ϵ3 −
ϵ1ϵ2
2
2(3 −ϵ1)
! Qsep = 0 . (69) (69) Comparing (69) with (58) we can now clearly identify two extra terms that appear
when assuming that each local patch evolves independently from each other, i.e., when
using the separate universe approach. These terms are O(ϵ1ϵ2) [55], being strongly depen-
dent on the inflationary regime, for example, they are O(ϵUSR
1
) in USR (where ϵ2 ≃−6)
and O
ϵSR
1
2
in SR.
In order to better quantify this difference we will solve (69) in a similar way as done
with (58) and compare the results. We can again write (69) as a total derivative as follows 2V
3a3Hb ˙φb
d
dt
3a3
Hb2 ˙φb
2V
˙Qsep
Hb −ϵ2
2 Qsep
= 0 . 4.1.2. K = 0 or Linear Separate Universe Approach (70) (70) Defining a “separate universe” comoving curvature perturbation as Defining a “separate universe” comoving curvature perturbation as Rsep
c
≡−Hb
˙φb Qsep ,
(71)
we can write (70) as
3a3
˙φb2
2V
= C′
1 ,
(72) Rsep
c
≡−Hb
˙φb Qsep ,
(71)
3a3
˙φb2
2V
= C′
1 ,
(72) Rsep
c
≡−Hb
˙φb Qsep ,
(71) (71) we can write (70) as 3a3
˙φb2
2V
= C′
1 ,
(72) (72) Universe 2022, 8, 334 16 of 39 16 of 39 where C′
1 is a constant. Hence, the solution for Qsep is: where C′
1 is a constant. Hence, the solution for Qsep is: where C′
1 is a constant. Hence, the solution for Qsep is: Qsep = C′
2
˙φb
Hb + C′
1
˙φb
Hb
Z 1
a3ϵ1
−1
3a3
. (73) (73) We can see that the solutions for Qsep of (73) and for Q of (62) differ by an extra term in
the decaying mode, which is obviously due to the difference of O(ϵ1ϵ2) in the equation of
motion. The importance of this extra term depends on the inflationary regime, being more
important beyond SR, where the term proportional to C′
1 does not decay (see discussion
below (62)). Since the main difference between the linear separate universe approach and the
k →0 limit is the inclusion of the non-trivial information of the momentum constraint at
large scales, which is not taken into account in the former, one could think that the two
solutions will coincide if we impose the solution (73) to satisfy the momentum constraint;
however, this is not the case. In fact, in the following we are going to check that the mode
proportional to C′
1, which contains the new extra term, does not represent the k →0 limit
of some solution to the perturbations equations with k ̸= 0. This should not be a surprise
since the correct solution for the MS variable in the long wavelength limit is given by (62). In order to check that, let us rearrange equations (65) to write HbAsep −˙Dsep −
˙φb
2M2
PL
δφsep = −Hb ˙φb
2V
˙Qsep
Hb −ϵ2
2 Qsep
. 4.1.2. K = 0 or Linear Separate Universe Approach (74) (74) If we want solution (73) to be the k →0 solution to the perturbation equations with
k ̸= 0 we must be sure that our solution satisfies the momentum constraint (although
it is lost in the separate universe approach.) because, as we know, this constraint gives
non-trivial information even at large scales. What would be the momentum constraint in
the linear separate universe approach is HbAsep −˙Dsep −
˙φb
2M2
PL
δφsep = 0 ,
(75) (75) which, comparing with (74), immediately implies: ˙Qsep
Hb −ϵ2
2 Qsep = 0 . (76) (76) From condition (76) and the equation of motion (70) we can see that, if the momentum
constraint of (75) is satisfied at initial time, it will be always satisfied so one could think that
the problem of ignoring the momentum constraint in the linear separate universe approach
is simply solved by an appropriate choice of initial conditions; however, the only mode that
satisfies (76) is the mode proportional C′
2 in the solution (73), which means that the mode
proportional to C′
1 therein does not represent a k →0 solution of the perturbation equations
with k ̸= 0 and it must be set to zero. However, setting C′
1 = 0 means that we are losing the
term proportional to C1 in the correct solution of (62), which can be important beyond SR. Thus, imposing that the momentum constraint must be satisfied in the separate universe
approach does not give the correct solution for the long-wavelength limit of the MS variable
and hence we have to use the whole set of ADM equations in the long wavelength limit
to describe the correct dynamics (at all orders in SR parameters) of the MS variable at
super-horizon scales. Let us indicate here that although there exist some ways to recover
the correct k →0 limit of the MS variable using only the linear separate universe approach
(see [96,97]), we will not study them here because they are restricted to linear perturbation
theory and, as we will see later on, we are planning to use the long wavelength limit of
inhomogeneities beyond perturbation theory. 4.1.2. K = 0 or Linear Separate Universe Approach For clarity purposes, let us summarize the findings of this section until now: Universe 2022, 8, 334 17 of 39 17 of 39 •
Although both the k →0 and the linear separate universe approach consider local
homogeneous and isotropic universes, the k →0 limit allow some interaction between
them whereas the linear separate universe approach assume that they evolve inde-
pendently. Mathematically speaking, the linear separate universe approach ignore
possible non-local terms and equations with overall spatial derivatives, both present
in the k →0 limit. •
As a consequence, the equation of motion for Qsep differ by terms of O(ϵ1ϵ2) from
the equation of motion for Q in the long wavelength limit. This difference induces an
extra decaying term, namely: •
As a consequence, the equation of motion for Qsep differ by terms of O(ϵ1ϵ2) from
the equation of motion for Q in the long wavelength limit. This difference induces an
extra decaying term, namely: Qsep = Q(k →0) + C′
1
˙φb
Hb
Z
1
3a3 dt
(77) (77) •
The difference (77) always decays so one could think that it can be safely ignored;
however, its importance strongly depends on the inflationary regime. For example,
in USR we have Qsep −Q(k →0) ∼O(ϵ1) so if we want to be precise enough when
studying the long-wavelength limit of perturbation theory, we should use the k →0
limit rather than the linear separate universe approach. •
The difference (77) always decays so one could think that it can be safely ignored;
however, its importance strongly depends on the inflationary regime. For example,
in USR we have Qsep −Q(k →0) ∼O(ϵ1) so if we want to be precise enough when
studying the long-wavelength limit of perturbation theory, we should use the k →0
limit rather than the linear separate universe approach. •
The difference (77) always decays so one could think that it can be safely ignored;
however, its importance strongly depends on the inflationary regime. For example,
in USR we have Qsep −Q(k →0) ∼O(ϵ1) so if we want to be precise enough when
studying the long-wavelength limit of perturbation theory, we should use the k →0
limit rather than the linear separate universe approach. p
pp
•
Finally, imposing the momentum constraint to be satisfied in the separate universe
approach not only does not solve the difference between Qsep and Q(k →0), but it
makes it worse. 4.2. Linear Perturbation Theory and Pbhs In order to finish this section we will study the behaviour of the power spectrum of
the inflationary regimes that can lead to the formation of PBHs in the long wavelength
limit (The reader interested in PBHs as probes for the physics of the very early universe
in a more detailed way can read the following reviews: [98,99]). We will do that in a very
qualitative way and at leading order in SR parameters, this is why we will simply use the
kτ →0 limit of solution (51), where the initial conditions are properly specified. If we
expand the Hankel function of first order we have: lim
kτ→0 Qk = lim
kτ→0 e
i
2(ν+ 1
2)
√π
2a
√
−τH(1)
ν (−kτ) ≃−ie
i
2(ν+ 1
2) 2ν−1
a√π
√
−τ(−kτ)−νΓ[ν] ,
(78) (78) where Γ[ν] is the Euler gamma. where Γ[ν] is the Euler gamma. g
For the formation of PBH we are interested in the power spectrum of the comoving
curvature perturbation, i.e., For the formation of PBH we are interested in the power spectrum of the comoving
curvature perturbation, i.e., PRc =
k3
2π2
Hb
˙φb
!2
|Qk|2 ≃
Hb4
π3 ˙φb2
k
2
3−2ν 1
aHb
3−2ν
Γ[ν]2 ,
(79) (79) where we have used τ ≃−
1
aHb . aH
For USR and SR we have ν ≃3
2 and hence the power spectrum is roughly scale
invariant:
4
2 aH
For USR and SR we have ν ≃3
2 and hence the power spectrum is roughly scale
invariant:
4
2 PSR
Rc ≃PUSR
Rc
≃
Hb4
4π2 ˙φb2 =
Hb2
8π2M2
PLϵ1
. (80) (80) However, and although the k dependence of the comoving curvature power spectrum
for USR and SR is roughly the same, and hence nRc
s
−1 ∼O(ϵ1) for both, this is not true
for the time dependence. In fact we have PSR
Rc ∼constant ,
PUSR
Rc
∼e6Hbt . (81) (81) Universe 2022, 8, 334 18 of 39 18 of 39 An exponential growth of the power spectrum at super-horizon scales as it happens
in USR has important consequences for the formation of PBH. In fact, by definition of the
power spectrum, PRc give us the variance of the probability distribution that follow the
amplitude of the perturbations
Rk1
c with characteristic wavenumber k1, therefore, a
growth in the power spectrum can be interpreted as a growth in the variance or, equivalently,
a spreading of the probability distribution. This means that high values for the amplitude of
the perturbations placed at the tail of the probability distribution are much more probables
if the power spectrum grows. Finally, and since the high values for the amplitude of the
curvature perturbations are the ones that collapse to form a PBH when they re-enter the
horizon, we can conclude that inflationary regimes beyond SR favor the creation of PBH. The argument given above is very hand-waving but it is enough to remark the impor-
tance of inflationary regimes beyond SR when talking about PBH. where Γ[ν] is the Euler gamma. However, if we want to
make actual predictions about the mass and abundances of PBH we need to study inflation-
ary dynamics in a much more precise way, in fact, the tail of the probability distribution
for the amplitude of perturbations, which is where the perturbations responsible for the
formation of PBH are located, is very sensitive to any small change in the power spectrum
(or in the higher order correlators as the bispectrum). This is the reason why a description of
the perturbations at all orders in SR is highly desirable, making the linear separate universe
approach not the best approximation to study the tail of the probability distribution of
the amplitude of perturbations generated during a inflationary regime beyond SR, i.e., to
study the formation of PBH. The reason is that, generically, Qsep −Q(k →0) ∼O(ϵ1) as
explained above. Finally, another problem arises when dealing with inhomogeneities large enough
to form a PBH, namely, non-linear or even non-perturbative effects can play an impor-
tant role, reason why we need to go beyond linear perturbation theory. The rest of the
review is devoted to the study of inhomogeneities generated during inflation beyond linear
perturbation theory. 5. Gradient Expansion Having a quantity whose leading order
is gradient expansion is O(σ−1) could seem problematic; however this is only telling us
that βi is generically a non-local quantity, in fact, its linearization give us the non-local
variable B (see (23)) studied in Section 4.1.1. Furthermore, we will see that βi always appear
t
th
ith
ti l d
i
ti
i
th
ti
f
ti where we are using the subscript (0) to remind the reader that we are at leading order
in gradient expansion It is important to remark here that the leading order in gradient
expansion of each quantity can be different, for example, the leading order for α and ζ is
O(σ0) whereas the leading order for βi is O(σ−1). Having a quantity whose leading order
is gradient expansion is O(σ−1) could seem problematic; however this is only telling us
that βi is generically a non-local quantity, in fact, its linearization give us the non-local
variable B (see (23)) studied in Section 4.1.1. Furthermore, we will see that βi always appear
together with a spatial derivative in the equations of motion. expansion of each quantity can be different, for example, the leading order for α and ζ is
O(σ0) whereas the leading order for βi is O(σ−1). Having a quantity whose leading order
is gradient expansion is O(σ−1) could seem problematic; however this is only telling us
that βi is generically a non-local quantity, in fact, its linearization give us the non-local
variable B (see (23)) studied in Section 4.1.1. Furthermore, we will see that βi always appear
together with a spatial derivative in the equations of motion. One could be worried about the fact that a homogeneous and isotropic metric con-
tains terms outside the diagonal; however, following [110] we know that a space-time is
homogeneous and isotropic if: 1. All constant time hypersurfaces Σt are constant curvature spaces. In our case the
hypersurfaces Σt are simply Euclidean and this condition is trivially satisfied. 1. All constant time hypersurfaces Σt are constant curvature spaces. In our case the
hypersurfaces Σt are simply Euclidean and this condition is trivially satisfied. 2. The extrinsic curvature of the hypersurfaces is homogeneous and isotropic. 5. Gradient Expansion The gradient expansion is a non-perturbative, in terms of the amplitude of the inhomo-
geneities, expansion of the ADM equations valid when the characteristic wavelength of the
inhomogeneities L is much larger that the Hubble horizon H−1
l
[100–109]. Since this is the
same approximation that we do when studying the long wavelength behaviour of the linear
perturbations, we will follow the same idea and we will take advantage of the fact that,
when L ≫H−1
l
, the universe is locally homogeneous and isotropic to define an expansion
parameter σ ≡
H−1
l
L
such that at leading order in σ, each local patch of the universe of size
(σH−1
l
) (we will call this scale the coarse-grained scale) is approximately described by a
FLRW universe. Higher order terms in the σ will instead capture local inhomogeneities. Contrary to the linear theory approach to cosmological perturbations, the gradient
expansion is valid for any amplitude of local over-densities as long as the patch is taken
small enough for the gradients to be negligible. Note that this assumption on which the
gradient expansion is based on implies that a patch can be found such that any spatial
gradient would only introduce an order σ. In other words, for any generic function X,
∂iX ∼X × O(σ). This is because a function which is approximately homogeneous in local
coordinates can be written as X(t, σxi) with σ ≪1. Thus, we have ∂iX(t, σxi) = σ
∂
∂(σxi) X(t, σxi) = σ
∂
∂(σxi) X(t, σxi)
σxi=0 + O(σ2) , and since
∂
∂(σxi) X(t, σxi)
σxi=0 c 0 can be of the same order as X(t, σxi) we can generically write: nce
∂
∂(σxi) X(t, σxi)
σxi=0 can be of the same order as X(t, σxi) we can generically write ∂iX ∼X × O(σ) . ∂iX ∼X × O(σ) . Universe 2022, 8, 334 19 of 39 19 of 39 When we were studying linear perturbation theory, the way of relating different
patches was to define a global background over which each patch represented a perturba-
tion. In this case we do not have a physical background metric because we are no longer
perturbing anything; however we will still define a fictitious global background metric
with coordinates t and xi, i.e., ds2
b = −dt2 + a(t)2δijdxidxj . 5. Gradient Expansion (82) (82) We are then interested in writing each local FLRW patch, which at leading order in
σ and in terms of local coordinates is simply (63), in terms of the coordinates t and xi. At
leading order in gradient expansion and considering only scalar perturbations (note that,
if we want to study inflation in a fully non-perturbative way, we should also take into
account vector and tensor perturbations. This is because, although they are independent
at linear order in perturbation theory, this is no longer true at higher orders. The reason
why we do not include vector and tensor perturbations here is because, although at this
level it would be straightforward, it is not possible when applying gradient expansion to
stochastic inflation as we will see) we have: ds2
l = −(0)α2dt2 + (0)γij
dxi + (0)βidt
dxj + (0)βj
,
(83) (83) with the conditions: with the conditions: 1. (0)α = (0)α(t) ,
2. (0)βi = b(t)xi ,
3. (0)γij = γ(t)δij = a(t)2e2 (0)ζ(t)δij , 1. (0)α = (0)α(t) ,
2. (0)βi = b(t)xi ,
3. (0)γij = γ(t)δij = a(t)2e2 (0)ζ(t)δij , where we are using the subscript (0) to remind the reader that we are at leading order
in gradient expansion It is important to remark here that the leading order in gradient
expansion of each quantity can be different, for example, the leading order for α and ζ is where we are using the subscript (0) to remind the reader that we are at leading order
in gradient expansion It is important to remark here that the leading order in gradient
expansion of each quantity can be different, for example, the leading order for α and ζ is
O(σ0)
h
th l
di
d
f
βi i O(σ−1) H
i
tit
h
l
di
d where we are using the subscript (0) to remind the reader that we are at leading order
in gradient expansion It is important to remark here that the leading order in gradient
expansion of each quantity can be different, for example, the leading order for α and ζ is
O(σ0) whereas the leading order for βi is O(σ−1). 5. Gradient Expansion Focusing
only in the scalar part of Mij we can then write ˜γij −δij ≃−2
∂i∂j −1
3δij∇2
C + O(σ2) ,
(87) (87) where C is a scalar function. This immediataly implies that
∂i∂j −1
3δij∇2
C ∼O(σ). Note that, as we will see later on, this condition is not in contradiction with ∇2C ∼O(σ0). 5. Gradient Expansion Using
the definition of extrinsic curvature of (12) together with the conditions for (0)α, (0)βi
and (0)γij specified below (83), we can see that the extrinsic curvature only depends
on time and hence this condition is also satisfied. Different functions for (0)α, (0)βi and (0)ζ will give different FLRW patches as long
as they satisfy the conditions given below (83). We can then relate the different locally
homogeneous and isotropic patches by knowing the different non-perturbative functions
for (0)α, (0)βi and (0)ζ that lead to each one of them. Of course, in the same way as in
perturbation theory, the value of (0)α, (0)βi and (0)ζ will depend on the gauge choice and
on the solution for the ADM equations. We can generalize the idea of writing a FLRW metric in terms of xi and t to writ-
ing quasi-FLRW metrics using the same coordinates. Following the ADM formalism
we can write a metric valid at all orders in gradient expansion. With the identification
γij = a(t)2e2ζ ˜γij we have: (84) ds2 = gµνdxµdxν = −α2dt2 + a(t)2e2ζ ˜γij(dxi + βidt)(dxj + βjdt) , Universe 2022, 8, 334 20 of 39 where the leading order in gradient expansion for each variable is: where the leading order in gradient expansion for each variable is: (0)α ∼O(σ0) ,
(0)ζ ∼O(σ0) ,
(0)βi ∼O(σ−1) ,
˜γij −δij ∼O(σ) ,
(0)φ ∼O(σ0) . (85) The last term has been added to take into account the expansion of the scalar field,
which is generically non-zero at the background level. It is important to realize here that
the condition ˜γij −δij ∼O(σ) implies a further condition on the scalar part of ˜γij, in fact,
using the expansion of the exponential of a matrix we can write The last term has been added to take into account the expansion of the scalar field,
which is generically non-zero at the background level. It is important to realize here that
the condition ˜γij −δij ∼O(σ) implies a further condition on the scalar part of ˜γij, in fact,
using the expansion of the exponential of a matrix we can write ˜γij = e−2Mij ≃δij −2Mij + O(σ2) . (86) (86) Now, Mij must be traceless by definition (see see the paragraph under (23)). Focusing
only in the scalar part of Mij we can then write Now, Mij must be traceless by definition (see see the paragraph under (23)). 5.1. O(σ0) in Gradient Expansion Having specified the leading order gradient expansion for each one of the non-
perturbative variables, we can now expand the ADM equations in terms of σ. As an
example we will expand the Hamiltonian constraint at O
σ0
using the spatially flat
gauge, i.e., (γf)ij = a2δij ,
(88) (88) where the subscript f stands for “flat”. Since in this gauge we also have R(3)
f
= 0 we can
write the Hamiltonian constraint from (14) as: −(Af)ij(Af)ij + 2
3K2
f −
2
M2
PL
(Tf)µν(nf)µ(nf)ν = 0 ,
(89) (89) where (nf)µ = gµν(nf)ν =
1
αf , −(βf)i
αf
. i where (nf)µ = gµν(nf)ν =
1
αf , −(βf)i
αf
.
Equation (89) can be written in terms of the metric variables αf and (βf)i of (84) and
the scalar field φf. Using the results for (Af)ij and Kf given by (18) and (17) respectively
we have: −1
4α2
f
δik∂j(βf)k + δjk∂i(βf)k −2
3δij∂k(βf)k
δik∂j(βf)k + δjk∂i(βf)k −2
3δij∂k(βf)k
+2
3
−3 Hb
αf
+ 1
αf
∂k(βf)k
!2
−
2
M2
PL
" ˙φ2
f
2α2
f
−
˙φf(βf)i∂iφf
α2
f
+ (βf)i(βf)j∂iφf∂jφf
2α2
f
+ ∂iφf∂iφf
2a2
+ V(φf)
#
= 0 ,
(90) (90) which is valid at all orders in gradient expansion. Keeping only the O(σ0) terms we get: 21 of 39 21 of 39 Universe 2022, 8, 334 2
3
−3 Hb
(0)αf
+ 1
αf
∂k
(0)βf
k
!2
−
2
M2
PL
(0) ˙φf
2
2
(0)αf
2 −
(0) ˙φf
(0)βf
i
(0)(∂iφf)
(0)αf
2
+
(0)βf
i
(0)βf
j
(0)(∂iφf) (0)
∂jφf
2
(0)αf
2
+ V(φf)
= 0 ,
(91) (91) where by (0)(∂iφf) we mean σ
∂
∂(σxi) φ(t, σxi)
σxi=0 as explained before. where by (0)(∂iφf) we mean σ
∂
∂(σxi) φ(t, σxi)
σxi=0 as explained before. Note that the first line of (90) is O(σ) because of the condition of (0)βi below Equation (83). 5.1. O(σ0) in Gradient Expansion Following this procedure, it is straightforward to write all the ADM equations at order
O(σ0); however, we know from linear perturbation theory that there are equations with
global spatial derivatives that play a role in the k →0 limit, which translated to the gradient
expansion way of thinking means that there are equations that contain an overall factor σ
and hence in order to extract its contribution at O(σ0) they must be written up to O(σ). We know that the momentum constraint is one of these equations so let us analyze it. In
spatially flat gauge, the momentum constraint (15) is: ∂j ˜Af
ij −2
3∂iKf =
1
M2
PL
Ji
(92) (92) If we expand (92) up to O(σ) we get:
(0)∂i
−3 Hb
αf
! =
1
M2
PL
−
1
(0)αf
(0) ˙φf (0)(∂iφf) +
(0)βf
k
(0)αf
(0)(∂kφf) (0)(∂lφf)
. (93) If we expand (92) up to O(σ) we get: −2
3 (0)∂i
−3 Hb
αf
! =
1
M2
PL
−
1
(0)αf
(0) ˙φf (0)(∂iφf) +
(0)βf
k
(0)αf
(0)(∂kφf) (0)(∂lφf)
. (93) (93) An important aspect regarding the gradient expansion is worthy to remark at this
point, note that, in the derivation of (93) we have used ∂j ˜Af
ij ∼O(σ), which seems in
contradiction with
˜Af
ij ∼O(σ) (as we saw when deriving the Hamiltonian constraint at
O(σ0)) and the fact that each spatial gradient introduces an order in σ. However, in this
case we have an exception due to the traceless nature of
˜Af
ij. Let us see why: from the
condition 2 below (83) we have (βf)i ≃b(t, σxl)xi and from (18) in spatially flat gauge: ∂j ˜Af
ij = δik∂j∂j(βf)k + ∂i∂k(βf)k −2
3∂i∂k(βf)k = 4
3∂i∂k(βf)k = 4∂ib(t, σxl) + O(σ2) , which is clearly O(σ). y
( )
The linear version of (93) obviously corresponds to the linear momentum constraint
in spatially flat gauge, i.e., ∂i
HbAf
= ∂i
˙φb
2M2
PL
δφf
! . (94) (94) As already stressed during Section 4.1.1, although (94) is an equation that appears at O(σ),
it contains information at O(σ0), namely HbAf =
˙φb
2M2
PL
δφf . 5.2. Non-Perturbative Separate Universe Approach 5.2. Non-Perturbative Separate Universe Approach The O(σ0) expression for the Hamiltonian constraint (91) seems very difficult to
solve, and we have just argued the difficulties that arise when trying to extract the O(σ0)
information from the O(σ) momentum constraint. This is why a further approximation in
addition to the O(σ0) gradient expansion is usually performed in the literature [20,111,112]. This new approximation is the separate universe approach, which assumes that each FLRW
patch of the universe evolve independently one from each other. We have already studied
the linear version of the separate universe approach in Section 4.1.2, where we have seen
that this assumption implies that neither non-local terms nor the momentum constraint
will be present, which is equivalent to state that βi ∼O(σ3) as done in [109]. Within this
approximation, the Hamiltonian constraint in spatially flat gauge of (91) takes a much
simpler form: Hb
(0)αsep
f
!2
=
1
3M2
PL
(0) ˙φsep
f
2
2
(0)αsep
f
2 + V
(0)φsep
f
,
(96) (96) which reminds us of the background Hamiltonian constraint (4). In fact, this coincidence
extends also to the equation of motion of the scalar field as one could expect since we are
assuming every FLRW to evolve independently from the others. A famous application
of the separate universe approach is the δN formalism in order to compute the uniform-
density curvature perturbation in a non perturbative way [70–73]. As a reminder, the linear
uniform-density curvature perturbation is defined as Rud ≡−Hb
˙ρb δρf , Rud ≡−Hb
˙ρb δρf , 2 where ρ is the energy density of the scalar field, which in inflation is ρ = 3M2
PL
Hb2
and
δρf is the perturbation of the energy density in spatially flat gauge. and In this section we have presented the non-perturbative generalization of the long
wavelength linear perturbation theory presented in Section 4, being the O(σ0) in gradient
expansion of Section 5.1 the generalization of the k →0 limit for scalar perturbations of
Section 4.1.1 and being the separate universe approach the non-linear generalization of
the k = 0 (or linear separate universe approach) case of Section 4.1.2. 5.1. O(σ0) in Gradient Expansion (95) (95) Unfortunately, things are not that easy when dealing with the fully non-perturbative
momentum constraint at O(σ), in fact, if we look at (93), we will easily realize that the
total spatial derivative is no longer present. How the O(σ0) information is encoded
in (93) in a fully non-perturbative way is beyond the scope of this review, neverthe- Universe 2022, 8, 334 22 of 39 22 of 39 less, it is very important to be aware that we must take this information into account
if we want to correctly describe the non-perturbative and long wavelength dynamics of
the inhomogeneities, otherwise we would be making a mistake equivalent as when us-
ing the linear separate universe approach of Section 4.1.2 instead of the k →0 limit of
Section 4.1.1. Nevertheless, due to its usefulness, in the following section we will present
the non-perturbative version of the linear separate universe approach. 6. Stochastic Approach to Inflation The stochastic approach to inflation combines the two approximations schemes pre-
sented until now to study the evolution of inhomogeneities in a non-perturbative way. The idea is to split the variables of interest (let us say X) into two parts: an infrared (IR)
part that contains all the inhomogeneities with characteristic wavelength larger that some
coarse-grained scale (σH)−1 (σ is the same parameter as the one used in gradient expan-
sion) and a ultraviolet (UV) part, which encompasses inhomogeneities with characteristic
scale smaller than (σH)−1 (or characteristic wavenumber k bigger than σaH). Since the UV part starts evolving well inside the Hubble horizon, we will assume that
it is perturbatively small. Thus, one can use linear perturbation theory to describe it, where
initial conditions are well defined. The IR part instead can be large; however, since the IR
part only contains long wavelengths, the gradient expansion can be used there. As we will
see, whenever an UV mode exits the coarse-grained scale, it will act as a kick for the IR part,
solving the initial condition problem of gradient expansion. Note that, although we will
only study stochastic inflation in the context of Einstein’s gravity, the only requirements for
the construction of a stochastic formalism are the possibility of a separation between IR and
UV modes and a well-behaved perturbative expansion. There is then no reason a priori
to think that the stochastic framework can not be applied to modifications of Einstein’s
gravity such as massive gravity [113,114]. The stochastic formalism is then a mathematical framework that, in principle, allows
us to study the inhomogeneities generated during inflation in a non-perturbative way,
reason why it is widely used when studying PBHs formation (see for example [115,116]). To see how stochastic inflation works and why it is called “stochastic” we will derive
the formalism step by step. From Section 5 it should be clear now that we will have
different stochastic equations depending on if we are using the separate universe approach
or the O(σ0) gradient expansion. The stochastic formalism that uses the separate universe
approach is the most widely used in the literature (see for example [10,14–19,23,27,35,37,38,
51–53,63,66,115,116]) so we will start with its derivation. After that, we will take advantage
of the stochastic equations just derived to present a stochastic formalism based on the
O(σ0) gradient expansion [65]. 5.2. Non-Perturbative Separate Universe Approach For this reason, it is
obvious that the O(σ0) gradient expansion and the separate universe approach will give
different “decaying” terms, being the ones of the O(σ0) gradient expansion the correct
ones, as it happened in linear theory. Using the results from linear theory again, we can conclude that the error made when
using the separate universe approach instead of the O(σ0) gradient expansion will be
generically of O(ϵ1), strongly depending on the inflationary regime. This conclusion is
deduced of course assuming that higher orders in perturbation theory will not spoil the
results at leading order, assumption that can be problematic if the inhomogeneity under
study is non-perturbative. We will finish this section by reminding the reader that setting initial conditions for
long wavelength perturbations is problematic because we cannot use the Bunch-Davies
vacuum, which is only well defined when
k
aH →∞. This is why, although the gradient
expansion is a very useful way to study inhomogeneities in a non-perturbative way, we
need some other tool to set the initial conditions. Universe 2022, 8, 334 23 of 39 23 of 39 6. Stochastic Approach to Inflation Before starting with the derivation and, since it is not as trivial as the spatially flat
gauge, let us present the gauge we will be working with: the uniform-N gauge. We define
the number of e-folds N as the integrated expansion rate of Σt hypersurfaces, i.e., N ≡−1
3
Z
Kdtl ,
(97) (97) where K ≡−3Hl (being Hl the local Hubble parameter) is the extrinsic curvature defined
in (12), which coincides with the expansion rate of Σt hypersurfaces as shown in Section 3
and tl is the local time of (63). In terms of the variables and coordinates of the ADM metric
(84) N can be written as:
N ≡
Z
Hb + ˙ζ −1
3 Diβi
dt . (98) (98) The uniform-N gauge is defined such that N coincides with the number of e-folds
defined in the global background of (82), i.e., N ≡R
Hbdt. From (98), this immediately
implies ζδN = 0 and (βδN)i = 0 (where the subscript δN specifies the gauge), or DδN = 0
and BδN = 0 in its linear limit (see Equation (23)). The reason behind this gauge choice will
be clarified along the following two subsections. 6.1. Stochastic Formalism Based on the Separate Universe Approach As we have already indicated, this is the stochastic formalism most widely used in the
literature due to its simplicity; however, as we will see, it has some problems. First of all,
it is very important to realize that in the separate universe approach, both the uniform-N
gauge and the spatially flat gauge are equivalent, which leads to many authors to use the Universe 2022, 8, 334 24 of 39 24 of 39 uniform-N gauge for the IR part and the spatially flat gauge for the UV part [60]. Let us
see why: uniform-N gauge for the IR part and the spatially flat gauge for the UV part [60]. Let us
see why: y
As we know, in the separate universe approach, due to the absence of non-local terms,
both ( ˜γsep)ij = δij and ∂i(βsep)i = 0 are automatically satisfied (see (66)). This means
that, under this assumption, we have to add the condition ∂i
βsep
f
i
= 0 to the spatially
flat gauge where ( ˜γf) = δij and ζf = 0. In the same way, we have to add the condition
˜γsep
δN
ij = δij to the uniform-N gauge, where ∂i(βδN)i = 0 and ζδN = 0. The main
consequence of this is that, under the separate universe condition, we can express all the
scalar fluctuations only in terms of the field inhomogeneities not only in the spatially flat
gauge, but also in the uniform-N gauge. This can be clearly seen when looking at the linear
“separate-universe” gauge invariant MS variable of (68). For non-linear generalizations of
this variables see [117,118]. Before continuing, let us remind the reader that the equivalence between these two
gauges in only valid under the separate universe approach, which, as seen in Section 4.1.2,
it generically fails at O(ϵ1). During this section, and although it is not compulsory, we will work using the number
of e-folds N ≡R
Hbdt as time variable (Note that we can use any time variable we want
because we will use the coordinates of a fictitious global background, i.e., the coordinates of
(84). If we would instead use local coordinates (as in (63)), we would be interested in using
an unperturbed time variable, being N the natural choice in the uniform-N gauge). 6.1. Stochastic Formalism Based on the Separate Universe Approach −3 Hb
αIR
∂
∂N
Hb
αIR
! −3
Hb
αIR
!2
+ DkDkαIR −
1
M2
PL
Hb
αIR
!2∂φIR
∂N
2
−V
φIR
= −3
Hb2
(αIR)3
∂αUV
∂N
+
9
Hb2
(αIR)4
∂αIR
∂N −
6Hb
(αIR)3
∂Hb
∂N
!
αUV −
6
Hb2
(αIR)3 αUV −∇2
a2 αUV +
1
M2
PL
2
Hb
αIR
!2 ∂φIR
∂N
∂φUV
∂N
−2
Hb2
(αIR)3
∂φIR
∂N
2
αUV −Vφ
φIR
φUV
. (101) (101) Note that this is the same as we did in linear perturbation theory of Section 4 but using
the metric (63) (or equivalently (84)) as background metric. Now, since the IR variables are well outside the Hubble horizon, we will use the
separate universe approach for them. Since αIR ∼O(σ0) and hence DkDkαIR ∼O(σ2)
we have: −3
Hb
(0)αIR
∂
∂N
Hb
(0)αIR
! −3
Hb
(0)αIR
!2
−
1
M2
PL
Hb
(0)αIR
!2
∂(0)φIR
∂N
!2
−V
(0)φIR
= −3
Hb2
(0)αIR
3
∂αUV
∂N
+
9
Hb2
(0)αIR
4
∂(0)αIR
∂N
−
6Hb
(0)αIR
3
∂Hb
∂N
!
αUV −
6
Hb2
(0)αIR
3 αUV
−∇2
a2 αUV +
1
M2
PL
2
Hb
(0)αIR
!2 ∂(0)φIR
∂N
∂φUV
∂N
−2
Hb2
(0)αIR
3
∂(0)φIR
∂N
!2
αUV −Vφ
(0)φIR
φUV
,
(102) (102) where we have inserted an extra subindex (0) to indicate that we are at leading order in
gradient expansion. Using Fourier analysis we can now define more rigorously the IR and UV modes. If
we choose the Heaviside theta as a window function (The choice of the Heaviside theta as
window function in the stochastic formalism is the most common one. However it can lead
to some problems as indicated in [28]) we have the following decomposition for a generic
function X. 6.1. Stochastic Formalism Based on the Separate Universe Approach Another
important aspect is that, in order not to overload notation, we will suppress the subscript
δN indicating the gauge we are using, such that in the following, unless otherwise stated, a
variable without a subscript that indicates the gauge is a variable in the uniform-N gauge. As indicated before, the stochastic formalism presented in this subsection is based on
the separate universe approach for the IR part and on linear perturbation theory for the UV
part. To illustrate this we will consider in detail the equation of motion for the trace the
extrinsic curvature (19) in uniform-N gauge, which can be written using the variables of
the ADM metric (84) as: −
Hb
2α
!2 ∂˜γij
∂N
∂˜γij
∂N −3
Hb
α
!2
+ DkDkα −
1
M2
PL
Hb
α
!2 ∂φ
∂N
2
−V(φ)
= 0 . (99) (99) The first thing to do is to split the variables of interest into their IR and UV part. In
this case we only have two variables to split: The first thing to do is to split the variables of interest into their IR and UV part. In
this case we only have two variables to split: α = αIR + αUV ,
φ = φIR + φUV . (100) (100) In (100) we are not considering
∂˜γij
∂N as a variable of interest not only because ˜γij = δij
in the separate universe approach, but also because
∂˜γij
∂N
∂˜γij
∂N ∼O(σ2) in gradient expansion
and quadratic in perturbation theory so it does not play any role even if we were using
O(σ0) gradient expansion. Due to the perturbative nature of the UV variables, we will expand (99) keeping only
linear terms in UV and isolate them in the right hand side of the equation getting 25 of 39 Universe 2022, 8, 334 −3 Hb
αIR
∂
∂N
Hb
αIR
! −3
Hb
αIR
!2
+ DkDkαIR −
1
M2
PL
Hb
αIR
!2∂φIR
∂N
2
−V
φIR
= −3
Hb2
(αIR)3
∂αUV
∂N
+
9
Hb2
(αIR)4
∂αIR
∂N −
6Hb
(αIR)3
∂Hb
∂N
!
αUV −
6
Hb2
(αIR)3 αUV −∇2
a2 αUV
+
1
M2
PL
2
Hb
αIR
!2 ∂φIR
∂N
∂φUV
∂N
−2
Hb2
(αIR)3
∂φIR
∂N
2
αUV −Vφ
φIR
φUV
. 6.1. Stochastic Formalism Based on the Separate Universe Approach XIR(t, x) ≡
Z
dk
(2π)3/2 Θ(σal(N)Hl(N) −k)X IR
k (t, x) ,
XUV(t, x) ≡
Z
dk
(2π)3/2 Θ(k −σal(N)Hl(N))X UV
k
(t, x) ,
(103) (103) where, similarly as in linear perturbation theory (see (47)),X UV
k
(t, x) is define as the follow-
ing hermitian operator: X UV
k
(t, x) = e−ik·xXk(N)ak + eik·xX∗
ka†
k ,
(104) (104) where Xk(N) is the solution of the evolution equation for the perturbation X over the local
background defined by (83) and ak and a†
k are the usual creation and annihilation operators
which follow the commutation relation given in (48). Note that, in the spirit of gradient expansion, the splitting is done in the local cosmolog-
ical coarse-grained scale (σHl)−1, which generically differs form the one of the background, Universe 2022, 8, 334 26 of 39 for example in uniform-N gauge we have Hl =
Hb
(0)αIR . Inserting the definition of XUV of
(103) into (102) we get: for example in uniform-N gauge we have Hl =
Hb
(0)αIR . Inserting the definition of XUV of
(103) into (102) we get: −3
Hb
(0)αIR
∂
∂N
Hb
(0)αIR
! −3
Hb
(0)αIR
!2
−
1
M2
PL
Hb
(0)αIR
!2
∂(0)φIR
∂N
!2
−V
(0)φIR
3
Hb2
(0)αIR
3
∂
∂N
σa
Hb
(0)αIR
! Z
dk
(2π)3/2 δ
k −σa
Hb
(0)αIR
! αUV
k
−
2
M2
PL
Hb
(0)αIR
!2 ∂(0)φIR
∂N
∂
∂N
σa
Hb
(0)αIR
! Z
dk
(2π)3/2 δ
k −σa
Hb
(0)αIR
! ϕUV
k
+
Z
dk
(2π)3/2 Θ
k −σa
Hb
(0)αIR
!(
−3
Hb2
(0)αIR
3
∂αUV
k
∂N
+
9
Hb2
(0)αIR
4
∂(0)αIR
∂N
−
6Hb
(0)αIR
3
∂Hb
∂N
!
αUV
k
−
6
Hb2
(0)αIR
3 αUV
k
+ k2
a2 αUV
k
1
M2
PL
2
Hb
(0)αIR
!2 ∂(0)φIR
∂N
∂ϕUV
k
∂N
−
2
Hb2
(0)αIR
3
∂(0)φIR
∂N
!2
αUV
k
−Vφ
(0)φIR
ϕUV
k
)
,
(105) (105) where αUV
k
and ϕUV
k
are operators defined as in (104). where αUV
k
and ϕUV
k
are operators defined as in (104). 6.1. Stochastic Formalism Based on the Separate Universe Approach If we now follow the
procedure for the evolution equation of the trace of the extrinsic curvature just explained
with the Hamiltonian constraint we get: 6
Hb
(0)αIR
!2
−
2
M2
PL
Hb
(0)αIR
!2
∂(0)φIR
∂N
!2
+ V
(0)φIR
=
2
M2
PL
Hb
(0)αIR
!2 ∂(0)φIR
∂N
ξ1 ,
(108)
2 6
Hb
(0)αIR
!2
−
2
M2
PL
Hb
(0)αIR
!2
∂(0)φIR
∂N
!2
+ V
(0)φIR
=
2
M2
PL
Hb
(0)αIR
!2 ∂(0)φIR
∂N
ξ1 ,
(108)
which can be solved for
Hb
(0)αIR
2
, i.e.,
Hb
(0)αIR
!2
=
V
(0)φIR
3M2
PL −1
2
∂(0)φIR
∂N
2
−
∂(0)φIR
∂N
ξ1
. (109) (108) Hb
(0)αIR
!2
=
V
(0)φIR
3M2
PL −1
2
∂(0)φIR
∂N
2
−
∂(0)φIR
∂N
ξ1
. (109) (109) Finally, the stochastic equation of motion for the field is obtained in the same way: ∂2 (0)φIR
∂N2
+
3 +
∂
∂N
Hb
(0)αIR
Hb
(0)αIR
∂(0)φIR
∂N
+
Vφ
(0)φIR
Hb
(0)αIR
2 ∂2 (0)φIR
∂N2
+
3 +
∂
∂N
Hb
(0)αIR
Hb
(0)αIR
∂(0)φIR
∂N
+
Vφ
(0)φIR
Hb
(0)αIR
2
= −∂ξ1
∂N −ξ2 −
3 +
∂
∂N
Hb
(0)αIR
Hb
(0)αIR
ξ1 +
∂(0)φIR
∂N
ξ3
(0)αIR ,
(110) = −∂ξ1
∂N −ξ2 −
3 +
∂
∂N
Hb
(0)αIR
Hb
(0)αIR
ξ1 +
∂(0)φIR
∂N
ξ3
(0)αIR ,
(110) (110) where ξ2 is defined similarly to ξ1 and ξ3: where ξ2 is defined similarly to ξ1 and ξ3: ξ2 ≡−∂
∂N
σa
Hb
(0)αIR
! Z
dk
(2π)3/2 δ
k −σa
Hb
(0)αIR
! ∂ϕUV
k
∂N
. (111) (111) As anticipated before, the usage and uniform-N gauge and the separate universe
approach ensures that all the scalar inhomogeneities are encoded in the scalar field. This
becomes clearer once we realize that we can write the system (106)–(110) in terms only of
the scalar field. 6.1. Stochastic Formalism Based on the Separate Universe Approach k
k
The right-hand side of (105) has two different terms: k
k
The right-hand side of (105) has two different terms: 1. The second integral (terms multiplying the Heaviside theta) is the evolution equation
for the extrinsic curvature linearized over a local FLRW patch defined by (0)αIR and
(0)φIR. Once the Bunch-Davies vacuum is chosen for that patch, this term will be
automatically satisfied so it can be consistently set to zero. Note that the solution of
this part equalized to zero is precisely what give us the functions Xk in (104). p
q
p
y
g
2. The first two integrals, proportional to a Dirac delta, can be seen as boundary con-
ditions and hence they will act as the initial conditions missing when using only
gradient expansion. 2. The first two integrals, proportional to a Dirac delta, can be seen as boundary con-
ditions and hence they will act as the initial conditions missing when using only
gradient expansion. We then get: We then get: −3
Hb
(0)αIR
∂
∂N
Hb
(0)αIR
! −3
Hb
(0)αIR
!2
−
1
M2
PL
Hb
(0)αIR
!2
∂(0)φIR
∂N
!2
−V
(0)φIR
= −3
Hb2
(0)αIR
3 ξ3 +
2
M2
PL
Hb
(0)αIR
!2 ∂(0)φIR
∂N
ξ1 ,
(106) 3 +
2
M2
PL
Hb
(0)αIR
! ∂(0)φIR
∂N
ξ1 ,
(106) (106) where we have defined ξ1 and ξ3 as: where we have defined ξ1 and ξ3 as: where we have defined ξ1 and ξ3 as: ξ1 ≡−∂
∂N
σa
Hb
(0)αIR
! Z
dk
(2π)3/2 δ
k −σa
Hb
(0)αIR
! ϕUV
k
,
ξ3 ≡−∂
∂N
σa
Hb
(0)αIR
! Z
dk
(2π)3/2 δ
k −σa
Hb
(0)αIR
! αUV
k
. (107) (107) Universe 2022, 8, 334
27 of 39 Universe 2022, 8, 334 27 of 39 For the moment we will not characterize the quantities ξ1 and ξ3. 6.1.1. Characterization of the Noises 6.1.1. Characterization of the Noises 6.1.1. Characterization of the Noises 6.1.1. Characterization of the Noises The interpretation of ξ1 and ξ2 (note that ξ3 no longer appears in the final equation
of motion) as classical noises is not trivial because they are, strictly speaking, quantum
operators. In order to see how they are effectively classical, we can compute the two-point
correlation function of ξ1 for example at equal space point, the result is: ⟨0|ξ1(N1)ξ1(N2)|0⟩=
∂
∂N
σa
Hb
(0)αIR
! σa
Hb
(0)αIR
!2
δφ(N1)
k=
σa
Hb
(0)αIR
! 2
δ(N1 −N2) ,
(114) (114) where we have used (104) together with the commutation relation (48). From (114) we
see that δφk, which is the solution for the field perturbation over the local patch of size
σa
Hb
(0)αIR
−1
, is evaluated at the coarse-grained scale, i.e., well outside the Hubble horizon. It can then be shown that at those scales, any perturbation that started from a coherent
vacuum state has evolved into a highly squeezed state [119,120], which means that we where we have used (104) together with the commutation relation (48). From (114) we
see that δφk, which is the solution for the field perturbation over the local patch of size
σa
Hb
(0)αIR
−1
, is evaluated at the coarse-grained scale, i.e., well outside the Hubble horizon. It can then be shown that at those scales, any perturbation that started from a coherent
vacuum state has evolved into a highly squeezed state [119,120], which means that we
can consider
δφ(N)
k=
σa
Hb
(0)αIR
! 2
as the power spectrum of a classical random variable, can consider
δφ(N)
k=
σa
Hb
(0)αIR
! as the power spectrum of a classical random variable, can consider
δφ(N)
k=
σa
Hb
(0)αIR
! ! whose time evolution is consistent with probabilities conserved along classical trajectories. whose time evolution is consistent with probabilities conserved along classical trajectories. Once this is clarified, we are now in position to describe ξ1 as a classical white noise
(Its “white” nature is due to the presence of δ(N1 −N2) in the two-point correlator. Note
that this is a consequence of the the choice of the Heaviside theta function as Window
function, any other choice would lead to coloured noises, which are much more difficult to
deal with, both analytically and numerically) with variance given in (114). 6.1. Stochastic Formalism Based on the Separate Universe Approach Inserting (106) and (109) into (110) and neglecting ξ2
i terms because they
are quadratic in perturbation theory we get: ∂2 (0)φIR
∂N2
+
3 −
1
2M2
PL
∂(0)φIR
∂N
!2
∂(0)φIR
∂N
+
3M2
PL −1
2
∂(0)φIR
∂N
!2
Vφ
(0)φIR
V
(0)φIR
−∂ξ1
∂N −ξ2 −
3 −
1
2M2
PL
∂(0)φIR
∂N
!2
−
1
M2
PL
∂(0)φIR
∂N
!2
−
Vφ
(0)φIR
V
(0)φIR
∂(0)φIR
∂N
!
ξ1 ,
(112) (112) which can be conveniently written if we use an auxiliary variable (0)πIR: (0)πIR =
∂(0)φIR
∂N
+ ξ1 ,
∂(0)πIR
∂N
+
3 −
(0)πIR2
2M2
PL
(0)πIR +
3M2
PL −
(0)πIR2
2
Vφ
(0)φIR
V
(0)φIR
= −ξ2 . (113) (113) Universe 2022, 8, 334 28 of 39 6.1.1. Characterization of the Noises Furthermore,
since the field fluctuations are Gaussian to a good level of approximation, the variance
computed in (114) is enough to fully characterize ξ1. Finally, in order to characterize the
system (113) we also need: ⟨0|ξ1(N1)ξ2(N2)|0⟩= ⟨0|ξ2(N1)ξ1(N2)|0⟩∗=
∂
∂N
σa
Hb
(0)αIR
! σa
Hb
(0)αIR
!2
δφ(N1)
k=
σa
Hb
(0)αIR
! ∂δφ∗(N1)
k=
σa
Hb
(0)αIR
! ∂N
δ(N1 −N2) ,
⟨0|ξ1(N1)ξ1(N2)|0⟩=
∂
∂N
σa
Hb
(0)αIR
! σa
Hb
(0)αIR
!2
∂δφ(N1)
k=
σa
Hb
(0)αIR
! ∂N
2
δ(N1 −N2) . (115) (115) The characterization of ξ1 and ξ2 as white noises give us now a intuitive picture of the
physics behind the stochastic formalism. As explained before, different functions for (0)αIR
and (0)φIR (in uniform-N gauge) describe the evolution of different FLRW patches, the way
of getting these different functions is now clear if we see ξ1 and ξ2 as random variables. For
example, the evolution of a specific patch, let us call it 1FLRW, will be given by 1
(0)αIR and
1
(0)φIR, whose specific form will be determined by the random values that the noises 1ξ1
and 1ξ2 will pick at each time step. Now, if we want to describe a second patch 2FLRW, we
just have to solve again the stochastic equation with different random values for the noises
2ξ1 and 2ξ2, always satisfying the statistics described by (114) and (115). Like this, we are
then able to describe the evolution of an ensemble of FLRW patches by solving many times
the same stochastic equation with different random values for the noises. The correlators
between these patches are then simply described by statistical moments of the IR variables. Universe 2022, 8, 334 29 of 39 29 of 39 The stochastic system is already fully characterized by (113)–(115); however, this
system is very difficult to solve because, in order to compute the variance of the noises ξ1
and ξ2, we need to solve the perturbation equations and compute δφk over a stochastic
background every time step, which makes the systen non-Markovian. Although there are
numerical algorithms capable of doing so [67], it would be very convenient if we were able
to write an analytical solution for δφk in terms of the IR variables, that would make the
system Markovian and easily solvable. 6.1.1. Characterization of the Noises Let us try to solve the equation for δφk over the
stochastic local background. We know that, as a consequence of the separate universe approach used here, the
linearized equation for the field perturbation in the uniform-N gauge will be the same as
the equation for the “separate-universe” gauge invariant variable Qsep defined in (68), in
other words, Qsep = δφf = δφδN under the separate universe assumption. Now, since the
linearized equation for Qsep only gives the correct solution for the true gauge-invariant MS
variable Q defined in (44) if we set ϵ1 = 0 as checked in Section 4.1.2, the equation we will
try to solve here is the linearized equation for the gauge invariant quantity Q over a local
background in which all the SR parameters have been set to zero (note that, in order for
this approximation to work we need −3
2ϵ2 −1
4ϵ2
2 ∼O(ϵ1) , which is true for SR and USR. This is because we are setting the combination of SR parameters multiplying Q in (45) to
zero), in other words, we will try to solve the equation for Q as if the local background
were an exact de-Sitter, which is: ∂2δφk
∂N2 + 3∂δφk
∂N +
(0)αIR2
k2
a2 Hb2 δφk = 0 ,
(116) (116) or, using the Hamiltonian constraint of (109): ∂2δφk
∂N2 + 3∂δφk
∂N + k2
a2
3M2
PL −( (0)πIR)
2
2
V
(0)φIR
δφk = 0 . (117) (117) Now, both the functions (0)πIR and V
(0)φIR
are stochastic so, in order to know
them we must already know the value for the variable ξ1. This makes Equation (117) not
analitically solvable, more concretely, the stochastic system of (113) is non-Markovian,
meaning that the value of the noises at each time N will depend on the whole evolution of
the stochastic patch up to N. The only option remaining to solve the stochastic system of (113) seems then to be
numerically; however, a further and very important approximation is usually done, which
consists on assuming that the IR (and stochastic) quantities do not differ much from
their global background (and deterministic) counterpart, namely YIRXUV ≃YbXUV +
O
XUV2
. Here XUV and YIR are any UV and IR functions, we then define Yb as the
equivalent background function of YIR. 6.1.1. Characterization of the Noises Under this approximation, the last term of (116) is: k2
(0)αIR
aHb
!2
δφk ≃
k
aHb
2
δφk + O
δφ2
k
,
(118) (118) where we have substituted (0)αIR by its background value, i.e., 1. Under this approximation
we can write a analytical solution for (116), that, once evaluated at coarse-grained scale,
will correspond to the long-wavelength limit of solution (51) with ν = 3
2, i.e., δφ(N)k=(σaHb)
2
=
Hb2
2
σaHb3 . (119) (119) Universe 2022, 8, 334 30 of 39 and therefore the correlators (114) and (115) can be written as follows: and therefore the correlators (114) and (115) can be written as follows: ⟨ξ1(N1)ξ1(N2)⟩=
Hb
2π
!2
δ(N1 −N2) ,
(120)
⟨ξ1(N1)ξ2(N2)⟩= ⟨ξ2(N1)ξ2(N2)⟩= 0 ,
(121) (120) (121) Under this approximation, the stochastic system of (113) simplifies considerably and
becomes a Markovian process with additive noises, meaning that ξ1 depends only on
time, which is true as long as we are using the global background to characterize it. The
system is: (0)πIR =
∂(0)φIR
∂N
+ Hb
2π ξ(N) ,
∂(0)πIR
∂N
+ 3 (0)πIR + M2
PL
Vφ
(0)φIR
V
(0)φIR
= 0 ,
(122) (122) where ⟨ξ(N1)ξ(N2)⟩= δ(N1 −N2) and we have dropped O(ϵ1) terms in order to be
consistent with the computation of the noises and the reasoning above (116). Note that in
SR the acceleration of the field is also of higher order in SR parameters so in this case the
stochastic equation would simplify even more: ∂(0)φIR
∂N
+ M2
PL
Vφ
(0)φIR
V
(0)φIR
= −Hb
2π ξ(N) . (123) (123) Although the approximation used in order to arrive to (122) seems very useful, it has
important consequences, in fact, it is equivalent to state that any YIR −Yb ∼O
XUV
. Thus, we immediately see that if this approximation holds, the system in (122) can only
reproduce the results of linear theory at leading order in SR parameters, and hence it does
not give any non-perturbative (or even non-linear) information. In fact, (122) is slightly
inconsistent. 6.1.1. Characterization of the Noises The point is that, by the same approximation adopted on the right hand side,
the left hand side should also be linearized (in fact the linearization of the left-hand side
of (122) is sometimes performed when recursive methods are used in order to solve the
stochastic system as it can be seen in [42–44]). This inconsistency can however give some
information when comparing the results from the stochastic formalism with the ones of
linear perturbation theory, in fact, since we know that the correlations functions calculated
with the stochastic system of (122) will coincide, up to second order in perturbation theory,
to the ones calculated in linear perturbation theory with QFT methods, any inconsistency
between the two approaches will signal the break-down of perturbation theory. pp
g
p
y
Since the stochastic Equation (116) and its deterministic counterpart with (0)αIR = 1
have the same structure, one could be tempted to write the solution of (116) in a similar
way as the solution (119) but substituting Hb by
Hb
(0αIR , i.e: δφ(N)
k=
σa
Hb
(0αIR
! 2
=
Hb
(0αIR
!2
1
2
σa
Hb
(0αIR
3 ,
(124) (124) Universe 2022, 8, 334 31 of 39 and hence the stochastic system of (113) would approximately be: and hence the stochastic system of (113) would approximately be: (0)πIR =
∂(0)φIR
∂N
+
Hb
2 (0)αIRπ ξ(N) ,
∂(0)πIR
∂N
+ 3 (0)πIR + 3M2
PL
Vφ
(0)φIR
V
(0)φIR
= 0 . (125) (125) The system of (125) represents a Markovian process with non-additive noises (the term
proportional to ξ does no longer depends only on time), which is, provided that (124) holds,
able to describe the inhomogeneities generated during inflation in a non-perturbative way. g
g
g
p
y
Unfortunately, we cannot trivially generalize the solution (119) for a deterministic equation
to a solution (124) for a stochastic equation, this is due to the differences between stochastic
and deterministic integrals (see for example [121]). This is why, although it is the most
common approach found in the literature, we will not trust (125) to describe non-linear
inflationary effects in this review. y
Having discarded this option, we are left with three different ways of solving the
stochastic system of (113): 1. 6.1.1. Characterization of the Noises We can use the system (113) where the noises are computed numerically over the
stochastic local background, for example, using the algorithm described in [67]. •
Pros: It describe non-linear inflationary dynamics. •
Cons: It is very difficult to solve due to the non-Markovianity of the process. Furthermore, it is only valid up to leading order in SR parameters due to the use
of the separate universe approach. 2. We can use the system of (122) •
Pros: It is a Markovian process with additive noises for which even analytical
solutions can be obtained (see Section VI of [65]). •
Cons: It only describes linear perturbations whenever they are approximately
described by solution (119), which is not the case during some interesting regimes
for PBH formation, such as a SR-USR transition where ν ̸= 3
2. It is then even less
precise than the linear separate universe approach. 3. Finally, we can solve the MS equation over a global background at all orders in SR
parameters, i.e., we can solve (45) and characterize the noises as in (114) with this
solution. This can be done because, as we have already indicated, under the separate
universe assumption Q = δφδN. Once the noises are characterized in this way, we can
use the stochastic system (113) and solve the dynamics. •
Pros: It is a Markovian process with additive noises able to describe the linear
dynamics of the inflationary perturbations even when they are not approximately
described by (119). y (
)
•
Cons: It is not capable of describing any non-linear effects and it is inconsistent
generically at leading order in ϵ1 due to the use of the separate universe approach. This inconsistency can be clearly seen in the term proportional to ξ1 in (112),
which in the background can be written as
3 −ϵ1 + ϵ1ϵ2
3−ϵ1
and not (3 −ϵ1) as it
should be if it came from the MS equation. In fact
3 −ϵ1 + ϵ1ϵ2
3−ϵ1
is precisely the
term that appears in the equation for Qsep (69) derived in Section 4.1.2, making it
clear that this inconsistency is a consequence of the separate universe approach. 6.1.1. Characterization of the Noises Due to the problems that the stochastic formalism presented in this section has (namely
its validity only up to leading order in ϵ1 due to the separate universe assumption and
its difficulty to describe non-linear dynamics), it does not seem very promising if we
want to describe the non-perturbative dynamics of the inohomogeneities generated during
inflationary regimes of interest for PBH formation with enough precision. However, in the Universe 2022, 8, 334 32 of 39 32 of 39 next subsection we will present a stochastic formalism (firstly presented in [65]) which is at
least able to reproduce linear perturbation theory at all orders in SR parameters, and with
the potential to correctly describe the non-perturbative dynamics of the inhomogeneities
during any inflationary regime. However, before doing so, and in order to finish this subsection, it is important to
know that the stochastic system of (113) can be straightforwardly derived by splitting into
a IR and an UV only the scalar field in the separate universe equations of (64). In this case
we would have used the metric (63) for the local patch instead of the metric (83) as we have
done here. We have not chosen this option in this review because of three main reasons: 1. It does not make it clear that we have used gradient expansion, and hence the problem
of not using the momentum constraint that we solve in the next subsection is difficult
to remark. 2. Since it only works if the time variable is unperturbed, it could lead us to think that
the number of e-folds N is the only allowed time variable for a stochastic formalism
that describes all the scalar inhomogeneities in terms of the inflaton field. On the
contrary, the derivation used in this review is valid for any time variable and makes
it clear that the description of inhomogeneities in terms solely of the inflaton field is
only a gauge choice. y
g
g
3. It does not explicitly obtains the linear equations used for the characterization of
δφk, more concretely, for example this derivation would miss the k2
a2 αUV
k
term that
multiplies the Heaviside theta in (105). 6.2. Stochastic Formalism Based on O(σ0) Gradient Expansion Splitting (127) into
IR and UV and using the decomposition of ˜γij explained around (87) to keep only O(σ)
terms in the IR part (remember that the O(σ0) information from the momentum constraint Universe 2022, 8, 334 33 of 39 can only be extracted if we write the momentum constraint up to O(σ)), we can write the
stochastic equation for the momentum constraint: can only be extracted if we write the momentum constraint up to O(σ)), we can write the
stochastic equation for the momentum constraint: (0)∂i
∂
∂N
1
3∇2CIR
−(0)∂iαIR
(0)αIR +
∂(0)φIR
∂N
(0)∂iφ
2M2
PL
= −∂iξ4 ,
(128) (128) where ξ4 is defined similarly to ξ1, ξ2 and ξ3, i.e., ξ2 ≡−∂
∂N
σa
Hb
(0)αIR
! Z
dk
(2π)3/2 δ
k −σa
Hb
(0)αIR
!
−k2
3 CUV
k
. (129) (129) With the addition of the stochastic Equation (128) to the system of (113) obtained
before, we have a stochastic formalism able to describe the non-linear evolution of scalar
inhomogeneities at all orders in SR parameters. With the addition of the stochastic Equation (128) to the system of (113) obtained
before, we have a stochastic formalism able to describe the non-linear evolution of scalar
inhomogeneities at all orders in SR parameters. However, it is not all good news: firstly, since the construction of the gradient expan-
sion in Section 5, we have been neglecting possible interactions scalar-tensor or scalar-vector,
reason why the stochastic formalism constructed here will not take these interactions into
account either. Secondly, we do not exactly know how to extract the O(σ0) information
form (128) in a fully non linear way. Finally, we do not know which is the combination of
(0)φIR and ∇2CIR that give us the correct and non-perturbative and gauge invariant quan-
tity that describe scalar inhomogeneities, i.e., we do not have a non-linear generalization
of the MS variable. The first issue is beyond the scope of this review. With respect to the
third one, it is true that a non-linear gauge invariant variable at leading order in gradient
expansion has been defined in [117,118] as: ∂iQNL ≡∂iφ + 1
α
∂φ
∂N ∂iζ However, the variable above does not include the term proportional to ∇2E in its
linearization. Reason why it can be only interpreted as a non-linear generalization of
Qsep. 6.2. Stochastic Formalism Based on O(σ0) Gradient Expansion As many times claimed during the review, the separate universe approach generically
fails to give the correct long-wavelength evolution of the inhomogeneities at O(ϵ1). The
O(σ0) gradient expanision solves this problem by including both non-local terms and the
momentum constraint, this is why in this section we will construct a stochastic formalism
based on O(σ0) gradient expansion. First of all, it is important to remark that some of the affirmations we did about the
uniform-N gauge at the beginning of Section 6.1 are no longer correct, more concretely, we
cannot longer study the scalar inhomogeneities in terms solely of the inflaton field. This is
clear form linear perturbation theory where the MS variable can be written as Q = δφf = δφδN −∂φb
∂N
1
3∇2EδN ,
(126) (126) so if we insist on using the uniform-N gauge we must also take into account the contribution
from E when studying scalar perturbations. We could also use spatially flat gauge in this case and forget about E; however, in
this case we should take into account all the terms proportional to
(0)βf
i
that appear for
example in (90), this is why we will keep using the uniform-N gauge, where βi = 0. One can easily check that the stochastic equations for the evolution of the extrinsic
curvature (106), for the Hamiltonian constraint (109) and for the evolution of the field (110)
do not change when including E and hence the stochastic system is still the one given
by (113). The only difference will then be given by the inclusion of the momentum
constraint (15), which, in uniform-N gauge can be written as: Dj
−Hb
2α
∂˜γij
∂N
! −2
3 DiK = −
1
M2
PLα
∂φ
∂N ∂iφ ,
(127) (127) where we have used the evolution Equation for ˜γij (18) to eliminate ˜Aij. 6.2. Stochastic Formalism Based on O(σ0) Gradient Expansion This is the reason we will not use the non-linear variable defined above and we
will solve the second and third issues, at least approximately, by imposing that both the
momentum constraint and the non-linear generalization of the MS variable match their
linear counterpart when the global background is subtracted. In this way, the O(σ0)
information of (128) can be straightforwardly extracted and the whole system of stochastic
equations based on the O(σ0) gradient expansion and hence valid at all orders in SR
parameters is: (0)πIR =
∂(0)φIR
∂N
+ ξ1 ,
∂(0)πIR
∂N
+
3 −
(0)πIR2
2M2
PL
(0)πIR +
3M2
PL −
(0)πIR2
2
Vφ
(0)φIR
V
(0)φIR
= −ξ2 ,
∂
∂N
1
3 (0)∇2CIR
−
Hb
v
u
u
u
t3M2
PL −( (0)πIR)
2
2
V
(0)φIR
−1
+
1
22
PL
∂(0)φIR
∂N
(0)φIR −φb
= −ξ4 ,
(130) (0)πIR =
∂(0)φIR
∂N
+ ξ1 ,
∂(0)πIR
∂N
+
3 −
(0)πIR2
2M2
PL
(0)πIR +
3M2
PL −
(0)πIR2
2
Vφ
(0)φIR
V
(0)φIR
= −ξ2 ,
∂
∂N
1
3 (0)∇2CIR
−
Hb
v
u
u
u
t3M2
PL −( (0)πIR)
2
2
V
(0)φIR
−1
+
1
22
PL
∂(0)φIR
∂N
(0)φIR −φb
= −ξ4 ,
(130) (130) where we have used the Hamiltonian constraint to eliminate (0)αIR in the last equation. Note that ξ1,ξ2 and ξ4 are constructed in the uniform-N gauge, which is no longer equiva-
lent to the spatially flat gauge. To see the gauge transformation between spatially flat and uniform-N gauges in linear
theory one can see [60], where it is claimed that the differences between those two gauges
is always of higher order in gradient expansion; however, this conclusion is reached by Universe 2022, 8, 334 34 of 39 considering the value of ϵ1 at horizon crossing (ϵ∗
1 there) to be constant with k, which is
generically not a good approximation beyond SR. 6.2. Stochastic Formalism Based on O(σ0) Gradient Expansion In fact in [67] it is shown numerically that
the difference between δφf and δφδN can be O(ϵ1) in regimes of interest for PBH formation,
in agreement with the differences between the separate universe approach and O(σ0)
gradient expansion remarked in this paper. Finally, as suggested in [122–124], we can define the non-linear counterpart of the MS
variable of (44) at leading order in gradient expansion as: QIR = (0)φIR −φb −
∂(0)φIR
∂N
1
3 (0)∇2CIR ,
(131) (131) where we remind the reader that (0)φIR and (0)∇2CIR are both in the uniform N gauge. 0 where we remind the reader that (0)φIR and (0)∇2CIR are both i The stochastic formalism based on the O(σ0) gradient expansion presented in this
section was introduced for the first time, although in a different gauge, in [65], where the
authors show numerically that, when computing the noises over a global background,
i.e., when using the approximation described around (118), the two point correlator com-
puted using the stochastic formalism perfectly matches with the one computed in linear
perturbation theory at all orders in SR parameters, as expected. This already represents an
important improvement with respect to any stochastic formalism based on the separate
universe approach as the one explained in Section 6.1. 6.3. Stochastic Formalism Versus Linear Perturbation Theory Once the stochastic formalism has been properly introduced, the simplest way to
know if there exists any important non-perturbative effect during inflation is to compare
the results obtained within the stochastic framework with the results coming from linear
perturbation theory explained in Section 4. In order to do so, it is crucial to realize that
the results coming from the stochastic formalism are in real space whereas the result for
the linear MS variable in terms of Hankel function of (51) is in Fourier space. Due to the
difficulty when trying to express stochastic correlators in Fourier space, we will compare
the stochastic two-point correlator with the linear two-point correlator, both in real space. As explained below (115), the correlators between different FLRW patches are de-
scribed by statistical moments of IR variables, so the two point correlator of the scalar
inhomogeneities is: ⟨(0)φIR
(0)φIR⟩= Var
(0)φIR
,
(132) (132) if we are using the separate universe approach or if we are using the separate universe approach or ⟨QIRQIR⟩= Var
QIR
,
(133) (133) if we are using O(σ0) in gradient expansion. if we are using O(σ0) in gradient expansion. if we are using O(σ0) in gradient expansion. O
th
th
h
d th t
i t
l t
i
li
t
b ti
th
i d fi
d On the other hand, the two-point correlator in linear perturbation theory is defined as
(see (52)): On the other hand, the two-point correlator in linear perturbation theory is defined as
(see (52)): ⟨Q(N)Q(N)⟩=
Z σa(N)Hb(N)
σa(N=0)Hb(N=0)
dk
k PQ(k, N) ,
(134) (134) where we are now using the power spectrum evaluated at the same spatial point PQ(k, N) ≡
k3
2π2 |Qk(N)|2 ,
(135) (135) where Qk in uniform-N gauge is given by (126). The limits of (134) correspond to the
selection of modes inside the coarse-grained scale defined by k = σa(N)Hb(N) and they
are necessary to correctly compare the two point correlators obtained in both formalisms,
in fact, in the stochastic formalism the IR part of the field recieves stochastic kicks from
N = 0 onwards. Thus the first k-mode from which the IR field recieves a kick is the one
with k = σa(N = 0)Hb(N = 0). 6.3. Stochastic Formalism Versus Linear Perturbation Theory Universe 2022, 8, 334 35 of 39 Whenever PQ(k, N) does not depend on N, one can do a very useful approximation,
which consist on evaluating the power spectrum at coarse-grained scale crossing, i.e., at
k = σaHb, and assume that this value does not change with time. This would allow us to
write (134) as:
N ⟨Q(N)Q(N)⟩=
Z N
0
P
k = σa(N′)Hb(N′)
dN′ ,
(136) (136) In this case one could write the power spectrum as the derivative with respect to the
number of e-folds N of the correlator in real space. PQ(k) =
d
dN ⟨Q(N)Q(N)⟩. (137) (137) However, and although it has been sometimes wrongly used for a time-dependent
power spectrum [33], this technique cannot be used if the power spectrum evolves with
time, which makes it only valid at zeroth order in O(ϵ1). Finally, and due to the difficulty in the definition of non-linear gauge invariant vari-
ables (remember for example that the “trivial” non-linear generalization of the MS vari-
able of (131) is not guaranteed to be the correct one), some authors have used the so-
called stochastic-δN formalism to compute the non-linear curvature perturbation, which
is very useful when dealing with PBHs, by studying the statistics in the number of
e-folds [36,41,46,50,51]. However, and similarly to what happens with the usual δN for-
malism, the stochastic-δN formalism uses the separate universe approach and hence it is
generically only valid at leading order in SR parameters. 7. Conclusions The stochastic approach to inflation seems the most promising way to study, in a
non-perturbative way, the probability distribution of the scalar inhomogeneities generated
during inflation and responsible for the creation of PBHs. However, as remarked during
the review, there is still a lot of work to do in that direction. Indeed, although very useful,
the most common and widely used stochastic formalism has some difficult problems to
solve, which can be summed up in three points: 1. As we have seen during the review, the stochastic formalism uses a gradient expansion
for the IR part and a perturbative expansion for the UV part in such a way that the IR
part, due to the large wavelength of the characteristic inhomogeneities that form this
sector, can be described as a local FLRW universe. This description, that we called
O(σ0) gradient expansion, relates the different local FLRW patches via non-local
terms and the momentum constraint, and describes at all orders in SR parameters the
correct dynamics of long wavelength scalar inhomogeneities. However, it presents
some problems such as the extraction of the O(σ0) information from the momentum
constraint. This is the reason why it is usually assumed that the different local FLRW universes
evolve independently from each other, which is an assumption known as the separate
universe approach. Under this approximation, the problem with the momentum
constraint disappears (the momentum constraint itself disappears); nevertheless,
we checked in Section 4.1.2 that this assumption fails to describe the correct long-
wavelength dynamics of scalar perturbations generically at O(ϵ1) already in its lin-
ear limit. For this reason, the stochastic formalism commonly used presented in
Section 6.1, which is based on the separate universe approach will fail to describe the
non-perturbative dynamics of the scalar inhomogeneities at O(ϵ1). p
y
g
(
)
A stochastic formalism that does not uses the separate universe approach can also
be constructed, as we did in Section 6.2. However, this option is not without its
difficulties, more concretely, it has problems when extracting the long wavelength
information from the momentum constraint and when describing the scalar inhomo-
geneities with a gauge invariant generalization of the MS variable. Universe 2022, 8, 334 36 of 39 2. On the other hand, the UV part of the inhomogeneities is assumed to behave pertur-
batively, having as a background the local FLRW background defined by the IR part. 7. Conclusions Since the UV part acts as a stochastic noise for the IR part and the IR part is necessary
to characterize the UV noises, the stochastic approach to inflation is generically a
non-Markovian process, meaning that the value of the noises depend on the whole
history of the local patch over which they are computed. Thi d
t
t
h
bl
h
l i
th
t
h
ti
ti 2. On the other hand, the UV part of the inhomogeneities is assumed to behave pertur-
batively, having as a background the local FLRW background defined by the IR part. Since the UV part acts as a stochastic noise for the IR part and the IR part is necessary
to characterize the UV noises, the stochastic approach to inflation is generically a
non-Markovian process, meaning that the value of the noises depend on the whole
history of the local patch over which they are computed. This does not represent a huge problem when solving the stochastic equations nu-
merically; however, in order to have analytical results we need to do some other
approximation that makes the process Markovian. This approximation consists of
computing the stochastic noises over the global background instead of over the local
one Unfortunately doing so is equivalent to assume that all the IR inhomogeneities history of the local patch over which they are computed. This does not represent a huge problem when solving the stochastic equations nu-
merically; however, in order to have analytical results we need to do some other
approximation that makes the process Markovian. This approximation consists of
computing the stochastic noises over the global background instead of over the local
one. Unfortunately, doing so is equivalent to assume that all the IR inhomogeneities
are linear in perturbation theory. This does not represent a huge problem when solving the stochastic equations nu-
merically; however, in order to have analytical results we need to do some other
approximation that makes the process Markovian. This approximation consists of
computing the stochastic noises over the global background instead of over the local
one. Unfortunately, doing so is equivalent to assume that all the IR inhomogeneities
are linear in perturbation theory. p
y
3. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 7. Conclusions Finally, and although we have not paid too much attention to this problem, any
stochastic approach that aims to describe the non-perturbative behaviour of scalar in-
homogeneities at the long wavelength limit should also take into account scalar-vector
and scalar-tensor interactions, which no longer decouple beyond linear perturba-
tion theory. 3. References 1. Starobinsky, A.A. A New Type of Isotropic Cosmological Models without Singularity. Phys. Lett. B 198 1. Starobinsky, A.A. A New Type of Isotropic Cosmological Models without Singularity. Phys. Lett. B 1980, 91, 99–102. [CrossRef]
2. Guth, A.H. The Inflationary Universe: A Possible Solution to the Horizon and Flatness Problems. Phys. Rev. D 1981, 23, 347–356. [CrossRef] 1. Starobinsky, A.A. A New Type of Isotropic Cosmological Models without Singularity. Phys. Lett. B 1980, 91, 99–102. [CrossRef]
2. Guth, A.H. The Inflationary Universe: A Possible Solution to the Horizon and Flatness Problems. Phys. Rev. D 1981, 23, 347–356. [CrossRef] y
yp
p
g
g
y
y
2. Guth, A.H. The Inflationary Universe: A Possible Solution to the Horizon and Flatness Problems. Phys. Rev. D 1981, 23, 347–356. [CrossRef] [
]
3. Aghanim, N.; Akrami, Y.; Ashdown, M.; Aumont, J.; Baccigalupi, C.; Ballardini, M.; Bay, A.J.; Barreiro, R.B.; Bartolo, N.; Basak, S.;
et al. [Planck], Planck 2018 results. VI. Cosmological parameters. Astron. Astrophys. 2020, 641, A6. Erratum in Astron. Astrophys. 2021, 652, C4. [CrossRef] 4. Akrami, Y.; Arroja, F.; Ashdown, M.; Aumont, J.; Baccigalupi, C.; Ballardini, M.; Banday, A.J.; Barreiro, R.B.; Bartolo, N.; Basak, N.;
et al. Planck 2018 results. X. Constraints on inflation. Astron. Astrophys. 2020, 641, A10. [CrossRef] 5. Carr, B.J.; Hawking, S.W. Black holes in the early Universe. Mon. Not. Roy. Astron. Soc. 1974, 168, 3 6. Blais, D.; Kiefer, C.; Polarski, D. Can primordial black holes be a significant part of dark matter? Phys. Lett. B 2002, 535, 11–16. [CrossRef] 7. Bean, R.; Magueijo, J. Could supermassive black holes be quintessential primordial black holes? Phys. Rev. D 2002, 66, 63505. [CrossRef] 8. Sasaki, M.; Suyama, T.; Tanaka, T.; Yokoyama, S. Primordial Black Hole Scenario for the Gravitational-Wave Event GW150914. Phys. Rev. Lett. 2016, 117, 61101. Erratum in Phys. Rev. Lett. 2018, 121, 59901. [CrossRef] 9. Germani, C.; Musco, I. Abundance of Primordial Black Holes Depends on the Shape of the Inflationary Power Spectrum. Phys. Rev. Lett. 2019, 122, 141302. [CrossRef] binsky, A.A.; Stochastic de Sitter (inflationary) stage in the early universe. Lect. Notes Phys. 1986, 246, 107–12
en, J.M.; Bublik, G.J. Quantum fluctuactions and inflation. Class. Quantum Grav. 1987, 4, 573. [CrossRef] arobinsky, A.A.; Stochastic de Sitter (inflationary) stage in the early universe. Lect. Notes Phys. Funding: This research received no external funding. Funding: This research received no external funding. Acknowledgments: I would like to thank Cristiano Germani for our weekly discussions and his
constant support and encouragement. I would also like to thank Misao Sasaki, Vincent Vennin and
Danilo Artigas for the many correspondences and help in understanding the gradient expansion
method and the stochastic formalism. I am supported by the Spanish MECD fellowship PRE2018-
086135. The group I belong to is also supported by the Unidad de Excelencia Maria de Maeztu Grants
No. MDM-2014-0369 and CEX2019-000918-M, and the Spanish national grants FPA2016-76005-C2-2-P,
PID2019-105614GB-C22 and PID2019-106515GB-I00. References Stochastic approach to inflation. 2. Classicality, coarse grain
1999, 59, 63512. [CrossRef] 28. Winitzki, S.; Vilenkin, A. Effective noise in stochastic description of inflation. Phys. Rev. D 2000, 61, 84008. [CrossRef] 29. Afshordi, N.; Brandenberger, R.H. Super Hubble nonlinear perturbations during inflation. Phys. Rev. D 2001, 63, 123505. [CrossRef] eshnizjani, G.; Afshordi, N. Coarse-grained back reaction in single scalar field driven inflation. J. Cosmol. Ast
11. [CrossRef] 31. Tsamis, N.C.; Woodard, R.P. Stochastic quantum gravitational inflation. Nucl. Phys. B 2005, 724, 295–328. [CrossRef]
32. Martin, J.; Musso, M. Solving stochastic inflation for arbitrary potentials. Phys. Rev. D 2006, 73, 43516. [CrossRef] 31. Tsamis, N.C.; Woodard, R.P. Stochastic quantum gravitational inflation. Nucl. Phys. B 2005, 724, 295–328. [CrossRef] Tsamis, N.C.; Woodard, R.P. Stochastic quantum gravitational inflation. Nucl. Phys. B 2005, 724, 295–328. M
ti
J M
M S l i
t
h
ti i flti
f
bit
t
ti l
Ph
R
D 2006 73 43516
[C 32. Martin, J.; Musso, M. Solving stochastic inflation for arbitrary potentials. Phys. Rev. D 2006, 73, 435 artin, J.; Musso, M. Solving stochastic inflation for arbitrary potentials. Phys. Rev. D 2006, 73, 43516. [Cross
b
h
fl
l
h
[
f] ;
,
g
y p
y
,
,
[
.E. Perturbations in stochastic inflation. J. Cosmol. Astropart. Phys. 2006, 7, 14. [CrossRef] J
g
y p
y
[
]
33. Kunze, K.E. Perturbations in stochastic inflation. J. Cosmol. Astropart. Phys. 2006, 7, 14. [CrossRef] p
y
34. Breuer, H.P.; Kunze, K.E. Stochastic inflation with coloured noise. AIP Conf. Proc. 2006, 841, 314. [C f
35. Finelli, F.; Marozzi, G.; Starobinsky, A.A.; Vacca, G.P.; Venturi, G. Generation of fluctuations during inflation: Comparison of
stochastic and field-theoretic approaches. Phys. Rev. D 2009, 79, 44007. [CrossRef] 36. Enqvist, K.; Nurmi, S.; Podolsky, D.; Rigopoulos, G.I. On the divergences of inflationary superhorizon perturbations. J. Cosmol. Astropart. Phys. 2008, 4, 25. [CrossRef] 37. Finelli, F.; Marozzi, G.; Starobinsky, A.A.; Vacca, G.P.; Venturi, G. Stochastic growth of quantum fluctuations during slow-roll
inflation. Phys. Rev. D 2010, 82, 64020. [CrossRef] 38. Clesse, S. Hybrid inflation along waterfall trajectories. Phys. Rev. D 2011, 83, 63518. [CrossRef] 8. Clesse, S. Hybrid inflation along waterfall trajectori 39. Martin, J.; Vennin, V. Stochastic Effects in Hybrid Inflation. Phys. Rev. D 2012, 85, 43525. [CrossRef] 40. Garbrecht, B.; Rigopoulos, G.; Zhu, Y. Infrared correlations in de Sitter space: Field theoretic versus stochastic approach. Phys. Rev. References 1986, 246, 107
ardeen J M ; Bublik G J Quantum fluctuactions and inflation Class Quantum Grav 1987 4 573
[CrossRef Starobinsky, A.A.; Stochastic de Sitter (inflationary) stage in the early universe. Lect. Notes Phys. 1986, 246, y,
;
(
y)
g
y
y
,
,
ardeen, J.M.; Bublik, G.J. Quantum fluctuactions and inflation. Class. Quantum Grav. 1987, 4, 573. [CrossRef 11. Bardeen, J.M.; Bublik, G.J. Quantum fluctuactions and inflation. Class. Quantum Grav. 1987, 4, 573. [C 11. Bardeen, J.M.; Bublik, G.J. Quantum fluctuactions and inflation. Class. Quantum Grav. 1987, 4, 573. [CrossRef]
12. Rey, S.J. Dynamics of Inflationary Phase Transition. Nucl. Phys. B 1987, 284, 706–728. [CrossRef] ey, S.J. Dynamics of Inflationary Phase Transition. Nucl. Phys. B 1987, 284, 706–728. [CrossRef] 12. Rey, S.J. Dynamics of Inflationary Phase Transition. Nucl. Phys. B 1987, 284, 706–728. [CrossRef] 13. Goncharov, A.S.; Linde, A.D.; Mukhanov, V.F. The Global Structure of the Inflationary Universe. Int. J. Mod. Phys. A 1987, 2
561–591. [CrossRef] ]
asaki, M. Stochastic Stage of an Inflationary Universe Model. Phys. Lett. B 1988, 205, 441–446. [CrossRef] Nambu, Y.; Sasaki, M. Stochastic Stage of an Inflationary Universe Model. Phys. Lett. B 1988, 205, 441–446 g
y
y
15. Nambu, Y.; Sasaki, M. Stochastic Approach to Chaotic Inflation and the Distribution of Universes. Phys. Lett. B 1989, 219, 240–246
[CrossRef] Y.; Sasaki, M. Stochastic Approach to Chaotic Inflation and the Distribution of Universes. Phys. Lett. B 1989, 21
f] 16. Kandrup, H.E. Stochastic inflation as a time dependent random walk. Phys. Rev. D 1989, 39, 2245. [CrossRef]
17. Nakao, K.i.; Nambu, Y.; Sasaki, M. Stochastic Dynamics of New Inflation. Prog. Theor. Phys. 1988, 88, 1041. [CrossRef] 17. Nakao, K.i.; Nambu, Y.; Sasaki, M. Stochastic Dynamics of New Inflation. Prog. Theor. Phys. 1988, 88, 1041. [CrossRef] 37 of 39 37 of 39 Universe 2022, 8, 334 18. Nambu, Y. Stochastic Dynamics of an Inflationary Model and Initial Distribution of Universes. Prog. Theor. Phys. 1989, 81, 1037. [CrossRef] [
]
19. Mollerach, S.; Matarrese, S.; Ortolan, A.; Lucchin, F. Stochastic inflation in a simple two field model. Phys. Rev. D 1991, 44,
1670–1679. [CrossRef] 20. Salopek, D.S.; Bond, J.R.; Nonlinear evolution of long wavelength metric fluctuations in inflationary models. Phys. Rev. D 1990,
42, 3936–3962. [CrossRef] [
]
21. Salopek, D.S.; Bond, J.R. Stochastic inflation and nonlinear gravity. Phys. Rev. D 1991, 43, 1005–1031. [CrossRef] [PubMed]
22. Habib, S. References Stochastic inflation: The Quantum phase space approach. Phys. Rev. D 1992, 46, 2408–2427. [CrossRef] [PubMed]
23. Linde, A.D.; Linde, D.A.; Mezhlumian, A. From the Big Bang theory to the theory of a stationary universe. Phys. Rev. D 1994, 49, 21. Salopek, D.S.; Bond, J.R. Stochastic inflation and nonlinear gravity. Phys. Rev. D 1991, 43, 1005–1031. [CrossRef] [PubMed]
22. Habib, S. Stochastic inflation: The Quantum phase space approach. Phys. Rev. D 1992, 46, 2408–2427. [CrossRef] [PubMed] g
y
22. Habib, S. Stochastic inflation: The Quantum phase space approach. Phys. Rev. D 1992, 46, 2408–2427. [CrossRef] [PubMed] 23. Linde, A.D.; Linde, D.A.; Mezhlumian, A. From the Big Bang theory to the theory of a stationary universe. Phys. Rev. D 1994, 49,
1783–1826. [CrossRef] [PubMed] 23. Linde, A.D.; Linde, D.A.; Mezhlumian, A. From the Big Bang theory to the theory of a stationary universe. Phys. Rev. D 1994, 49,
1783–1826. [CrossRef] [PubMed]
24. Starobinsky, A.A.; Yokoyama, J. Equilibrium state of a selfinteracting scalar field in the De Sitter background. Phys. Rev. D 1994, 1783 1826. [CrossRef] [PubMed]
24. Starobinsky, A.A.; Yokoyama, J. Equilibrium state of a selfinteracting scalar field in the De Sitter background. Phys. Rev. D 1994,
50, 6357–6368. [CrossRef] okoyama, J. Equilibrium state of a selfinteracting scalar field in the De Sitter background. Phys. Rev. D 1994
ssRef] 24. Starobinsky, A.A.; Yokoyama, J. Equilibrium state of a selfinteracting scalar field in the De Sitter ba
50, 6357–6368. [CrossRef] 25. Morikawa, M. Dissipation and fluctuation of quantum fields in expanding universes. Phys. Rev. D 1990, 42, 1027–1034. [CrossRef]
26. Hosoya, A.; Morikawa, M.; Nakayama, K. Stochastic dynamics of scalar field in the inflationary universe. Int. J. Mod. Phys. A
1989, 4, 2613–2625. [CrossRef] 25. Morikawa, M. Dissipation and fluctuation of quantum fields in expanding universes. Phys. Rev. D 1990, 42, 1027–1034. [CrossRef] 5. Morikawa, M. Dissipation and fluctuation of quant 25. Morikawa, M. Dissipation and fluctuation of quantum fields in expanding universes. Phys. Rev. D 1990, 42, 1027–1034. [CrossRef]
26
Hosoya A ; Morikawa M ; Nakayama K Stochastic dynamics of scalar field in the inflationary universe Int J Mod Phys A 26. Hosoya, A.; Morikawa, M.; Nakayama, K. Stochastic dynamics of scalar field in the inflationary universe. Int. J. Mod. Phys. A
1989, 4, 2613–2625. [CrossRef] .; Montemayor, R.; Sisterna, P. Stochastic approach to inflation. 2. Classicality, coarse graining and noises. P
63512. [CrossRef] 27. Casini, H.; Montemayor, R.; Sisterna, P. References D 2020, 102, 1 Firouzjahi, H.; Nassiri-Rad, A.; Noorbala, M. Stochastic nonattractor inflation. Phys. Rev. D 2020, 102, 1235
Ando, K.; Vennin, V. Power spectrum in stochastic inflation. J. Cosmol. Astropart. Phys. 2021, 4, 57. [CrossR 62. Ando, K.; Vennin, V. Power spectrum in stochastic inflation. J. Cosmol. Astropart. Phys. 2021, 4, 57. 63. Ballesteros, G.; Rey, J.; Taoso, M.; Urbano, A. Stochastic inflationary dynamics beyond slow-roll and consequences for primordial
black hole formation. J. Cosmol. Astropart. Phys. 2020, 8, 43. [CrossRef] p
y
64. Vennin, V. Stochastic inflation and primordial black holes. arXiv 2009, arXiv:2009.08715 . 65. Cruces, D.; Germani, C. Stochastic inflation at all order in slow-roll parameters: Foundations. Phys. Rev. D 2022, 105, 23533. [CrossRef] 66. Pattison, C.; Vennin, V.; Wands, D.; Assadullahi, H. Ultra-slow-roll inflation with quantum diffusion. J. Cosmol. Astropart. Phys. 2021, 2021, doi: 10.1088/1475-7516/2021/04/080 . [CrossRef] 67. Figueroa, D.G.; Raatikainen, S.; Rasanen, S.; Tomberg, E. Implications of stochastic effects for primordial black hole production in
ultra-slow-roll inflation. J. Cosmol. Astropart. Phys. 2021, 2021, 80. [CrossRef] p
y
De, A. Smooth coarse-graining and colored noise dynamics in stochastic inflation. arXiv 2022, arXiv:2204.03 68. Mahbub, R.; De, A. Smooth coarse-graining and colored noise dynamics in stochastic inflation. arXiv 2 g
g
y
69. Gihman, I.; Skorohod, A. Stochastic Differential Equations; Springer: Berlin/Heidelberg, Germany; New York, NY, USA, 1972. 70
S
bi
k
A A D
i
f Ph
T
i i
i
h N
I fl
i
U i
S
i
d G
i
f P
b
i
Ph 69. Gihman, I.; Skorohod, A. Stochastic Differential Equations; Springer: Berlin/Heidelberg, Germany; N korohod, A. Stochastic Differential Equations; Springer: Berlin/Heidelberg, Germany; New York, NY, USA, 1 69. Gihman, I.; Skorohod, A. Stochastic Differential Equations; Springer: Berlin/Heidelberg, Germany; New York, NY, USA, 1972. 70
St
bi
k
A A D
i
f Ph
T
iti
i
th N
I flti
U i
S
i
d G
ti
f P
t
b ti
Ph A.A. Dynamics of Phase Transition in the New Inflationary Universe Scenario and Generation of Perturbation
117, 175–178. [CrossRef] 70. Starobinsky, A.A. Dynamics of Phase Transition in the New Inflationary Universe Scenario and Gene
Lett. B 1982, 117, 175–178. [CrossRef] 71. Sasaki, M.; Stewart, E.D. A General analytic formula for the spectral index of the density perturbations produced during inflation. Prog. Theor. Phys. 1996, 95, 71–78. [CrossRef] 72. Lyth, D.H.; Rodriguez, Y. References D 2014, 89, 63506. [CrossRef] 41. Fujita, T.; Kawasaki, M.; Tada, Y.; Takesako, T. A new algorithm for calculating the curvature perturbations in stochastic inflation. J. Cosmol. Astropart. Phys. 2013, 12, 36. [CrossRef] 42. Levasseur, L.P. Lagrangian formulation of stochastic inflation: Langevin equations, one-loop correcti
approach. Phys. Rev. D 2013, 88, 83537. [CrossRef] 42. Levasseur, L.P. Lagrangian formulation of stochastic inflation: Langevin equations, one-loop corrections and a proposed recursive
approach. Phys. Rev. D 2013, 88, 83537. [CrossRef] pp
43. Levasseur, L.P.; Vennin, V.; Brandenberger, R. Recursive Stochastic Effects in Valley Hybrid Inflation. Phys. Rev. D 2013, 88, 83538. [CrossRef] Donough, E. Backreaction and Stochastic Effects in Single Field Inflation. Phys. Rev. D 2015, 91, 63513 44. Levasseur, L.P.; McDonough, E. Backreaction and Stochastic Effects in Single Field Inflation. P
[CrossRef] 45. Garbrecht, B.; Gautier, F.; Rigopoulos, G.; Zhu, Y. Feynman Diagrams for Stochastic Inflation and Quantum Field Theory in de
Sitter Space. Phys. Rev. D 2015, 91, 63520. [CrossRef] 46. Fujita, T.; Kawasaki, M.; Tada, Y. Non-perturbative approach for curvature perturbations in stochastic δN formalism. J. Cosmol. Astropart. Phys. 2014, 10, 30. [CrossRef] sky, A.A. Correlation Functions in Stochastic Inflation. Eur. Phys. J. C 2015, 75, 413. [CrossRef] 47. Vennin, V.; Starobinsky, A.A. Correlation Functions in Stochastic Inflation. Eur. Phys. J. C 2015, 75, 413. 47. Vennin, V.; Starobinsky, A.A. Correlation Functions in Stochastic Inflation. Eur. Phys. J. C 2015, 75, 413. [CrossRef]
48
Onemli VK Vacuum Fluctuations of a Scalar Field during Inflation: Quantum versus Stochastic Analysis Phys Rev D 2015 91 48. Onemli, V.K. Vacuum Fluctuations of a Scalar Field during Inflation: Quantum versus Stochastic Analy
103537. [CrossRef] 49. Burgess, C.P.; Holman, R.; Tasinato, G. Open EFTs, IR effects \& late-time resummations: systematic corrections in stochastic
inflation. J. High Energy Phys. 2016, 1, 153. [CrossRef] g
gy
y
50. Vennin, V.; Assadullahi, H.; Firouzjahi, H.; Noorbala, M.; Wands, D. Critical Number of Fields in Stochastic Inflation. Phys. Rev. Lett. 2017, 118, 31301. [CrossRef] 51. Assadullahi, H.; Firouzjahi, H.; Noorbala, M.; Vennin, V.; Wands, D. Multiple Fields in Stochastic Inflation. J. Cosmol. Astropart. Phys. 2016, 16, 43. [CrossRef] y
52. Pattison, C.; Vennin, V.; Assadullahi, H.; Wands, D. Quantum diffusion during inflation and primordial black holes. J. Cosmol. Astropart. Phys. 2017, 10, 46. [CrossRef] 38 of 39 Universe 2022, 8, 334 53. Firouzjahi, H.; Nassiri-Rad, A.; Noorbala, M. Stochastic Ultra Slow Roll Inflation. J. Cosmol. Astropart. Phys. y
Bardeen, J.M. Gauge Invariant Cosmological Perturbations. Phys. Rev. D 1980, 22, 1882–1905. [CrossRef] References 2019, 1, 40. [CrossRef]
54. Pattison, C.; Vennin, V.; Assadullahi, H.; Wands, D. The attractive behaviour of ultra-slow-roll inflation. J. Cosmol. Astropart. Phys. 2018, 8, 48. [CrossRef] 53. Firouzjahi, H.; Nassiri-Rad, A.; Noorbala, M. Stochastic Ultra Slow Roll Inflation. J. Cosmol. Astropart. Phys. 2019, 1, 40. [CrossRef]
54
Pattison C ; Vennin V; Assadullahi H ; Wands D The attractive behaviour of ultra-slow-roll inflation J Cosmol Astropart Phys 53. Firouzjahi, H.; Nassiri-Rad, A.; Noorbala, M. Stochastic Ultra Slow Roll Inflation. J. Cosmol. Astropart. Phys. 2019, 1, 40. [CrossRef] 54. Pattison, C.; Vennin, V.; Assadullahi, H.; Wands, D. The attractive behaviour of ultra-slow-roll inflation. J. Cosmol. Astropart. Phys. 2018, 8, 48. [CrossRef] [
]
55. Cruces, D.; Germani, C.; Prokopec, T. Failure of the stochastic approach to inflation beyond slow-roll. 2019, 3, 48. [CrossRef] [
]
56. Kitajima, N.; Tada, Y.; Takahashi, F. Stochastic inflation with an extremely large number of e-folds. Phys. Lett. B 2020, 800, 135097. [CrossRef] [
]
57. Prokopec, T.; Rigopoulos, G. ∆N and the stochastic conveyor belt of ultra slow-roll inflation. Phys. Rev. D 2021, 104, 83505. [CrossRef] [
]
58. Kuhnel, F.; Freese, K. On Stochastic Effects and Primordial Black-Hole Formation. Eur. Phys. J. C 2019, 79, 954. [CrossRef]
59
Ezquiaga J M ; García Bellido J ; Vennin V The exponential tail of inflationary fluctuations: consequences for primordial black reese, K. On Stochastic Effects and Primordial Black-Hole Formation. Eur. Phys. J. C 2019, 79, 954. [CrossRe [
]
58. Kuhnel, F.; Freese, K. On Stochastic Effects and Primordial Black-Hole Formation. Eur. Phys. J. C 2019, 59. Ezquiaga, J.M.; García-Bellido, J.; Vennin, V. The exponential tail of inflationary fluctuations: conseque
holes. J. Cosmol. Astropart. Phys. 2020, 3, 29. [CrossRef] 60. Pattison, C.; Vennin, V.; Assadullahi, H.; Wands, D. Stochastic inflation beyond slow roll. J. Cosmol. Astropart. Phys. 2019, 7, 31. [CrossRef] [
]
61. Firouzjahi, H.; Nassiri-Rad, A.; Noorbala, M. Stochastic nonattractor inflation. Phys. Rev. D 2020, 102, 123504. [CrossRef]
62. Ando, K.; Vennin, V. Power spectrum in stochastic inflation. J. Cosmol. Astropart. Phys. 2021, 4, 57. [CrossRef] 61. Firouzjahi, H.; Nassiri-Rad, A.; Noorbala, M. Stochastic nonattractor inflation. Phys. Rev. D 2020, 102, 123504. [CrossRef]
62
A d
K V
i
V P
t
i
t
h
ti i flti
J C
l A t
t Ph
2021 4 57 [C
R f] 61. Firouzjahi, H.; Nassiri-Rad, A.; Noorbala, M. Stochastic nonattractor inflation. Phys. Rev. References The Inflationary prediction for primordial non-Gaussianity. Phys. Rev. Lett. 2005, 95, 121302. [CrossRef]
[PubMed] 73. Sugiyama, N.S.; Komatsu, E.; Futamase, T. δN formalism. Phys. Rev. D 2013, 87, 23530. [CrossRef] 74. Boyanovsky, D.; de Vega, H.J.; Holman, R.; Lee, D.S.; Singh, A. Dissipation via particle production in scalar field theories. Phys. Rev. D 1995, 51, 4419–4444. [CrossRef] 75. Carr, B.; Kohri, K.; Sendouda, Y.; Yokoyama, J. Constraints on primordial black holes. Rept. Prog. Phys. 2021, 84, 116902. [CrossRef] [PubMed] 76. Atal, V.; Germani, C. The role of non-gaussianities in Primordial Black Hole formation. Phys. Dark Univ. 2019, 24, 100275. [CrossRef] [
]
77. Motohashi, H.; Hu, W. Primordial Black Holes and Slow-Roll Violation. Phys. Rev. D 2017, 96, 6350 78. Germani, C.; Sheth, R.K. Nonlinear statistics of primordial black holes from Gaussian curvature perturbations. Phys. Rev. D 2020,
101, 63520. [CrossRef] 79. Germani, C.; Prokopec, T. On primordial black holes from an inflection point. Phys. Dark Univ. 2017, 18, 6–10. [CrossRef]
80. Kinney, W.H. Horizon crossing and inflation with large eta. Phys. Rev. D 2005, 72, 23515. [CrossRef] 79. Germani, C.; Prokopec, T. On primordial black holes from an inflection point. Phys. Dark Univ. 2017, 1 ani, C.; Prokopec, T. On primordial black holes from an inflection point. Phys. Dark Univ. 2017, 18, 6–10. [C
W H H
i
i
d i flti
ith l
t
Ph
R
D 2005 72 23515 [C
R f] ani, C.; Prokopec, T. On primordial black holes from an inflection point. Phys. Dark Univ. 2017, 18, 6–10. [C
y, W.H. Horizon crossing and inflation with large eta. Phys. Rev. D 2005, 72, 23515. [CrossRef] 79. Germani, C.; Prokopec, T. On primordial black holes from an inflection point. Phys. Dark Univ. 2017, 18, 6 10. [CrossRef]
80. Kinney, W.H. Horizon crossing and inflation with large eta. Phys. Rev. D 2005, 72, 23515. [CrossRef] 80. Kinney, W.H. Horizon crossing and inflation with large eta. Phys. Rev. D 2005, 72, 23515. [CrossRef 81. Martin, J.; Motohashi, H.; Suyama, T. Ultra Slow-Roll Inflation and the non-Gaussianity Consistency Relation. Phys. Rev. D 2013
87, 23514. [CrossRef] 82. Arnowitt, R.L.; Deser, S.; Misner, C.W. The Dynamics of general relativity. Gen. Rel. Grav. 2008, 40, 199 82. Arnowitt, R.L.; Deser, S.; Misner, C.W. The Dynamics of general relativity. Gen. Rel. Grav. 2008, 40, 1997. [CrossRef]
83. Palenzuela, C. Introduction to Numerical Relativity. Front. Astron. Space Sci. 2020, 7, 58. [CrossRef] 82. References Arnowitt, R.L.; Deser, S.; Misner, C.W. The Dynamics of general relativity. Gen. Rel. Grav. 2008, 40, 199
83. Palenzuela, C. Introduction to Numerical Relativity. Front. Astron. Space Sci. 2020, 7, 58. [CrossRef] ,
;
,
;
,
y
g
y
,
,
[
]
83. Palenzuela, C. Introduction to Numerical Relativity. Front. Astron. Space Sci. 2020, 7, 58. [CrossRef] 84. Kodama, H.; Sasaki, M. Cosmological Perturbation Theory. Prog. Theor. Phys. Suppl. 1984, 78, 1–166. [C 85. Mukhanov, V.F.; Feldman, H.A.; Brandenberger, R.H. Theory of cosmological perturbations. Part 1. Classical perturbations. Part
2. Quantum theory of perturbations. Part 3. Extensions. Phys. Rept. 1992, 215, 203–333. [CrossRef] g
p
y
p
87. Durrer, R. Cosmological perturbation theory. Lect. Notes Phys. 2004, 653, 31–70. [CrossRef] 87. Durrer, R. Cosmological perturbation theory. Lect. Notes Phys. 2004, 653, 31–70. [CrossRef] 88. Riotto, A. Inflation and the theory of cosmological perturbations. ICTP Lect. Notes Ser. 2003, 14, 317–4 89. Bruni, M.; Matarrese, S.; Mollerach, S.; Sonego, S. Perturbations of space-time: Gauge transformations and gauge invariance at
second order and beyond. Class. Quant. Grav. 1997, 14, 2585–2606. [CrossRef] 39 of 39 39 of 39 Universe 2022, 8, 334 91. Bunch, T.S.; Davies, P.C.W. Quantum Field Theory in de Sitter Space: Renormalization by Point Splitting. Proc. Roy. Soc. Lond. A
1978, 360, 117. [CrossRef] 91. Bunch, T.S.; Davies, P.C.W. Quantum Field Theory in de Sitter Space: Renormalization by Point Splitting. Proc. Roy. Soc. Lond. A
1978, 360, 117. [CrossRef] 92. Polarski, D.; Starobinsky, A.A. Spectra of perturbations produced by double inflation with an intermediate matter dominated
stage. Nucl. Phys. B 1992, 385, 623–650. [CrossRef] 93. Wands, D.; Malik, K.A.; Lyth, D.H.; Liddle, A.R. A New approach to the evolution of cosmological perturbations on large scales. Phys. Rev. D 2000, 62, 43527. [CrossRef] y
94. Lyth, D.H.; Wands, D. Conserved cosmological perturbations. Phys. Rev. D 2003, 68, 103515. [CrossRe 95. Artigas, D.; Grain, J.; Vennin, V. Hamiltonian formalism for cosmological perturbations: the separate-universe approach. J. Cosmol. Astropart. Phys. 2022, 2, 1. [CrossRef] p
y
96. Kodama, H.; Hamazaki, T. Evolution of cosmological perturbations in the long wavelength limit. Phys. Rev. D 1998, 57, 7177–7185. [CrossRef] 97. Sasaki, M.; Tanaka, T. Superhorizon scale dynamics of multiscalar inflation. Prog. Theor. Phys. 1998, 99
98
Khl
M Y P i
di l Bl
k H l
R
A
A
h
1
10 495 528
[C
R f] 97. Sasaki, M.; Tanaka, T. References Gradient expansion approach to nonlinear superhorizon perturbations. Prog. Theor. Phys. 2007, 117,
633–654. [CrossRef]
109. Tanaka, Y.; Sasaki, M. Gradient expansion approach to nonlinear superhorizon perturbations. II. A Single scalar field. Prog. Theor. 633 654. [CrossRef]
109. Tanaka, Y.; Sasaki, M. Gradient expansion approach to nonlinear superhorizon perturbations. II. A Single scalar field. Prog. Theor. Phys. 2007, 118, 455–473. [CrossRef] 109. Tanaka, Y.; Sasaki, M. Gradient expansion approach to nonlinear superhorizon perturbations. II. A Sing
Phys. 2007, 118, 455–473. [CrossRef] [
]
, M.O. Killing vector fields and a homogeneous isotropic universe. Phys. Usp. 2016, 59, 689–700. [CrossRef] 110. Katanaev, M.O. Killing vector fields and a homogeneous isotropic universe. Phys. Usp. 2016, 59, 689–700. [CrossRef] 111. Rigopoulos, G.I.; Shellard, E.P.S. The separate universe approach and the evolution of nonlinear superhorizon cosmological
perturbations. Phys. Rev. D 2003, 68, 123518. [CrossRef] a, Y. Anisotropic separate universe and Weinberg’s adiabatic mode. J. Cosmol. Astropart. Phys. 2021, 7, 51 112. Tanaka, T.; Urakawa, Y. Anisotropic separate universe and Weinberg’s adiabatic mode. J. Cosmol
[CrossRef] 113. de Rham, C.; Gabadadze, G.; Tolley, A.J. Resummation of Massive Gravity. Phys. Rev. Lett. 2011, 106, 231101 [CrossRef] [PubMed]
114
Kodama H Arraut I Stability of the Schwarzschild de Sitter black hole in the dRGT massive gravity theory Prog Theor Exp 113. de Rham, C.; Gabadadze, G.; Tolley, A.J. Resummation of Massive Gravity. Phys. Rev. Lett. 2011, 106, 231 113. de Rham, C.; Gabadadze, G.; Tolley, A.J. Resummation of Massive Gravity. Phys. Rev. Lett. 2011, 106, 231101 [CrossRef] [PubMed]
114. Kodama, H.; Arraut, I. Stability of the Schwarzschild–de Sitter black hole in the dRGT massive gravity theory. Prog. Theor. Exp. Phys. 2014, 2014, 23E02. [CrossRef] y
y
y
114. Kodama, H.; Arraut, I. Stability of the Schwarzschild–de Sitter black hole in the dRGT massive gravity theory. Prog. Theor. Exp. Phys. 2014, 2014, 23E02. [CrossRef] ellido, J. Massive Primordial Black Holes from Hybrid Inflation as Dark Matter and the seeds of Galaxies
2, 23524. [CrossRef] y
115. Clesse, S.; García-Bellido, J. Massive Primordial Black Holes from Hybrid Inflation as Dark Matter a
Phys. Rev. D 2015, 92, 23524. [CrossRef] 115. Clesse, S.; García-Bellido, J. Massive Primordial Black Holes from Hybrid Inflation as Dark Matter and the seeds of Galaxies. Phys. Rev. D 2015, 92, 23524. [CrossRef]
116. Tada, Y.; Vennin, V. Statistics of coarse-grained cosmological fields in stochastic inflation. J. Cosmol. Astropart. Phys. 2022, 2, 21. Phys. Rev. References Superhorizon scale dynamics of multiscalar inflation. Prog. Theor. Phys. 1998, 99, 763–782. [CrossRef]
98. Khlopov, M.Y. Primordial Black Holes. Res. Astron. Astrophys. 2010, 10, 495–528. [CrossRef] p
y
98. Khlopov, M.Y. Primordial Black Holes. Res. Astron. Astrophys. 2010, 10, 495–528. [CrossRef] 98. Khlopov, M.Y. Primordial Black Holes. Res. Astron. Astrophys. 2010, 10, 495–528. [CrossRef] 99. Dolgov, A.D. Massive and supermassive black holes in the contemporary and early Universe and problems in cosmology and
astrophysics. Usp. Fiz. Nauk. 2018, 188, 121–142. [CrossRef] p y
p
100. Lyth, D.H.; Malik, K.A.; Sasaki, M. A General proof of the conservation of the curvature perturbation. 2005, 5, 4. [CrossRef] .A.; Sasaki, M. A General proof of the conservation of the curvature perturbation. J. Cosmol. Astropart. Phys
f] 101. Deruelle, N.; Langlois, D. Long wavelength iteration of Einstein’s equations near a space-time singularity. Phys. Rev. D 1995, 52),
2007–2019. [CrossRef] [
]
102. Iguchi, O.; Ishihara, H.; Soda, J. Inhomogeneity of spatial curvature for inflation. Phys. Rev. D 1997, 55, 3337–3345. [CrossRef]
103
Kh l t ik
I M K
h hik A Y M
t lli i M St
bi
k
A A Q
ii
t
i
l ti
f th Ei
t i
ti [
]
102. Iguchi, O.; Ishihara, H.; Soda, J. Inhomogeneity of spatial curvature for inflation. Phys. Rev. D 1997, 55 102. Iguchi, O.; Ishihara, H.; Soda, J. Inhomogeneity of spatial curvature for inflation. Phys. Rev. D 1997, 55, 3337–3345. [CrossRef]
103. Khalatnikov, I.M.; Kamenshchik, A.Y.; Martellini, M.; Starobinsky, A.A. Quasiisotropic solution of the Einstein equations near a
cosmological singularity for a two fluid cosmological model. J. Cosmol. Astropart. Phys. 2003, 3, 1. [CrossRef] 104. Parry, J.; Salopek, D.S.; Stewart, J.M. Solving the Hamilton-Jacobi equation for general relativity. Phys. Rev. D 1994, 49, 2872–2881. [CrossRef] [PubMed] 105. Soda, J.; Ishihara, H.; Iguchi, O. Hamilton-Jacobi equation for Brans-Dicke theory and its long wavelength solution. Prog. Theor. Phys. 1995, 94, 781–794. [CrossRef] 106. Nambu, Y.; Taruya, A. Application of gradient expansion to inflationary universe. Class. Quant. Grav. 1996, 13, 705–714. [CrossRef] 107. Leach, S.M.; Sasaki, M.; Wands, D.; Liddle, A.R. Enhancement of superhorizon scale inflationary curvature perturbations. Phys. Rev. D 2001, 64, 23512. [CrossRef] M. Gradient expansion approach to nonlinear superhorizon perturbations. Prog. Theor. Phys. 2007, 117 108. Tanaka, Y.; Sasaki, M. Gradient expansion approach to nonlinear superhorizon perturbations. Prog. Theor. Phys. 2007, 117,
633–654. [CrossRef] 108. Tanaka, Y.; Sasaki, M. References D 2015, 92, 23524. [CrossRef]
116. Tada, Y.; Vennin, V. Statistics of coarse-grained cosmological fields in stochastic inflation. J. Cosmol. Astropart. Phys. 2022, 2, 21. [C
R f] y
,
,
[
]
116. Tada, Y.; Vennin, V. Statistics of coarse-grained cosmological fields in stochastic inflation. J. Cosmol. Astropart. Phys. 2022, 2, 21. [CrossRef] 117. Langlois, D.; Vernizzi, F. Evolution of non-linear cosmological perturbations. Phys. Rev. Lett. 2005, 95, 91303. [CrossRef] 118. Rigopoulos, G.I.; Shellard, E.P.S. Non-linear inflationary perturbations. J. Cosmol. Astropart. Phys. 2005, 10, 6. [CrossRef]
119. Kiefer, C.; Polarski, D. Why do cosmological perturbations look classical to us? Adv. Sci. Lett. 2009, 2, 164–173. [CrossRef] G.I.; Shellard, E.P.S. Non-linear inflationary perturbations. J. Cosmol. Astropart. Phys. 2005, 10, 6. [CrossRef]
larski, D. Why do cosmological perturbations look classical to us? Adv. Sci. Lett. 2009, 2, 164–173. [CrossRe 119. Kiefer, C.; Polarski, D. Why do cosmological perturbations look classical to us? Adv. Sci. Lett. 2009, 2, 1 120. Grishchuk, L.P.; Sidorov, Y.V. Squeezed quantum states of relic gravitons and primordial density fluctu
42, 3413–3421. [CrossRef] 121. Kloeden, P.E.; Platen, E. Numerical Solution of Stochastic Differential Equations; Springer: Berlin/Heidelberg, Germany, 1992. [CrossRef] 122. Takamizu, Y.i.; Kobayashi, T. Nonlinear superhorizon curvature perturbation in generic single-field inflation. Prog. Theor. Exp. Phys. 2013, 2013, 063E03. [CrossRef] 123. Takamizu, Y.I.; Mukohyama, S.; Sasaki, M.; Tanaka, Y. Non-Gaussianity of superhorizon curvature perturbations beyond δ N
formalism. J. Cosmol. Astropart. Phys. 2010, 6, 19. [CrossRef] 124. Wang, J. Construction of the conserved ζ via the effective action for perfect fluids. Annals Phys. 2015, 362, 223–238. [CrossRef]
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English
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Action prediction modulates both neurophysiological and psychophysical indices of sensory attenuation
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Frontiers in human neuroscience
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ORIGINAL RESEARCH ARTICLE published: 28 February 2014
doi: 10.3389/fnhum.2014.00115 published: 28 February 2014
doi: 10.3389/fnhum.2014.00115 Frontiers in Human Neuroscience INTRODUCTION are perceived as less intense than externally generated stimuli, a
phenomenon known as sensory attenuation. Sensory attenuation
has been demonstrated in the somatosensory (Blakemore et al.,
1998), the auditory (Sato, 2008) and the visual domain (Cardoso-
Leite et al., 2010; Roussel et al., 2013). Cardoso-Leite et al. (2010),
for example, studied the influence of the congruency between
anticipated and actual action effect (tilted Gabor stimuli) on the
detection of the latter. Detection performance in the congruent
condition (i.e., when the effect anticipated by the action and the
true effect matched) was worse than in the neutral and incongru-
ent conditions, demonstrating sensory attenuation. Studies inves-
tigating sensory attenuation as a perceptual phenomenon have
been complemented by studies investigating neurophysiological
correlates of anticipated action effects (e.g., Schafer and Marcus,
1973; Blakemore et al., 1998; Bäss et al., 2008; Aliu et al., 2009;
Gentsch and Schütz-Bosbach, 2011; Hughes and Waszak, 2011;
Hughes et al., 2013b). To give an example, Bäss et al. (2008) found
a reduced auditory N1 component for action-triggered tones
compared to externally triggered tones, suggesting that cortical
activity was attenuated for the former. The ability to produce intended effects in the environment
is an important aspect of action control. The ideomotor the-
ory of action claims that bidirectional associations are formed
between an action’s motor code and the ensuing sensory effects. These associations can be used to select an action by anticipat-
ing or internally activating their perceptual consequences (e.g.,
Greenwald, 1970; Prinz, 1997; Elsner and Hommel, 2001; Herwig
et al., 2007; Waszak et al., 2012). A similar approach has also been
employed in the development of forward models of action, which
claim that an efference copy, generated during action selection,
is used to predict future behavioral state of the system as well
as the sensory consequences of that behavior (Wolpert et al.,
1995; Wolpert and Miall, 1996). Many computational models also
include inverse models that provide the motor command which,
given the particular current state, would result in a desired end
state, for example, a particular sensory effect (Wolpert et al.,
1995). These principles of action prediction have been investigated
using a number of different methodologies. One line of evidence
comes from paradigms studying processing of anticipated action
effects. Cedric Roussel 1,2*, Gethin Hughes 3 and Florian Waszak1,2 1 Laboratoire de Psychologie de la Perception, Université Paris Descartes, Sorbonne Paris Cité, Paris, France
2 Laboratoire Psychologie de la Perception, Centre National de la Recherche Scientifique-Université Paris Descarte
3 Department of Psychology, University of Essex, Colchester, UK Sensory attenuation refers to the observation that stimuli that are predicted based
on one’s action are attenuated. This phenomenon has primarily been observed as
a neurophysiological phenomenon, with reduced Event-Related Potential (ERP) (e.g.,
Bäss et al., 2008) and BOLD (e.g., Blakemore et al., 1998). However, psychophysical
investigations (e.g., Sato, 2008; Cardoso-Leite et al., 2010; Roussel et al., 2013) have
confirmed that action prediction also influences the perception of sensory action effects. The present study recorded both neurophysiological and psychophysical measures in a Sensory attenuation refers to the observation that stimuli that are predicted based
on one’s action are attenuated. This phenomenon has primarily been observed as
a neurophysiological phenomenon, with reduced Event-Related Potential (ERP) (e.g.,
Bäss et al., 2008) and BOLD (e.g., Blakemore et al., 1998). However, psychophysical
investigations (e.g., Sato, 2008; Cardoso-Leite et al., 2010; Roussel et al., 2013) have
confirmed that action prediction also influences the perception of sensory action effects. The present study recorded both neurophysiological and psychophysical measures in a
single experiment, to confirm whether the two phenomena are related. In addition, by
measuring the ERP modulations of both stimulus contrast and prediction congruency, we
sought to directly relate the neurophysiological phenomenon to the magnitude of sensory
processing in the brain. Participants performed left- and right-hand voluntary actions that
were previously associated with the letters A and H. In the test phase, participants were
presented with these same two letters, at one of two possible contrasts. Participants
were required to report which of the two possible contrasts had been presented. We
observed both reduced contrast discrimination (in line with Roussel et al., 2013) and
a reduced ERP response for congruent action-effects. Furthermore, our congruency
modulation was observed on the same component that differed as a function of stimulus
contrast. Taken together these results strongly suggest that neurophysiological indices of
sensory attenuation reflect reduced sensory processing of voluntary action effects. *Correspondence: *Correspondence:
Cedric Roussel, Laboratoire
Psychologie de la Perception, Centre
National de la Recherche
Scientifique-Université Paris
Descartes, UMR 8158, Centre
Biomédical des Saints-Pères, 45 rue
des Saints-Pères, 75006 Paris, France
e-mail: cedric_roussel@live.fr Keywords: action prediction, effect prediction, psychophysycs, neurophysiology, contrast discrimination Reviewed by: Reviewed by:
Amanda Therrien, Johns Hopkins
University School of Medicine, USA
Mikkel C. Vinding, Aarhus University,
Denmark Edited by: Harriet Brown, University College
London, UK HUMAN NEUROSCIENCE HUMAN NEUROSCIENCE Keywords: action prediction, effect prediction, psychophysycs, neurophysiology, contrast discrimination HUMAN NEUROSCIENCE HUMAN NEUROSCIENCE ORIGINAL RESEARCH ARTICLE
bli h d
F b INTRODUCTION Notably, it has been shown that self-generated stimuli Both attenuated phenomenological and neurophysiological
responses are usually interpreted along the same lines in terms February 2014 | Volume 8 | Article 115 | 1 www.frontiersin.org www.frontiersin.org Neurophysiological and psychophysical indices of sensory attenuation Roussel et al. of forward models (e.g., Miall and Wolpert, 1996), as if they
reflect the same mechanism. At the same time, this mechanism
has usually been considered to be relatively low-level (“sen-
sory”). However, a systematic investigation of the relationship
between sensory attenuation as a perceptual phenomenon, on
the one side, and as a neurophysiological phenomenon, on the
other side, is missing. As a consequence, it is impossible to
tell how the attenuation of perceptual awareness is related to
the attenuation of cortical responses that have been observed
a number of times in separate experiments. Moreover, con-
cerning the locus of the effect, differences in cortical responses
between conditions with and without effect anticipation cannot
always be unequivocally attributed to sensory processing. Often
they may also be caused by other differences in attentional and
cognitive processing (cf., Waszak et al., 2012; Hughes et al.,
2013a). As a consequence, different studies do not always con-
verge to the same conclusions. For example, Bäss et al. (2008)
observed attenuated fronto-central negativity when comparing
action-triggered vs. externally triggered auditory stimuli. They
concluded that early sensory processing in the auditory cortex
is reduced. By contrast, Hughes and Waszak (2011) compared
Event-Related Potential (ERPs) to action-triggered vs. exter-
nally triggered visual stimuli. They observed an increased, not
a decreased, visual P1 component. In this study, attenuated
cortical responses were observed in a frontoparietal network,
starting 150 ms after stimulus. This result would be in line
with the findings of Del Cul et al. (2007) showing that sub-
jective thresholds of visual stimuli is reflected in later process-
ing in a fronto-parietal network, rather than in early visual
areas. components and reduced awareness of the action effects
interrelate. MATERIALS AND METHODS
STIMULI The stimuli were two white letters (A and H) presented within
a virtual square of 3.3◦of visual angle and displayed on a 24 inch
LED monitor at a 60 Hz refresh. These two stimuli were presented
at two different contrast values (C0 and C1, determined for each
subject; see Section Contrast Determination Phase) at the center
of the screen. In the test phase we used a uniform noise texture to
increase perceptual variance. This noise texture was re-sampled
on each screen refresh with always the same number of white and
black pixels. The mean luminance of the noise was equal to the
gray background. CONTRAST DETERMINATION PHASE In order to determine individual contrast values C0 and C1 yield-
ing a discrimination d′ of about 1.5, every participant completed
a psychophysical staircase converging on 90% correct responses
in a letter identification task (A vs. H). This correct response
rate was used to ensure that the stimuli were supraliminal and
that we could then independently manipulate discrimination. We
used the resulting contrast value as the referential contrast C0 in a
Two Alternative Forced Choice (2AFC) paradigm (with constant
stimuli ranging from C0 to C0 + 12%) in order to calculate
the contrast value of C1 yielding 80% correct responses in a
luminance discrimination task (C0 vs. C1). For an ideal observer,
this contrast yields a discrimination d′ of around 1.5 (Macmillan
and Creelman, 1991). This phase lasted on average 5 min. The aim of the current experiment was to shed new light
on two interrelated questions. First, we investigated whether
neurophysiological indices of sensory attenuation reflect early,
low-level or later, higher-level mechanisms. Second, we explored
how neurophysiological and perceptual indices of sensory atten-
uation relate. To do so, using Electro Encephalography (EEG),
we adapted a luminance discrimination protocol that has been
used before successfully to assess perceptual sensitivity and
response bias of anticipated and unanticipated visual action
effects (Roussel et al., 2013). Roussel et al. made participants
learn an association between left and right key presses and the
presentation of the letters A and H, respectively. They then
made participants perform left and right key presses that ran-
domly triggered presentation of either an H or and A at one
of two possible contrasts. Participants were required to make
discrimination judgment between the two contrasts. They showed
contrast discrimination to be worse when the prediction (H or
A, as learned during the association phase of the experiment)
matches the true stimulus. Importantly, this paradigm does not
only manipulate whether an action effect is predicted or not, but
also the action effects’ physical energy (contrast, as we used visual
stimuli). It, thus, allows us to test whether or not prediction influ-
ences the same early components in the EEG as physical stimulus
energy. If this is the case, then the effect of prediction (sensory
attenuation) is likely an early, low-level phenomenon. Moreover,
assessing both psychophysical and neurophysiological measures
of sensory attenuation enables us to tell how neurophysiological Frontiers in Human Neuroscience TEST PHASE Participants fixated at a square at the center of the screen, just as
in the association phase. They were asked to produce, at random,
right and left key presses. Again, the key presses triggered presen-
tation of letter stimuli 200 ms after the key press. In this phase,
however, Hs and As were presented randomly after each key
press, such that 47.5% of the generated stimuli were congruent
with the previous association (i.e., the letter corresponded to
the one associated to that key press in the association phase),
and 47.5% were incongruent. On the remaining 5% of trials, no
stimulus was presented. The stimuli appeared randomly (but in
equal proportions) with the luminance C0 or the luminance C1. Participants were told that there were two categories of luminance
ranging from the value 0–49 for the C0 category and from 51
to 100 for the C1 category. In order to maintain this uncertainty
about the contrast on 5% of trials stimuli appeared with a random
contrast between C0 – 15% contrast and C1 + 15% contrast. After
the stimulus had disappeared participants were required to judge
the luminance value of the stimulus on a luminance response bar. On this bar participants could place the cursor on the perceived
contrast value with values of 49 and under corresponding to C0
and 51 and over corresponding to C1. Participants completed 3
tests blocks of 44 trials (on average 25 min) before running a re-
association phase composed of one of each type of association
block (10 min on average). Thereafter, they ran another tests
blocks. In total participants responded to 264 test trials. The
experiment lasted on average 1.30 h. Participants could take short
breaks prior to each of the association phases. An ANOVA with the factors congruency (congruent, incon-
gruent) and stimulus contrast (C0, C1) was run on ERPs aver-
aged for each participant. Since our task involved visual stimuli,
we focused our analysis on a region of interest at the occip-
ital electrodes (O1 Oz O2). Since our stimuli were degraded
and presented in a continuous stream of background visual
noise, we postulated that this might influence the latency of
the visual response. Therefore we inspected the ERPs over our
region of interest to determine the time window corresponding
to an apparent peak for the visual stimulus. TEST PHASE The time window
for analysis was selected around the peak of this visual com-
ponent. Importantly, this time-window was not selected based
on the difference between our conditions but rather on the
presence of the component itself. Analysis of the modulation
of this component by contrast, would then provide further jus-
tification for the time-window, since stimulus contrast should
influence the magnitude of the visual response. It is impor-
tant to note that the main comparison of interest—the congru-
ency effect—was orthogonal to the contrast effect, and therefore
the selection of the time-window would not unduly bias this
comparison. EEG RECORDING AND DATA PREPROCESSING have to generate an action given a desired outcome, the action-
effect relationship will be encoded particularly strongly. EEG was recorded with 64 electrodes (actiCAP, Brain Prod-
uctsGmbH, Germany). The EEG was digitized at 500 Hz. EEG
analysis was conducted using EEGLAB (Delorme and Makeig,
2004) and custom-built Matlab scripts. The data were resampled
offline to a 250 Hz sample rate, low band-pass filtered at 45 Hz
to remove line noise. Epochs were generated from −500 to
980 ms relative to stimulus onset, with a 200 ms prestimulus
baseline correction. Initial artifact rejection was conducted in a
semiautomatic manner (in EEGLAB) by rejecting epochs with
activity above 100 µV or below −100 µV, as well as rejecting
trials where activity at any time point for any electrode was
more than 5 standard deviations from the mean activity for
that epoch. Any channels that contributed to the rejection of
many epochs were considered for removal and later interpola-
tion. Frontal channels that showed large amplitude blink activity
were also excluded from further analysis during this this first
semiautomatic artifact rejection. Ocular artifact correction was
conducted in EEGLAB in Matlab using independent component
analysis (Delorme and Makeig, 2004). Following removal of eye
blinks and eye movements, noisy channels were replaced by
an interpolated weighted average from surrounding electrodes. Data were then re-referenced to the common average. A final
round of semiautomatic artifact rejection with a threshold of +/−
80 µV was used to remove any remaining artifacts. All ERPs are
presented with a low-pass filter of 20 Hz for visual presentation
purposes. The association phase consisted of three FreeAsso blocks and
two MemoryAsso blocks. Each FreeAsso block contained 50 trials. Each MemoryAsso block contained 30 sequences of, on average, 5
items. Each Participant ran three FreeAsso and two MemoryAsso
blocks. This phase took on average 25 min. ANALYSIS OF DISCRIMINATION PERFORMANCE The luminance discrimination task was considered to be a yes/no
protocol, with C1 being the target. That is, a C1 response to a
C1 stimulus is a hit, a C1 response to a C0 stimulus is a false
alarm, etc. According to Signal Detection Theory (Green and
Swets, 1966), d′ (perceptual sensitivity) and c (response bias) are
calculated using d′ = z(hit rate) −z(false alarm rate) and c =
−0.5 × [z(hit rate) + z(false alarm rate)])1. Since participants
provided their judgments of contrast using a continuous scale
from 1 to 100 this allowed us to analyze not only their overall
contrast judgment (C0 or C1) but also their rating of the per-
ceived intensity (contrast) of the stimulus. We split the ratings
into 10 bins to compute ROC (receiver operating characteristic)
curves for each participant. The area under the curve, A′ was
calculated such that A′ = 1/2 ∗6(Fi+1 −Fi)(Hi+1 + Hi), with
F and H being False alarms and Hits respectively (Macmillan
and Creelman, 1991). D′, c and A were calculated separately for
congruent and incongruent trials. ASSOCIATION PHASE Participants fixated on a 3.34◦visual angle square located at the
center of the screen. They were asked to press with their right/left
index finger one of two keys (right and left on a response pad),
each key press triggering presentation of a visual effect (A or H). The key-letter mapping was counterbalanced across participants. The letters appeared 200 ms after the key press at full contrast in
the square at the center of the screen. There were two types of association phase. First, in the
free association blocks (“FreeAsso”) a sequence of 50 actions
(left/right) was freely generated by the participants at a pace of
about 1 key press per second. The experimenter demonstrated the
pace to the participants before the experiment. We also measured
the time participants needed for each block to control their pace. In 5% of the trials the visual effect was a W. In these catch trials,
the participant had to press both buttons within 1 s of the appear-
ance of the stimulus. Catch trials were meant to ensure that par-
ticipants paid attention to the effect stimuli. Second, in memory
association blocks (“MemoryAsso”), random lists of As and Hs
were presented to the participants (the average list size was 5 going
from 3 to 8 items adapted to the participants performance with a
simple 1up 1down rule). The lists were presented via headphones
as spoken letters. After the lists were presented, participants had
to reproduce the sequence by pressing the corresponding button
sequence. This phase was meant to foster the learning of the
action-effect associations. We reasoned that when participants Frontiers in Human Neuroscience February 2014 | Volume 8 | Article 115 | 2 www.frontiersin.org www.frontiersin.org www.frontiersin.org Neurophysiological and psychophysical indices of sensory attenuation Roussel et al. Frontiers in Human Neuroscience Neurophysiological and psychophysical indices of sensory attenuation Neurophysiological and psychophysical indices of sensory attenuation Roussel et al. FIGURE 1 | ROC curves. Mean of participants ROC curves for congruent
and incongruent trials. For calculation detail of the ROC see Analysis of
Discrimination Performance section. FIGURE 2 | Contrast effect: ERP and Topography. This figure presents
the mean ERPs on O1 Oz O2 for C0 (in blue) and C1 (in red) from −500 to
980 ms relative to stimulus apparition. The blued surface represents the
analysis time window (from 180 to 320 ms, centered on the pic around 250
ms). In the top left corner the topography of the difference (C1 – C0) is
presented for the analysis time window. * p < 0.05. FIGURE 1 | ROC curves. Mean of participants ROC curves for congruent
and incongruent trials. For calculation detail of the ROC see Analysis of
Discrimination Performance section. FIGURE 2 | Contrast effect: ERP and Topography. This figure presents
the mean ERPs on O1 Oz O2 for C0 (in blue) and C1 (in red) from −500 to
980 ms relative to stimulus apparition. The blued surface represents the
analysis time window (from 180 to 320 ms, centered on the pic around 250
ms). In the top left corner the topography of the difference (C1 – C0) is
presented for the analysis time window. * p < 0.05. FIGURE 2 | Contrast effect: ERP and Topography. This figure presents
the mean ERPs on O1 Oz O2 for C0 (in blue) and C1 (in red) from −500 to
980 ms relative to stimulus apparition. The blued surface represents the
analysis time window (from 180 to 320 ms, centered on the pic around 250
ms). In the top left corner the topography of the difference (C1 – C0) is
presented for the analysis time window. * p < 0.05. FIGURE 1 | ROC curves. Mean of participants ROC curves for congruent
and incongruent trials. For calculation detail of the ROC see Analysis of
Discrimination Performance section. participants had action-effect mapping 1 (left →A, right →H),
and 8 had mapping 2 (8 women, 7 men; mean age = 24 years,
SEM = 3.69 years). stimulus onset. This peak appears to be greater for C1 than
C0, such that it likely reflects processing of the visual stimulus
(a delayed visual N1 component) (Ciesielski and French, 1989;
Mangun and Hillyard, 1991; Johannes et al., 1995). RESULTS In order to ensure that the data were equivalent between the EEG
and the behavioral analysis only trials free from EEG artifacts were
analyzed. The amount of rejected data was less than 10% of the
total number of trials. PSYCHOPHYSICAL RESULTS We analyzed our data dependent on participants’ contrast dis-
crimination. Discrimination performance (d′) was lower in the
congruent condition (d′ congruent: 1.22, SD = 0.46) than in
the incongruent (d′ incongruent: 1.42, SD = 0.45) condition. A
one factor repeated measures analysis of variance including the
factor of congruency showed this effect of congruency on d′ to be
significant (F(1,14) = 5.36, p = 0.03). At the same time, the criterion
(c) was not different in the two congruency conditions (1 factor
repeated measures ANOVA; F(1,14) = 0.165, p = 0.69). A bias free
measure of sensitivity (A′) also confirms our finding. Sensitivity
appears to be better for incongruent trials (A′: 0.81, SD = 0.075)
than for congruent trials (A′: 0.79, SD = 0.067) (F(1,14) = 4.670, p
= 0.04) (see Figure 1). In contrast the criterion (bias) appeared
not to differ between conditions (Congruent: 0.39, SD = 0.46;
Incongruent: 0.41, SD = 0.54; F(1,14) = 0.165, p = 0.691). In line with the aim of the current experiment, it appears that
this visual ERP peaks as a function of congruent motor prediction. Since we also observed attenuated sensitivity for congruent trials
in the behavioral analysis presented above, this provides evidence
that neural and behavioral measures of sensory attenuation are
likely related. Neurophysiological and psychophysical indices of sensory attenuation To quantify
these effects we took the average amplitude of each condition
in a 140 ms time window centered on this peak (180–320 ms). A repeated measure analysis of variance including the factor
of contrast and congruency revealed a significant main effect
of Contrast (F(1,14) = 6.54, p = 0.023), confirming significantly
greater amplitude for C1 (mean = −1.67; std = 1.55, CI: +/−
1.21) compared to C0 (mean = −0.81; std = 1.8, CI: +/−1.63). The topography of this difference is consistent with modulation
of an occipital ERP component. We also observed a significant
main effect of Congruency (F(1,14) = 11.36, p = 0.005), such that
our visual component was of significantly smaller amplitude in
the congruent condition (mean = −0.99; std = 1.57, CI: +/−
1.24), compared to the incongruent condition (mean = −1.50;
std = 1.64, CI: +/−1.36). The topography of this difference is also
consistent with a modulation of visual processing as a function of
action prediction. However the interaction between both factors
was not significant (F(1,14) = 0.02, p = 0.885). EEG RESULTS In this section we focus on the effect of motor prediction on the
neurophysiological indices of visual processing to determine the
degree to which behavioral and neural sensory attenuation are
related. The ERPs and the topographies for the different condi-
tions are presented in Figures 2, 3. A large negative deflection is
apparent in all the waveforms, peaking at around 250 ms after PARTICIPANTS Nineteen participants took part in the experiment. They were
naive to the purpose of the experiment. Four of these nineteen
participants were excluded from the analysis as their luminance
discrimination d′s were below 0.5 (for 2 of them) or because the
ratio of right left key presses during the test phases exceeded a
75% 25% ratio (for one of them). One was rejected because of
the poor quality of the EEG recordings. Seven of the remaining Frontiers in Human Neuroscience February 2014 | Volume 8 | Article 115 | 3 www.frontiersin.org www.frontiersin.org www.frontiersin.org Frontiers in Human Neuroscience DISCUSSION FIGURE 3 | Congruency effect: ERP and Topography. This figure presents
the mean ERPs on O1 Oz O2 for congruent (in blue) and incongruent (in
red) from −500 to 980 ms relative to stimulus apparition. The blued surface
represents the analysis time window (from 180 to 320 ms, centered on the
pic around 250 ms). In the top left corner the topography of the difference
(Incongruent—Congruent) is presented for the analysis time window. * p <
0.05. be observed in the visual domain. More importantly, one of
the aims of the experiment presented above was to investigate
the locus of sensory attenuation. This was done by way of
comparing the influence of prediction and stimulus contrast,
respectively, on the ERPs triggered by visual action effects. We
observed that an N1 component was clearly modulated by stimu-
lus contrast, with larger contrasts resulting in a larger deflection. Importantly, congruency affected the very same component, with
congruent trials resulting in smaller amplitudes than incongruent
trials. As concerns the effect of congruency on ERPs, we observed
that the contrast-sensitive visual component was significantly
smaller in the congruent condition compared to the incongruent
condition. We, thus, observed, to our knowledge for the first
time, sensory attenuation in psychophysical and neurophysiolog-
ical indices at the same time, suggesting that the two measures
of sensory attenuation are likely related. However, note that
the psychophysical effect corresponds to an interaction between
contrast and congruency: the discrimination between the two
contrast levels is more difficult in congruent than in incon-
gruent trials. If sensory attenuation assessed with psychophys-
ical methods were a direct reflection of the ERPs assessed at
the same time, we would have expected to see an interaction
between these two factors in our ERP data as well. However,
this was not the case. Of course, it might be that the ERP
data simply lack sufficient statistical power. However, it is also
possible that psychophysical and ERP indices of sensory atten-
uation (at least those assessed in the current experiment) are
not in a simple one-to-one relationship. Perception might be
dependent not only on early cortical responses, but also on later
processing and/or recurrent processing, tweaking the relationship
between perceptual measures and observable neurophysiological
measures. Of course, our data cannot show which processing stage
precisely the two factors manipulated in the current experiment
influence. DISCUSSION First of all, our experiment shows that neurophysiological sen-
sory attenuation is a phenomenon that is not restricted to
the auditory and somatosensory modality, but that it can also February 2014 | Volume 8 | Article 115 | 4 www.frontiersin.org www.frontiersin.org Neurophysiological and psychophysical indices of sensory attenuation Roussel et al. FIGURE 3 | Congruency effect: ERP and Topography. This figure presents
the mean ERPs on O1 Oz O2 for congruent (in blue) and incongruent (in
red) from −500 to 980 ms relative to stimulus apparition. The blued surface
represents the analysis time window (from 180 to 320 ms, centered on the
pic around 250 ms). In the top left corner the topography of the difference
(Incongruent—Congruent) is presented for the analysis time window. * p <
0.05. the fact that the stimuli used in the current experiment were
not presented with a sharp onset, but embedded in dynamic
pixel noise, such that the detection of a pattern is more time-
consuming. This evidence of reafferent attenuation in the visual modality
is also important regarding to the literature of saccadic sup-
pression (Bridgeman et al., 1975; Deubel et al., 1996) thought
to rely on a “corollary discharge” from the motor command
affecting the perceptual network (Sperry, 1950; Paus et al., 1995). Nonetheless the link between sensory attenuation and saccadic
suppression must be more thoroughly investigated since some
essential differences separate both phenomena, notably the timing
of the effect. For example sensory attenuation has been shown
to occur on the final consequence of the action (Blakemore
et al., 1998) while saccadic suppression has been shown to
occur during the saccade (Bridgeman et al., 1975; Deubel et al.,
1996). The second aim of the present study was to explore how neuro-
physiological and perceptual indices of sensory attenuation relate. We used a luminance discrimination protocol to assess perceptual
sensitivity and response bias of anticipated and unanticipated
visual action effects, assessing EEG activity at the same time. The
psychophysical results show that discrimination performance (d′,
A′) was better in the incongruent condition than in the congruent
condition. At the same time, the criterion was not different in
the two congruency conditions. The results, thus, are in line with
the findings of Roussel et al. (2013) and Cardoso-Leite et al. (2010). They show that contrast sensitivity is reduced when a
motor act provides an accurate prediction of the ensuing visual
stimulus. Frontiers in Human Neuroscience www.frontiersin.org REFERENCES Aliu, S. O., Houde, J. F., and Nagarajan, S. S. (2009). Motor-induced suppression
of the auditory cortex. J. Cogn. Neurosci. 21, 791–802. doi: 10.1162/jocn.2009. 21055 Miall, R. C., and Wolpert, D. M. (1996). Forward models for physiological
motor control. Neural Netw. 9, 1265–1279. doi: 10.1016/S0893-6080(96)0
0035-4 Bach, M., and Ullrich, D. (1997). Contrast dependency of motion-onset and
pattern-reversal VEPs: interaction of stimulus type, recording site and response
component. Vision Res. 37, 1845–1849. doi: 10.1016/s0042-6989(96)00317-3 Paus, T., Marrett, S., Worsley, K. J., and Evans, A. C. (1995). Extra-retinal modula-
tion of cerebral blood flow in the human visual cortex: implications for saccadic
suppression. J. Neurophysiol. 74, 2179–2183. Bäss, P., Jacobsen, T., and Schroger, E. (2008). Suppression of the auditory N1
event-related potential component with unpredictable self-initiated tones: evi-
dence for internal forward models with dynamic stimulation. Int. J. Psychophys-
iol. 70, 137–143. doi: 10.1016/j.ijpsycho.2008.06.005 Prinz, W. (1997). Perception and action planning. Eur. J. Cogn. Psychol. 9, 129–154. doi: 10.1080/713752551 Roussel, C., Hughes, G., and Waszak, F. (2013). A preactivation account of sensory
attenuation. Neuropsychologia 51, 922–929. doi: 10.1016/j.neuropsychologia. 2013.02.005 Blakemore, S. J., Wolpert, D. M., and Frith, C. D. (1998). Central cancellation of
self-produced tickle sensation. Nat. Neurosci. 1, 635–640. doi: 10.1038/2870 Sato, A. (2008). Action observation modulates auditory perception of the con-
sequence of others’ actions. Conscious. Cogn. 17, 1219–1227. doi: 10.1016/j. concog.2008.01.003 Bridgeman, B., Hendry, D., and Stark, L. (1975). Failure to detect displacement
of the visual world during saccadic eye movements. Vision Res. 15, 719–722. doi: 10.1016/0042-6989(75)90290-4 Schadow, J., Lenz, D., Thaerig, S., Busch, N. A., Frund, I., Rieger, J. W., et al. (2007). Stimulus intensity affects early sensory processing: visual contrast modulates
evoked gamma-band activity in human EEG. Int. J. Psychophysiol. 66, 28–36. doi: 10.1016/j.ijpsycho.2007.05.010 Cardoso-Leite, P., Mamassian, P., Schutz-Bosbach, S., and Waszak, F. (2010). A new
look at sensory attenuation. Action-effect anticipation affects sensitivity, not
response bias. Psychol. Sci. 21, 1740–1745. doi: 10.1177/0956797610389187 Ciesielski, K. T., and French, C. N. (1989). Event-related potentials before and after
training: chronometry and lateralization of visual N1 and N2. Biol. Psychol. 28,
227–238. doi: 10.1016/0301-0511(89)90002-1 Schafer, E. W., and Marcus, M. M. (1973). Self-stimulation alters human sensory
brain responses. Science 181, 175–177. doi: 10.1126/science.181.4095.175 Sperry, R. W. (1950). Neural basis of the spontaneous optokinetic response
produced by visual invasion. J. Comp. Physiol. Psychol. 43, 482–489. doi: 10. 1037/h0055479 Del Cul, A., Baillet, S., and Dehaene, S. (2007). ACKNOWLEDGMENTS Johannes, S., Munte, T. F., Heinze, H. J., and Mangun, G. R. (1995). Luminance and
spatial attention effects on early visual processing. Brain Res. Cogn. Brain Res. 2,
189–205. doi: 10.1016/0926-6410(95)90008-x The research leading to these results has received funding from the
European Research Council under the European Union’s Seventh
Framework Programme (FP7/2007-2013)/ERC grant agreement
no. 263067. The research leading to these results has received funding from the
European Research Council under the European Union’s Seventh The research leading to these results has received funding from the
European Research Council under the European Union’s Seventh
Framework Programme (FP7/2007-2013)/ERC grant agreement
no. 263067. Macmillan, N. A., and Creelman, C. D. (1991). Detection Theory: A User’s Guide. Mahwah, New Jersey: Lawrence Erlbaum Associates, Inc. Mangun, G. R., and Hillyard, S. A. (1991). Modulations of sensory-evoked brain
potentials indicate changes in perceptual processing during visual-spatial prim-
ing. J. Exp. Psychol. Hum. Percept. Perform. 17, 1057–1074. doi: 10.1037/0096-
1523.17.4.1057 DISCUSSION The current paradigm differs in important aspects
from other studies investigating visual evoked potentials. Notably,
we presented stimuli in visual noise resampled at each screen
refresh. The actions’ effects were, therefore, not presented with
a sharp onset. Moreover, in our experiment, stimuli were trig-
gered by an action. It is therefore difficult to compare our
results to ERPs found in previous studies. However, previous
research seems to suggest that contrast-dependent processes take
place rather early in the visual processing stream (e.g., Schadow
et al., 2007), while later components are rather modulated by
motion and form perception (Bach and Ullrich, 1997; Göpfert
et al., 1998). Importantly, our experiment allowed us to directly
compare the effect of contrast and prediction. As it demon-
strates that motor prediction influences the same processing
stage as visual contrast, we assume that motor prediction as
manipulated in our experiment influences an early processing
stage that is otherwise still modulated by basic stimulus-features. This interpretation is corroborated by the fact that the N1
component in question has an occipital topography. That this
component has a relatively late latency is probably due to In conclusion, in our experiment ERP effects of visual sensory
attenuation were found to correspond to contrast-dependent
processing stages. We conclude that motor prediction, thus, influ-
ences quite early processes. Moreover, we demonstrated that both February 2014 | Volume 8 | Article 115 | 5 www.frontiersin.org www.frontiersin.org www.frontiersin.org Neurophysiological and psychophysical indices of sensory attenuation Roussel et al. psychophysical and ERP indices of sensory attenuation can be
observed in the visual modality. However, the exact relationship
between the two types of measure needs to be further clarified, as
there are not only commonalities, but also differences. Hughes, G., Desantis, A., and Waszak, F. (2013b). Attenuation of auditory N1
results from identity-specific action-effect prediction. Eur. J. Neurosci. 37, 1152–
1158. doi: 10.1111/ejn.12120 Hughes, G., and Waszak, F. (2011). ERP correlates of action effect prediction
and visual sensory attenuation in voluntary action. Neuroimage 56, 1632–1640. doi: 10.1016/j.neuroimage.2011.02.057 REFERENCES Brain dynamics underlying the
nonlinear threshold for access to consciousness. PLoS Biol. 5:e260. doi: 10. 1371/journal.pbio.0050260 Waszak, F., Cardoso-Leite, P., and Hughes, G. (2012). Action effect anticipation:
neurophysiological basis and functional consequences. Neurosci. Biobehav. Rev. 36, 943–959. doi: 10.1016/j.neubiorev.2011.11.004 Delorme, A., and Makeig, S. (2004). EEGLAB: an open source toolbox for analysis
of single-trial EEG dynamics including independent component analysis. J. Neurosci. Methods 134, 9–21. doi: 10.1016/j.jneumeth.2003.10.009 Wolpert, D. M., Ghahramani, Z., and Jordan, M. I. (1995). An internal model
for sensorimotor integration. Science 269, 1880–1882. doi: 10.1126/science.756
9931 Deubel, H., Schneider, W. X., and Bridgeman, B. (1996). Postsaccadic target
blanking prevents saccadic suppression of image displacement. Vision Res. 36,
985–996. doi: 10.1016/0042-6989(95)00203-0 Wolpert, D. M., and Miall, R. C. (1996). Forward models for physiological
motor control. Neural Netw. 9, 1265–1279. doi: 10.1016/s0893-6080(96)00
035-4 Elsner, B., and Hommel, B. (2001). Effect anticipation and action control. J. Exp. Psychol. Hum. Percept. Perform. 27, 229–240. doi: 10.1037/0096-1523.27.1.229 Gentsch, A., and Schütz-Bosbach, S. (2011). I did it: unconscious expectation of
sensory consequences modulates the experience of self-agency and its functional
signature. J. Cogn. Neurosci. 23, 3817–3828. doi: 10.1162/jocn_a_00012 Gentsch, A., and Schütz-Bosbach, S. (2011). I did it: unconscious expectation of
sensory consequences modulates the experience of self-agency and its functional sensory consequences modulates the experience of self-agency and its functional
signature. J. Cogn. Neurosci. 23, 3817–3828. doi: 10.1162/jocn_a_00012 Conflict of Interest Statement: The authors declare that the research was con-
ducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest. Conflict of Interest Statement: The authors declare that the research was con-
ducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest. signature. J. Cogn. Neurosci. 23, 3817–3828. doi: 10.1162/jocn_a_00012 Göpfert, E., Müller, R., Breuer, D., and Greenlee, M. W. (1998). Similarities and
dissimilarities between pattern VEPs and motion VEPs. Doc. Ophthalmol. 97,
67–79. doi: 10.1023/A:1001888618774 Received: 05 November 2013; accepted: 17 February 2014; published online: 28
February 2014. Green, D. M., and Swets, J. A. (1996). Signal Detection Theory and Psychophysics. New York: Wiley. Citation: Roussel C, Hughes G and Waszak F (2014) Action prediction modulates both
neurophysiological and psychophysical indices of sensory attenuation. Front. Hum. Greenwald, A. G. (1970). Sensory feedback mechanisms in performance control:
with special reference to the ideo-motor mechanism. Psychol. Rev. 77, 73–99. doi: 10.1037/h0028689 Neurosci. 8:115. Received: 05 November 2013; accepted: 17 February 2014; published online: 28
February 2014. REFERENCES doi: 10.3389/fnhum.2014.00115 f
This article was submitted to the journal Frontiers in Human Neuroscience. This article was submitted to the journal Frontiers in Human Neuroscience. This article was submitted to the journal Frontiers Herwig, A., Prinz, W., and Waszak, F. (2007). Two modes of sensorimotor integra-
tion in intention-based and stimulus-based actions. Q. J. Exp. Psychol. (Hove)
60, 1540–1554. doi: 10.1080/17470210601119134 Hughes, G., Desantis, A., and Waszak, F. (2013a). Mechanisms of intentional
binding and sensory attenuation: the role of temporal prediction, temporal
control, identity prediction and motor prediction. Psychol. Bull. 139, 133–151. doi: 10.1037/a0028566 February 2014 | Volume 8 | Article 115 | 6 Frontiers in Human Neuroscience www.frontiersin.org www.frontiersin.org
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Abnormal Development of Dendrites in Adult-Born Rat Hippocampal Granule Cells Induced by Cyclophosphamide
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Frontiers in cellular neuroscience
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Abnormal Development of Dendrites
in Adult-Born Rat Hippocampal
Granule Cells Induced by
Cyclophosphamide Lin Wu 1, Dandan Guo 1, Qi Liu 1, Fei Gao 1, Xiaochen Wang 1, Xueying Song 1, Fuwu Wang 2
and Ren-Zhi Zhan 1* 1Department of Physiology, Shandong University School of Medicine, Jinan, China, 2Department of Histology and
Embryology, Shandong University School of Medicine, Jinan, China Although
development
of
cognitive
decline
in
cancer
patients
who
receive
chemotherapy is common, the underlying mechanism(s) remains to be identified. As
abnormalities in adult hippocampal neurogenesis may serve as substrate for cognitive
dysfunction, the present study examines the effect of cyclophosphamide (CPP), a
widely prescribed chemotherapeutic agent, on dendritic development of adult-born
hippocampal granule cells in the rat. CPP was intraperitoneally injected into male
Sprague-Dawley rats once a week for four consecutive weeks. Four weeks and
1 week after the last dose of CPP, Morris water maze test and doublecortin (DCX)
immunohistochemistry were carried out to determine the effects of CPP on cognitive
function and the rate of hippocampal neurogenesis, respectively. Adult newborn
hippocampal granule cells were labeled at the same day as the first dose of CPP
and were examined 10 weeks after labeling. Results showed that cognitive decline
induced by CPP was associated with both suppressed adult hippocampal neurogenesis
and abnormal development of dendrites of newborn granule cells. The abnormalities
of dendrites in newborn granule cells after CPP exposure included less dendritic
branching, shorter total dendritic length, thinner and torturous dendritic shafts with
intermittent appearances of varicosities, and lower spine densities of stubby and
thin types along dendritic shafts, but an increased density of mushroom-like spines. Adult-born granule cells in the presence of CPP, a widely used anti-cancer medication,
display abnormal dendritic morphologies and fewer dendritic spines which may underlie
cognitive dysfunction. Citation: Wu L, Guo D, Liu Q, Gao F, Wang X,
Song X, Wang F and Zhan R-Z
(2017) Abnormal Development of
Dendrites in Adult-Born Rat
Hippocampal Granule Cells Induced
by Cyclophosphamide. Front. Cell. Neurosci. 11:171. doi: 10.3389/fncel.2017.00171 Edited by:
Antonio Gambardella,
Magna Græcia University, Italy Edited by:
Antonio Gambardella,
Magna Græcia University, Italy Reviewed by:
Jennifer Larimore,
Agnes Scott College, United States
Carmelo Sgobio,
German Center for
Neurodegenerative Diseases (HZ),
Germany *Correspondence:
Ren-Zhi Zhan
zhan0001@gmail.com *Correspondence:
Ren-Zhi Zhan
zhan0001@gmail.com Received: 09 March 2017
Accepted: 06 June 2017
Published: 21 June 2017 Keywords: adult neurogenesis, chemotherapy, cognition, dendrite, granule cell, hippocampus, spine Keywords: adult neurogenesis, chemotherapy, cognition, dendrite, granule cell, hippocampus, spine Edited by: Edited by:
Antonio Gambardella,
Magna Græcia University, Italy ORIGINAL RESEARCH
published: 21 June 2017
doi: 10.3389/fncel.2017.00171 Doses and Delivery of CPP y
CPP monohydrate (Sigma, St. Louis, MO, USA) was dissolved
in sterile saline to make a 50 mg/ml of stock solution. The stock
solution was stored at −80◦C until use. CPP was intraperitoneally
injected into animals once a week for four consecutive weeks
at either 25 mg/kg or 50 mg/kg. Animals received equivalent
amount of saline in a schedule same to CPP-treated ones were
used as controls. As a subcortical structure, the hippocampus functions in
learning, memory formation and mood regulation. Granule
cells in the hippocampal dentate gyrus are continuously to
be generated throughout life in this brain region from neural
stem/progenitor cells that reside in the subgranular zone. Ultimately, nearly a quarter of newborn granule cells survive
to become mature neurons under normal conditions (Christian
et al., 2014). Studies have shown that newborn granule cells
integrate into the pre-existing dentate network and participate
in almost all of the known functions of the hippocampus
(Clelland et al., 2009; Sahay et al., 2011; Aimone et al., 2014;
Christian et al., 2014; Opendak and Gould, 2015). The rate
of adult hippocampal neurogenesis is under intense regulatory
control of internal and external environments (Aimone et al.,
2014). Studying in animals has shown that cyclophosphamide
(CPP), a commonly used chemotherapeutic agent, suppresses
adult hippocampal neurogenesis (Yang et al., 2010; Briones
and Woods, 2011; Lyons et al., 2011; Christie et al., 2012;
Dietrich et al., 2015). Given the fact that it takes 6∼8 weeks for
newborn granule cells to mature (Aimone et al., 2014; Christian
et al., 2014; Bergami et al., 2015), a better understanding of
detrimental effects of chemotherapeutic agents on cognitive
function should focus not only on the rate of adult neurogenesis
but also on the integration of newborn granule cells. The
normal integration process needs newborn granule cells to be
positioned appropriately, to have fully developed dendrites, and
to innervate onto right targets. Maturation of newborn granule
cells may be delayed by reduced physical or exploring activities
(Ambrogini et al., 2010) which are present during and even
after chemotherapy (Irwin et al., 2004). In the present study,
we explored possible abnormalities of dendritic morphologies
of newborn granule cells of the rat hippocampus after exposure
to CPP. INTRODUCTION Systemic cancer treatment is associated with cognitive decline that manifests as impairments in
learning, memory formation, attention, information processing speed and executive functioning
(Anderson-Hanley et al., 2003; Minisini et al., 2004; Vardy and Tannock, 2007; Janelsins et al.,
2011; Dietrich et al., 2015; Iyer et al., 2015; Ono et al., 2015; Wefel et al., 2015). Multiple mechanisms June 2017 | Volume 11 | Article 171 Frontiers in Cellular Neuroscience | www.frontiersin.org 1 Cyclophosphamide and Dendritic Development Wu et al. including biological and neuropsychiatric responses to cancers
(Andreotti et al., 2015; Wefel et al., 2015), toxic effects of
chemotherapeutic agents (Ahles and Saykin, 2007; Hutchinson
et al., 2012; Dietrich et al., 2015) and side effects of adjuvant
hormones (Rugo and Ahles, 2003; Castellon et al., 2004) have
been proposed for the development of cognitive dysfunction. Although both direct and indirect detrimental effects of
chemotherapeutic agents on brain cells may contribute to
chemotherapy-induced cognitive dysfunction, specific pathways
have yet to be identified (Ahles and Saykin, 2007; Dietrich et al.,
2015). and during the experiments. In total, 34 animals were used in the
present study; two animals died after CPP monohydrate (CPP)
injection (all were in the 3rd week after initiation of injection). Three sets of experiments were conducted in 32 animals:
14 for Morris water maze testing, six for assessment of adult
neurogenesis with doublecortin (DCX) immunohistochemistry
and 12 for dendritic measurements following intradentate
retroviral vector injection. Transcardial Perfusion and Tissue
Preparation Each animal was deeply anesthetized with an intraperitoneal
injection of sodium pentobarbital (80 mg/kg). After complete
paralysis, the animal was perfused through the aorta with 50 ml
heparinized saline, followed by 300 ml of 4% paraformaldehyde
in 0.1 M phosphate-buffered saline (PBS, pH 6.8) over a period of
40 min. The brain was removed from the skull upon completion
of perfusion, and then post-fixed in the same fixative at 4◦C
overnight. Thereafter, the brain was sequentially immersed in
10% sucrose in 0.1 M PBS for 4 h, 15% sucrose in 0.1 M PBS
for 8 h, and finally 20% sucrose in 0.1 M PBS at 4◦C overnight. Tissue block including the hippocampus was made with the
aid of an appropriate brain matrix (WPI, Sarosata, FI, USA). After the tissue block was firmly embedded with a medium that
consisted of 30% (w/v) chicken egg albumin (Sigma, St. Louis,
MO, USA), 0.5% (w/v) gelatin, and 0.9% (v/v) glutaraldehyde
in 0.1 M PBS as described previously (Zhan and Nadler, 2009),
coronal sections were cut into 40 µm or 110 µm in thickness
(depending on experimental aims) with a vibratome (VT1000 S,
Leica Biosystems, Wetzlar, Germany). Doublecortin (DCX) Immunohistochemistry
The effect of CPP on the rate of neurogenesis was compared
in saline- and CPP-injected rats (three animals each group)
by staining and counting DCX-positive newborn granule cells. Brain sections were prepared 1 week after the final dose of
CPP. Sections (40 µm in thickness) localized between 3.0 mm
and 3.6 mm posterior to the bregma were serially cut. Sections
with the order of number 3, 9 and 15 were subjected to DCX
immunohistochemical staining with the free-floating procedure. After the collected sections were washed with 0.1 M PBS, they
were incubated in a solution that consisted of 30% methanol
and 3% H2O2 in water for 30 min to inactivate endogenous
peroxidase. After rinsing, sections were incubated in a blocking
solution that consisted of 2.5% BSA (Sigma, St. Louis, MO,
USA), 0.2% Triton X-100 and 5% normal donkey serum in
0.1 M PBS at 4◦C for 2.5 h to minimize non-specific reactions. Without rinsing, the sections were then incubated with a goat Behavioral Assessments For animals to be used for behavioral studies, open field test
assessing general locomotor activity was conducted to qualify
animals before CPP exposure. To this aim, a 90 cm × 90 cm
white board, divided into 36 equal squares by red lines was
used as a field apparatus. Each side of the board was fenced to
prevent animals to jump out. Animals were individually placed
in the central square and allowed to explore the area freely. The
numbers of crossing (squares crossed), rearing (standing on hind
limbs), grooming (washing face) and urination were recorded
for 5 min. After each trial, the apparatus was cleaned with
ethanol. Qualified rats were randomly divided into two groups,
receiving intraperitoneal injection of saline (control group) or
CPP (CPP group); both were carried out once a week for a
consecutive 4 weeks. Four weeks after the last injection, the
Morris water maze was conducted to assess animals’ spatial
learning and memory abilities. The water maze task was carried
out for seven consecutive days in which the first 6 days were used
to train the animals to find the hidden platform, whereas the last
day was used to probe how well the animals remembered the
position of the platform. A cylindrical pool (120 cm in diameter),
filled with water (22–25◦C) was placed in a quiet testing room. The cylindrical pool was equally divided into four quadrants with
a removable platform (10 cm in diameter) to be located in the
center of the first quadrant. The platform was set 2 cm below
the surface of water to make the platform invisible to animals. A digital camera was mounted above the pool and linked to an
automatic tracking system (SMART polyvalent video-tracking
system, Panlab, Spain) to track and record the performance of
individual animals. Several bright-colored cues posted on the
wall of the maze were used to help animals to find the platform. In the training sessions, each rat was put into the pool from Construction of CAG–GFP Retroviral
Vector Vector
Concentrated CAG–GFP retroviral vector was made from an
established stable virus-producing platinum-E cell line (Gao
et al., 2015) through two-step ultraspeed centrifugation detailed
below. After the frozen cells were thawed quickly in a 37◦C water
bath, 1 ml of cell suspension was immediately transferred into a
10 ml tube that contained 4 ml of Dulbecco’s modified eagle’s
medium (DMEM). The cell suspension mixed with DMEM
was centrifuged at 1000 rpm for 5 min. After discarding the
supernatant, the cell pellet was re-suspended with 2 ml of M10
(10% serum-contained DMEM). The resulted cell suspension
was transferred into a 10 ml culture dish in which 8 ml culture
medium was pre-added. After mixing, cells were cultured in a
5% CO2 incubator at 37◦C. Upon ∼90% confluence was reached,
culture medium was removed and the cells were rinsed twice
with autoclaved PBS. After cells were digested in 1 ml of 0.25%
trypsin-contained solution for 3–4 min, 4 ml of M10 was added
into the dish. After thorough mixing, 2 ml of cell suspension was
added into an 8 ml M10-contained 10 ml culture dish. At this
stage, 12 dishes were prepared each time. Dishes were cultured in
a 5% CO2 incubator at 37◦C and the supernatant was collected
24 and 48 h after culture. Supernatants collected from all dishes
were pooled together and centrifuged (18,000 rpm) at 4◦C for
2 h. The pellet was suspended with 1 ml Dulbecco’s phosphate-
buffered saline and then centrifuged (12,000 rpm) at 4◦C for
1.5 h. After removal of supernatant, the pellet was suspended in
30 µl of Dulbecco’s phosphate-buffered saline. After being kept
at 4◦C overnight, the viral suspension was gently mixed, divided
into 5–10 µl aliquots, and kept at −80◦C until use. Animals After several washes with
0.1 M PBS, immunoreactions were visualized by an incubation
with 3,3-diaminobenzidine, enhanced by the addition of 0.08%
ammonium nickel sulfate (Vector Labs., Burlingame, CA, USA). The reaction was terminated by incubating the section with
water. Sections were then flatly mounted on slide glasses and
left to be air-dried for at least an hour. After dehydration
with a series of graded ethanol solutions, the sections were
then cleared in xylene and coverslipped with neutral balsam. DCX-positive cells in the dentate gyrus were counted and the
lengths of the superior and inferior pyramidal blade of the
dentate gyrus were measured in each section by using Image-Pro-
Plus (Media Cybernetics, Rockville, MD, USA). The density of
DCX-positive cells was calculated by dividing the number of cells
with the total length of granule cell layer and expressed as the
number of cells/mm. Values in three sections each animal were
averaged. the four different quadrants with its head facing the wall to
prevent the animal seeing the platform directly. If a rat could
find the platform within 60 s, it would be allowed to stay onto
the platform for 20 s before removal from the pool. Instead, if
a rat failed to find the platform within 60 s, it would be guided
onto the platform. The time taken to find the hidden platform
(latency to escape) was recorded. On the 7th day, the platform
was removed and each rat was put into the pool from a start
point (the opposite quadrant of the first quadrant) to probe its
memory. The rat was allowed to swim freely in the pool for 60 s
and the path of navigation was traced. Swimming speed and the
time spent in the target quadrant and non-target quadrants were
collected. Animals All animal experiments were approved by the Animal Ethics
Committee of Shandong University School of Medicine and
performed according to the guides for the care and use of
laboratory animals set by National Research Council (US). Male
Sprague-Dawley rats (160–180 g, 6–8 weeks old) purchased from
Beijing HFK Bioscience Company (Beijing, China) were housed
in a university animal facility with a 12 h light/dark cycle and
free access to food and water, and were weighed weekly before June 2017 | Volume 11 | Article 171 Frontiers in Cellular Neuroscience | www.frontiersin.org 2 Cyclophosphamide and Dendritic Development Wu et al. Wu et al. anti-DCX (Santa Cruz, San Jose, CA, USA) that was diluted
with the blocking solution (1:400) at 4◦C overnight. After three
washes with 0.1 M PBS, the sections were incubated with
biotinylated donkey anti-goat IgG (Jackson ImmunoResearch,
West Grove, PA, USA) in a dilution of 1:400 at 4◦C for 1.5 h. After rinsing with 0.1 M PBS, the sections were incubated
with the avidin-peroxidase complex (Vector Labs., Burlingame,
CA, USA) at 4◦C for 60 min. After several washes with
0.1 M PBS, immunoreactions were visualized by an incubation
with 3,3-diaminobenzidine, enhanced by the addition of 0.08%
ammonium nickel sulfate (Vector Labs., Burlingame, CA, USA). The reaction was terminated by incubating the section with
water. Sections were then flatly mounted on slide glasses and
left to be air-dried for at least an hour. After dehydration
with a series of graded ethanol solutions, the sections were
then cleared in xylene and coverslipped with neutral balsam. DCX-positive cells in the dentate gyrus were counted and the
lengths of the superior and inferior pyramidal blade of the
dentate gyrus were measured in each section by using Image-Pro-
Plus (Media Cybernetics, Rockville, MD, USA). The density of
DCX-positive cells was calculated by dividing the number of cells
with the total length of granule cell layer and expressed as the
number of cells/mm. Values in three sections each animal were
averaged. anti-DCX (Santa Cruz, San Jose, CA, USA) that was diluted
with the blocking solution (1:400) at 4◦C overnight. After three
washes with 0.1 M PBS, the sections were incubated with
biotinylated donkey anti-goat IgG (Jackson ImmunoResearch,
West Grove, PA, USA) in a dilution of 1:400 at 4◦C for 1.5 h. After rinsing with 0.1 M PBS, the sections were incubated
with the avidin-peroxidase complex (Vector Labs., Burlingame,
CA, USA) at 4◦C for 60 min. Frontiers in Cellular Neuroscience | www.frontiersin.org Enhancement of GFP Fluorescence with
Anti-GFP Antibody for Dendritic Analyses Anti GFP Antibody for Dendritic Analyses
For
dendritic
measurements,
transcardial
perfusion
was
performed 10 weeks after retroviral vector injection. Coronally
cut hippocampal sections (110 µm in thickness) in which
GFP-labeled granule cell(s) presented were subjected to GFP
immunostaining by the free-floating procedure. Sections were
first washed with 1XPBS, and then incubated in a blocking
solution that consisted of 2.5% BSA, 0.2% Triton X-100 and
5% goat serum in 1XPBS to minimize non-specific reactions. Thereafter, sections were incubated with a mouse anti-GFP
(Invitrogen, Carlsbad, CA, USA) in a dilution of 1:1000 at
4◦C overnight. After three washes with 1XPBS, the sections
were incubated with an Alexa fluor 488-conjugated secondary
antibody (Invitrogen, Carlsbad, CA, USA) in a dilution of 1:600. After another three washes with 1XPBS, sections were mounted
on glass slides with 75% glycerol in PBS and coverslipped. Sections in which GFP was immunofluorescently amplified were
scanned with the Zeiss 780 laser scanning microscope (Carl
Zeiss, Jena, Germany). Images yielded were used for quantitative
measurements of dendrite length, the complexity of dendritic
arborization and dendrite spine. Statistical Analysis Data are expressed as mean ± standard error of the mean (SEM). Two-group data are compared with unpaired Student’s t-test. One-way analysis of variance (ANOVA) was used to compare
the means of three or more groups. Follow one-way ANOVA,
Dunnett’s test was used for individual comparisons. Latency to
escape was analyzed with repeated measures ANOVA, whereas
data for Sholl analysis were compared with two-way ANOVA. A
p ≤0.05 is considered to be significantly different. Measurements of Dendrite Length and
Complexity Complexity
To measure dendrite length and the complexity of dendritic
arborization, individual GFP-labeled cells residing in the
suprapyramidal blade were z-stacked under a 20× objective
with a z-series interval of 1 µm and zoom 1. A 3-dimensional
image created from the z-series file each cell was compressed
into a 2-demnesional image. Only cells with relatively intact
dendritic arborizations were quantitatively analyzed further. After GFP-positive soma and dendritic arborization were
manually traced under ImageJ1 with the NeuronJ plugin2, the
length of each dendritic segment was measured. Total dendrite
length was calculated by summing the lengths of all segments. After tracing, the complexity of dendritic arborization for
individual cells was carried out by using Sholl analysis with the
‘‘Sholl plugin’’. For Sholl analysis, the interval between concentric
circles was 25 µm with the center point at the center of soma. For
each rat, 3 cells were randomly picked for either of analysis and
the values were averaged to represent the animal. 1http://imagej.nih.gov/ij/
2http://www.imagescience.org/meijering/software/neuronj// Intradentate Injection of CAG–GFP
Retroviral Vector After an animal was anesthetized with an intraperitoneal
injection of sodium pentobarbital (25 mg/kg) and ketamine
(60 mg/kg), the animal was placed on a stereotaxic frame
suitable for rats (Stoelting, Wood Dale, IL, USA). After the
skin was sterilized with a sequential application of 7.5%
povidone iodine and 75% alcohol, the scalp was incised and
a skull hole on the right side was drilled. One microliter
of CAG–GFP retroviral vector was injected into the right
dentate gyrus at a rate of 0.1 µl/min using a 1 µl Hamilton June 2017 | Volume 11 | Article 171 Frontiers in Cellular Neuroscience | www.frontiersin.org 3 Wu et al. Cyclophosphamide and Dendritic Development Quantification of Dendritic Spines syringe (Bonaduz, GR, Switzerland) that was connected to an
automatic injection pump (Stoelting, Wood Dale, IL, USA). The injection site was located 3.2 mm posterior from the
bregma and 2.5 mm right to the midline, in a depth of
2.4 mm (measured from the surface of the cortex). The
needle was left in the place for additional 15 min after the
completion of injection to prevent possible backflow before
removal. All surgical procedures were performed under sterile
conditions. Animals were returned to cages after recovery from
anesthesia. Quantification of Dendritic Spines
z-stacks for dendritic segments located in the middle molecular
layer were carried by using a 63× oil objective. The other
parameters set for z-stacks were as follows: 0.12 µm of
z-series
thickness,
zoom
4,
and
a
spatial
resolution
of
0.03 µm × 0.03 µm × 0.12 µm. A 3-dimensional image was
reconstructed from individual z-stack file. Spines were classified
into stubby, thin or mushroom type based on spine length,
head diameter to neck width ratio, and the size of head specific
to dentate granule cells (Tyler and Pozzo-Miller, 2003; Boda
et al., 2004; Zhao et al., 2014). A stubby spine had a length
that was similar to the diameter of the neck as well as the
diameter of the head. A mushroom spine displayed a great head
diameter to neck width ratio in addition to a head surface area
that was ≥0.4 µm2. The head surface area was estimated by
using the function 0.25 × π × dx (µm) × dy (in µm); dx and
dy were the lengths of shortest and longest axes of the spine
head which were measured under ImageJ manually (Zhao et al.,
2014). A spine that could not be classified into either stubby
type or mushroom form with or without a bulbous head was
referred to as the thin type. The numbers of total and spines in
different forms on each segment were counted and the densities
of total and individual types were calculated by dividing the
numbers of spines with the length of corresponding dendritic
segment. Six to nine dendritic segments (30–50 µm in length
each) from at least three cells each animal located in the middle
molecular layer were randomly scanned and analyzed. Total,
stubby, thin and mushroom spine densities from all counted
segments each animal were averaged and used to represent the
animal. Long-Lasting Cognitive Decline after CPP
Exposure Is Associated with a Suppression
of Hippocampal Neurogenesis Morris water
maze test revealed that 4-week CPP treatment significantly
reduced learning and memory abilities, even though the test
was held 4 weeks after the last dose of CPP. Times spent for
finding the platform were analyzed with repeated measures
ANOVA (Figure 1A). Since Chi-analysis revealed a violation
of sphericity, data were analyzed with Huynh-Feldt correction. Statistical significances were found between the two groups
(F(1) = 22.9, p = 0.0004) and at different time points (F(5) = 16.1,
p < 0.0001) but no significant interaction between ‘‘group’’ and
‘‘time’’ (F(5) = 0.84, p = 0.5247). At the first day of training, the
time needed to find the platform (latency to escape) between
the control (Control) and CPP groups was similar; however, as
training days increased, animals in the control group spent less
time finding the platform. A day after completion of training,
the animals were tested on how firm they intended to find the
platform for escape. As shown in Figure 1B, the swimming
speeds were not statistically different between the control and
CPP groups. However, animals in the control group stayed
significantly longer in the target quadrant searching for the
platform, whereas the same trend did not exist in the CPP-treated
group (Figures 1C,D). in Figure 2A, at both doses, CPP significantly reduced the
density of DCX-positive cells in the hippocampal dentate gyrus. The density of DCX-positive cells was reduced significantly
from 46.0 ± 4.1 cells/mm in the control group (n = 3)
to 31.2 ± 3.3 cells/mm in 25 mg/kg group (n = 3) and
23.8 ± 0.9 cells/mm in 50 mg/kg group (n = 3), respectively
(Figure 2B). CPP Exposure Results in Less Dendritic
Arborization and Complexity in Newborn
Granule Cells At 10 weeks after retroviral vector injection, GFP-labeled cells
from control animals were found to reside in the border
between the granule cell layer and the hilus with apical dendrites
extending throughout the molecular layer and tips ending at the
hippocampal fissure (Figure 3A). The morphology of dendritic
tree of newborn granule cells obtained from CPP-treated animals
appeared different from control rats in at least two aspects:
they appeared to be beading (Figure 3B) and branched to a
lesser extent (Figure 3C). Total dendrite length in the CPP
group is 1279.0 ± 62.5 µm (n = 6), which is significantly
shorter than 1921.0 ± 110.5 µm (n = 6) observed in the control
group (p = 0.005, by unpaired t-test; Figure 3D). Sholl analysis
revealed that dendritic arborization of newborn granule cells
after CPP treatment is significantly less complex than that in
the control group (F(14,140) = 39.7, p < 0.0001), as shown in
Figure 3E. Noticeably, the number of intersection indicating
dendritic complexity was significantly less in CPP-treated group
in dendritic segments (between 135 µm and 270 µm away from
the soma). To determine if cognitive dysfunction induced by CPP is
associated with a slower rate of adult hippocampal neurogenesis,
we performed DCX immunohistochemistry. In addition to
50 mg/kg of CPP, a smaller dose was tested also. As shown FIGURE 1 | Reduced learning and memory abilities after cyclophosphamide
(CPP) treatment revealed by Morris water maze test. Animals were tested
4 weeks after the last dose of CPP (50 mg/kg). Error bars are standard error of
the mean (SEM). (A) Latency to escape recorded during the training days. ∗p < 0.01 (t = 2.82 at 4th and 2.99 at 6th, df = 72), in comparison with
“control” at the same time revealed by by Dunnett post hoc test following
repeated measures analysis of variance (ANOVA). (B) Swimming speed
recorded in the testing day. (C) Time spent in the target quadrant in the testing
day. ∗p < 0.05–0.01 (df = 24), in comparison with that in the target quadrant. Comparisons were done with Dunnett’s test following one-way ANOVA. TQ,
NT1, NT2 and NT3 indicate the target quadrant, non-target quadrant 1,
non-target quadrant 2 and non-target quadrant 3, respectively. (D) Time spent
in the target quadrant between the control (Control) and CPP groups in the
testing day (t = 2.81, df = 12). CPP Reduces Total Dendritic Spines but
Increases Mushroom-Like Spines ∗p < 0.01 (t = 2.82 at 4th and 2.99 at 6th, df = 72), in comparison with
“control” at the same time revealed by by Dunnett post hoc test following
repeated measures analysis of variance (ANOVA). (B) Swimming speed
recorded in the testing day. (C) Time spent in the target quadrant in the testing
day. ∗p < 0.05–0.01 (df = 24), in comparison with that in the target quadrant. Comparisons were done with Dunnett’s test following one-way ANOVA. TQ,
NT1, NT2 and NT3 indicate the target quadrant, non-target quadrant 1,
non-target quadrant 2 and non-target quadrant 3, respectively. (D) Time spent
in the target quadrant between the control (Control) and CPP groups in the
testing day (t = 2.81, df = 12). Long-Lasting Cognitive Decline after CPP
Exposure Is Associated with a Suppression
of Hippocampal Neurogenesis pp
p
g
To observe the effect of CPP on cognitive function, a dose of
50 mg/kg reported by a previous study (Christie et al., 2012)
was applied. Before Morris water maze testing, body weight
was measured and open field test was conducted in individual
animals. Body weight was significantly lower in CPP group
(379.0 ± 9.7 g, n = 7) in comparison with the control group
(430.9 ± 4.8 g, n = 7; p < 0.005 via unpaired t-test). Open field
test revealed that rats in CPP group (n = 7) had a trend to be
less active than control group (n = 7). Events summed up from
the number of crossing, rearing, glooming and urination in a 1http://imagej.nih.gov/ij/
2http://www.imagescience.org/meijering/software/neuronj// June 2017 | Volume 11 | Article 171 Frontiers in Cellular Neuroscience | www.frontiersin.org 4 Cyclophosphamide and Dendritic Development Wu et al. Wu et al. 5 min-period was 51.3 ± 19.7 in CPP group and 107.0 ± 20.7 in
the control group (p = 0.075, unpaired t-test). Morris water
maze test revealed that 4-week CPP treatment significantly
reduced learning and memory abilities, even though the test
was held 4 weeks after the last dose of CPP. Times spent for
finding the platform were analyzed with repeated measures
ANOVA (Figure 1A). Since Chi-analysis revealed a violation
of sphericity, data were analyzed with Huynh-Feldt correction. Statistical significances were found between the two groups
(F(1) = 22.9, p = 0.0004) and at different time points (F(5) = 16.1,
p < 0.0001) but no significant interaction between ‘‘group’’ and
‘‘time’’ (F(5) = 0.84, p = 0.5247). At the first day of training, the
time needed to find the platform (latency to escape) between
the control (Control) and CPP groups was similar; however, as
training days increased, animals in the control group spent less
time finding the platform. A day after completion of training,
the animals were tested on how firm they intended to find the
platform for escape. As shown in Figure 1B, the swimming
speeds were not statistically different between the control and
CPP groups. However, animals in the control group stayed
significantly longer in the target quadrant searching for the
platform, whereas the same trend did not exist in the CPP-treated
group (Figures 1C,D). 5 min-period was 51.3 ± 19.7 in CPP group and 107.0 ± 20.7 in
the control group (p = 0.075, unpaired t-test). CPP Reduces Total Dendritic Spines but
Increases Mushroom-Like Spines After revealing abnormalities in the dendritic tree of newborn
granule cells after CPP treatment, we then quantified spines in
dendritic segments of the middle molecular layer. In the control
group, dense protrusions in variable shapes appeared along the
relatively regular dendritic shaft (Figure 4A, left panel). CPP
treatment resulted in unusual appearances of dendrites—shafts
appeared to be thinner with intermittent varicosities, and in
addition, fewer protrusions were present (Figure 4A, right
panel). Total spine density in the control group (Control) is
∼1.2 spines per µm length (n = 5), which was significantly denser
than 0.7 spines per µm length (n = 5) in CPP-treated group
(Figure 4B). In contrast to the density of total, stubby and thin
spines, the density of mushroom-like spines, defined as having
a ≥0.4 µm2 in head surface area with shorter neck, increased
significantly in CPP-treated animals compared to the control
animals (Figure 4B). FIGURE 1 | Reduced learning and memory abilities after cyclophosphamide
(CPP) treatment revealed by Morris water maze test. Animals were tested
4 weeks after the last dose of CPP (50 mg/kg). Error bars are standard error of
the mean (SEM). (A) Latency to escape recorded during the training days. ∗p < 0.01 (t = 2.82 at 4th and 2.99 at 6th, df = 72), in comparison with
“control” at the same time revealed by by Dunnett post hoc test following
repeated measures analysis of variance (ANOVA). (B) Swimming speed
recorded in the testing day. (C) Time spent in the target quadrant in the testing
day. ∗p < 0.05–0.01 (df = 24), in comparison with that in the target quadrant. Comparisons were done with Dunnett’s test following one-way ANOVA. TQ,
NT1, NT2 and NT3 indicate the target quadrant, non-target quadrant 1,
non-target quadrant 2 and non-target quadrant 3, respectively. (D) Time spent
in the target quadrant between the control (Control) and CPP groups in the
testing day (t = 2.81, df = 12). FIGURE 1 | Reduced learning and memory abilities after cyclophosphamide
(CPP) treatment revealed by Morris water maze test. Animals were tested
4 weeks after the last dose of CPP (50 mg/kg). Error bars are standard error of
the mean (SEM). (A) Latency to escape recorded during the training days. DISCUSSION In addition to confirming that persistent cognitive decline
induced by CPP is associated with a suppression of adult
hippocampal neurogenesis, we found that CPP treatment leads June 2017 | Volume 11 | Article 171 Frontiers in Cellular Neuroscience | www.frontiersin.org 5 Wu et al. Wu et al. Cyclophosphamide and Dendritic Development FIGURE 2 | CPP reduces doublecortin (DCX)-positive cells in the hippocampal dentate gyrus. (A) Representative images showing DCX-positive cells in sections
obtained from the control (left panel) and CPP-treated rats (middle and right panels). Upper row: imaged under a lower objective; Bottom row: areas indicated by the
black boxes in the upper row were visualized under a higher magnification lens. (B) The densities of DCX-positive cells in the absence (Control) and the presence of
two doses of CPP. The number of animals is three in each group. ∗p < 0.05 and ∗∗p < 0.01 (df = 6), in comparison with “Control” by Dunnett’s test following
one-way ANOVA. FIGURE 2 | CPP reduces doublecortin (DCX)-positive cells in the hippocampal dentate gyrus. (A) Representative images showing DCX-positive cells in sections
obtained from the control (left panel) and CPP-treated rats (middle and right panels). Upper row: imaged under a lower objective; Bottom row: areas indicated by the
black boxes in the upper row were visualized under a higher magnification lens. (B) The densities of DCX-positive cells in the absence (Control) and the presence of
two doses of CPP. The number of animals is three in each group. ∗p < 0.05 and ∗∗p < 0.01 (df = 6), in comparison with “Control” by Dunnett’s test following
one-way ANOVA. FIGURE 2 | CPP reduces doublecortin (DCX)-positive cells in the hippocampal dentate gyrus. (A) Representative images showing DCX-positive cells in sections
obtained from the control (left panel) and CPP-treated rats (middle and right panels). Upper row: imaged under a lower objective; Bottom row: areas indicated by the
black boxes in the upper row were visualized under a higher magnification lens. (B) The densities of DCX-positive cells in the absence (Control) and the presence of
two doses of CPP. The number of animals is three in each group. ∗p < 0.05 and ∗∗p < 0.01 (df = 6), in comparison with “Control” by Dunnett’s test following
one-way ANOVA. to remarkable abnormal development of dendrites in newborn
granule cells. DISCUSSION (B) Newborn granule cells obtained from a rat treated with CPP (scale
bar = 50 µm). (C) A comparison of the number of dendritic branches between the control (Control) and CPP groups. Each data point represents one animal in which
the value was averaged from three newborn granule cells. Data are compared by unpaired student’s t-test. (D) A comparison of total dendrite length between the
control (Control) and CPP groups. Each data point represents one animal in which the value was averaged from three newborn granule cells. Data are compared by
unpaired student’s t-test. (E) Sholl analysis compares the complexity of dendritic arborization between the control (Control) and CPP groups. The numbers of
animals studied are shown inside the figure. For each animal, the value was averaged from three cells. Statistical comparisons were done with two-way ANOVA. Sidak’s multiple comparisons were used for individual comparisons. ∗Indicates p < 0.05∼0.001 as compared to the control (df = 150). FIGURE 3 | Representative images and statistical comparisons show the number of branch, total dendrite length and the complexity of arborization in the absence
(Control) and presence of CPP. The first dose of CPP (50 mg/kg) was given at the same day as the CAG-GFP retroviral vector labeling and was continued for
consecutive 4 weeks. Animals in the control group received equivalent amounts of saline. Animals in both groups were sacrificed 10 weeks after the injection of the
retroviral vector. (A) A newborn granule cell obtained from a control rat (scale bar = 50 µm). (B) Newborn granule cells obtained from a rat treated with CPP (scale
bar = 50 µm). (C) A comparison of the number of dendritic branches between the control (Control) and CPP groups. Each data point represents one animal in which
the value was averaged from three newborn granule cells. Data are compared by unpaired student’s t-test. (D) A comparison of total dendrite length between the
control (Control) and CPP groups. Each data point represents one animal in which the value was averaged from three newborn granule cells. Data are compared by
unpaired student’s t-test. (E) Sholl analysis compares the complexity of dendritic arborization between the control (Control) and CPP groups. The numbers of
animals studied are shown inside the figure. For each animal, the value was averaged from three cells. Statistical comparisons were done with two-way ANOVA. Sidak’s multiple comparisons were used for individual comparisons. DISCUSSION Multiple clinical surveys have provided solid evidence to show
that chemotherapy can lead to cognitive decline, although the to remarkable abnormal development of dendrites in newborn
granule cells. Multiple clinical surveys have provided solid evidence to show
that chemotherapy can lead to cognitive decline, although the FIGURE 3 | Representative images and statistical comparisons show the number of branch, total dendrite length and the complexity of arborization in the absence
(Control) and presence of CPP. The first dose of CPP (50 mg/kg) was given at the same day as the CAG-GFP retroviral vector labeling and was continued for
consecutive 4 weeks. Animals in the control group received equivalent amounts of saline. Animals in both groups were sacrificed 10 weeks after the injection of the
retroviral vector. (A) A newborn granule cell obtained from a control rat (scale bar = 50 µm). (B) Newborn granule cells obtained from a rat treated with CPP (scale
bar = 50 µm). (C) A comparison of the number of dendritic branches between the control (Control) and CPP groups. Each data point represents one animal in which
the value was averaged from three newborn granule cells. Data are compared by unpaired student’s t-test. (D) A comparison of total dendrite length between the
control (Control) and CPP groups. Each data point represents one animal in which the value was averaged from three newborn granule cells. Data are compared by
unpaired student’s t-test. (E) Sholl analysis compares the complexity of dendritic arborization between the control (Control) and CPP groups. The numbers of
animals studied are shown inside the figure. For each animal, the value was averaged from three cells. Statistical comparisons were done with two-way ANOVA. Sidak’s multiple comparisons were used for individual comparisons. ∗Indicates p < 0.05∼0.001 as compared to the control (df = 150). FIGURE 3 | Representative images and statistical comparisons show the number of branch, total dendrite length and the complexity of arborization in the absence
(Control) and presence of CPP. The first dose of CPP (50 mg/kg) was given at the same day as the CAG-GFP retroviral vector labeling and was continued for
consecutive 4 weeks. Animals in the control group received equivalent amounts of saline. Animals in both groups were sacrificed 10 weeks after the injection of the
retroviral vector. (A) A newborn granule cell obtained from a control rat (scale bar = 50 µm). DISCUSSION ∗Indicates p < 0.05∼0.001 as compared to the control (df = 150). June 2017 | Volume 11 | Article 171 6 Frontiers in Cellular Neuroscience | www.frontiersin.org Cyclophosphamide and Dendritic Development Wu et al. in the present study on the basis of the same dose which
has been used in a previous study (Christie et al., 2012) and
this dose resulted in nearly 50% reduction in DCX-labeled
newborn granule cells (Figure 2). Since CPP was applied for a
consecutive 4 weeks and dendritic morphological changes were
examined more than 2 months after birth, dendritic changes
observed in the present study are considered to be chronic
response. To avoid the influence of intradentate retroviral vector
injection on behavioral tests and behavioral training on dendritic
developments, cognitive function, alteration in hippocampal
neurogenesis and dendritic morphologies were not studied in
the same set of animals rather than three separated sets of
animals. FIGURE 4 | CPP reduces total but increases mushroom-like dendritic spines
in adult-born granule cells. (A) Images of dendritic segments in 10 week-old
newborn granule cells in a control animal (left panel) and a CPP
(50 mg/kg)-treated animal (CPP, right panel). Mushroom-like spines are
indicated by arrowheads. Scale bar = 5 µm. Note that dendritic segment
obtained from a CPP-treated animal appears to be thinner with irregular
varicosities (right panel). (B) Bar graphs show that CPP treatment reduces
total, thin and stubby dendritic spines but increases mushroom-like spines. Error bars are SEMs. Statistical comparisons are done with unpaired t-test. “n” indicates the number of animals. “a”, “b”, “c” and “d” indicates p values at
0.045 (t = 2.37, df = 8), 0.038 (t = 2.48, df = 8), 0.043 (t = 2.41, df = 8) and
0.009 (t = 3.49, df = 8), respectively. The Morris Water Maze was an established method for
testing hippocampus-dependent learning, acquisition of spatial
memory and long-term spatial memory. The substrate for
those hippocampus-dependent cognitive deficits during and
after CPP treatment has been firmly established. A recent study
revealed that CPP treatment was associated with abnormal
dendritic structures in hippocampal principal cells (Acharya
et al., 2015). By combining retroviral labeling and confocal
imaging, CPP was found to cause an abnormal development
of dendrites in newborn granule cells. DISCUSSION The characteristics of
dendritic abnormality include fewer branches and shorter total
length of dendrites, irregularity of dendritic shafts especially
intermediate dendritic varicosities along the thinner and
tortuous shafts, and sparser dendritic spines but increased
mushroom-like spines. Because the morphology of dendrites
and spines attached to dendritic shafts are critical in receiving
excitatory projections and in cellular signal integration (Šišková
et al., 2014), the abnormalities observed after CPP treatment
are expected to result in lower excitability and insufficient
integration of newborn granule cells, which could contribute
to the development of cognitive decline. In comparison with
dendrites of newborn granule cells in the controlled animals,
dendrites of newborn granule cells in the presence of CPP
appeared to be intermediately thinner and torturous. Since
these features were not obvious in pre-existing granule cells in
a similar treatment regimen (Acharya et al., 2015), dendritic
changes in newborn granule cells are unlikely to be caused by
direct toxic effect but developmental defect. While total spine
density (due to decreased thin and stubby spines) was found
to be lower in CPP-treated animals, the mushroom-like spine
density was rather increased. As larger spines are generally
considered to be composed by more excitatory amino acid
receptors (Matsuzaki et al., 2001; Noguchi et al., 2005) and
transformed from small spines in response to sensory stimuli
(Grutzendler et al., 2002; Yasumatsu et al., 2008), the increase in
mushroom-like spines after CPP treatment is puzzling. Several
mechanisms may underlie the increased density in mushroom
spines in the presence of CPP. First, it is possible that the
increased density of mushroom-like spines after CPP exposure
might have been a compensatory response, since persistent lack
of excitatory synapses is known to result in increased head
diameter and strength of remaining spines after naturation
(Turrigiano, 2007). In addition, changes in dendritic geometry
may alter integration of excitatory inputs, resulting in only FIGURE 4 | CPP reduces total but increases mushroom-like dendritic spines
in adult-born granule cells. (A) Images of dendritic segments in 10 week-old
newborn granule cells in a control animal (left panel) and a CPP
(50 mg/kg)-treated animal (CPP, right panel). Mushroom-like spines are
indicated by arrowheads. Scale bar = 5 µm. Note that dendritic segment
obtained from a CPP-treated animal appears to be thinner with irregular
varicosities (right panel). (B) Bar graphs show that CPP treatment reduces
total, thin and stubby dendritic spines but increases mushroom-like spines. Error bars are SEMs. DISCUSSION Statistical comparisons are done with unpaired t-test. “n” indicates the number of animals. “a”, “b”, “c” and “d” indicates p values at
0.045 (t = 2.37, df = 8), 0.038 (t = 2.48, df = 8), 0.043 (t = 2.41, df = 8) and
0.009 (t = 3.49, df = 8), respectively. underlying mechanism remains to be fully elucidated (Anderson-
Hanley et al., 2003; Minisini et al., 2004; Vardy and Tannock,
2007; Janelsins et al., 2011; Jim et al., 2012; Lindner et al., 2014;
Dietrich et al., 2015; Wefel et al., 2015). Animal studies have
found that chemotherapeutic agents, including CPP, profoundly
slow the rate of adult hippocampal neurogenesis (Mustafa
et al., 2008; Seigers et al., 2008, 2009, 2010; Yang et al., 2010;
Briones and Woods, 2011; Lyons et al., 2011; Christie et al.,
2012; ElBeltagy et al., 2012; Nokia et al., 2012). Because adult
hippocampal neurogenesis is implicated in functions that overlap
with those general functions of hippocampus (Clelland et al.,
2009; Sahay et al., 2011; Aimone et al., 2014; Christian et al.,
2014; Opendak and Gould, 2015), in addition to studying the
rate of neurogenesis, studying the intrinsic electrophysiological
properties and integration of newborn granule cells under the
influence of chemotherapeutic agents is needed. For newborn
granule cells to be integrated into the existing neural network
properly, normal developments of dendrites and dendritic spines
are essential. The integration process takes 6∼8 weeks in mice
(Aimone et al., 2014; Christian et al., 2014; Bergami et al., 2015),
and it may be even longer in other species (Kohler et al., 2011),
making this process potentially susceptible to environmental
changes. underlying mechanism remains to be fully elucidated (Anderson-
Hanley et al., 2003; Minisini et al., 2004; Vardy and Tannock,
2007; Janelsins et al., 2011; Jim et al., 2012; Lindner et al., 2014;
Dietrich et al., 2015; Wefel et al., 2015). Animal studies have
found that chemotherapeutic agents, including CPP, profoundly
slow the rate of adult hippocampal neurogenesis (Mustafa
et al., 2008; Seigers et al., 2008, 2009, 2010; Yang et al., 2010;
Briones and Woods, 2011; Lyons et al., 2011; Christie et al.,
2012; ElBeltagy et al., 2012; Nokia et al., 2012). Frontiers in Cellular Neuroscience | www.frontiersin.org DISCUSSION Because adult
hippocampal neurogenesis is implicated in functions that overlap
with those general functions of hippocampus (Clelland et al.,
2009; Sahay et al., 2011; Aimone et al., 2014; Christian et al.,
2014; Opendak and Gould, 2015), in addition to studying the
rate of neurogenesis, studying the intrinsic electrophysiological
properties and integration of newborn granule cells under the
influence of chemotherapeutic agents is needed. For newborn
granule cells to be integrated into the existing neural network
properly, normal developments of dendrites and dendritic spines
are essential. The integration process takes 6∼8 weeks in mice
(Aimone et al., 2014; Christian et al., 2014; Bergami et al., 2015),
and it may be even longer in other species (Kohler et al., 2011),
making this process potentially susceptible to environmental
changes. Clinical dosages of CPP vary according to the regimens of
treatment. For intravenous injection, 40–50 mg/kg of CPP has
been recommended for a consecutive 4 days (Cyclophosphamide
FDA Packet Insert, 2013) in which several cycles of treatment
may be given. Fifty milligram per kilogram of CPP was chosen for
the examinations of cognitive functions and dendritic changes June 2017 | Volume 11 | Article 171 Frontiers in Cellular Neuroscience | www.frontiersin.org 7 Cyclophosphamide and Dendritic Development Wu et al. Wu et al. few spines to be more active and consequently larger. Indeed,
increases in mushroom-like dendritic spines have been observed
in later stages of Alzheimer’s disease-like pathology (Dickstein
et al., 2010) and in Fyn defect mice (Babus et al., 2011) as well. It remains unknown if direct damage to CA1 pyramidal cells by
CPP might have affected the development of newborn granule
cells. (Bergami et al., 2008; Chan et al., 2008; Wang et al., 2015). CPP-induced decrease in total spine density with an increase
in mushroom spine density is also opposite to what has been
observed after BDNF application (Tyler and Pozzo-Miller, 2003). It is reasonable to consider that the abnormal development of
dendrites in newborn granule cells in the presence of CPP might
have been at least partially mediated by the down-regulation
of BDNF expression. However, future studies are needed
to examine if endogenously generated or externally applied
BDNF could rescue detrimental developmental changes caused
by CPP. The development of dendrites in newborn granule cells is
under strong regulatory control of the internal and external
environments (Aimone et al., 2014). REFERENCES Bergami, M., Masserdotti, G., Temprana, S. G., Motori, E., Eriksson, T. M.,
Göbel, J., et al. (2015). A critical period for experience-dependent remodeling
of adult-born neuron connectivity. Neuron 85, 710–717. doi: 10.1016/j.neuron. 2015.01.001 Acharya, M. M., Martirosian, V., Chmielewski, N. N., Hanna, N., Tran, K. K.,
Liao, A. C., et al. (2015). Stem cell transplantation reverses chemotherapy-
induced cognitive dysfunction. Cancer Res. 74, 676–686. doi: 10.1158/0008-
5472.CAN-14-2237 Bergami, M., Rimondini, R., Santi, S., Blum, R., Götz, M., and Canossa, M. (2008). Deletion of TrkB in adult progenitors alters newborn neuron integration into
hippocampal circuits and increases anxiety-like behavior. Proc. Natl. Acad. Sci. U S A 105, 15570–15575. doi: 10.1073/pnas.0803702105 Ahles, T. A., and Saykin, A. J. (2007). Candidate mechanisms for chemotherapy-
induced cognitive changes. Nat. Rev. Cancer 7, 192–201. doi: 10.1038/nrc2073 Boda,
B.,
Alberi,
S.,
Nikonenko,
I.,
Node-Langlois,
R.,
Jourdain,
P.,
Moosmayer, M., et al. (2004). The mental retardation protein PAK3 contributes
to synapse formation and plasticity in hippocampus. J. Neurosci. 24,
10816–10825. doi: 10.1523/JNEUROSCI.2931-04.2004 Aimone, J. B., Li, Y., Lee, S. W., Clemenson, G. D., Deng, W., and Gage, F. H. (2014). Regulation and function of adult neurogenesis: from genes to cognition. Physiol. Rev. 94, 991–1026. doi: 10.1152/physrev.00004.2014 Ambrogini, P., Cuppini, R., Lattanzi, D., Ciuffoli, S., Frontini, A., and Fanelli, M. (2010). Synaptogenesis in adult-generated hippocampal granule cells is affected
by behavioral experiences. Hippocampus 20, 799–810. doi: 10.1002/hipo. 20679 Briones, T. L., and Woods, J. (2011). Chemotherapy-induced cognitive
impairment
is
associated
with
decreases
in
cell
proliferation
and
histone modifications. BMC Neurosci. 12:e124. doi: 10.1186/1471-2202
-12-124 Anderson-Hanley, C., Sherman, M. L., Riggs, R., Agocha, V. B., and Compas, B. E. (2003). Neuropsychological effects of treatments for adults with cancer: a
meta-analysis and review of the literature. J. Int. Neuropsychol. Soc. 9, 967–982. doi: 10.1017/s1355617703970019 Castellon, S. A., Ganz, P. A., Bower, J. E., Petersen, L., Abraham, L.,
and Greendale, G. A. (2004). Neurocognitive performance in breast
cancer
survivors
exposed
to
adjuvant
chemotherapy
and
tamoxifen. J. Clin. Exp. Neuropsychol. 26,
955–969. doi:
10.1080/138033904905
10905 Andreotti, C., Root, J. C., Ahles, T. A., McEwen, B. S., and Compas, B. E. (2015). Cancer, coping, and cognition: a model for the role of stress reactivity in
cancer-related cognitive decline. Psychooncology 24, 617–623. doi: 10.1002/
pon.3683 Chan, J. P., Cordeira, J., Calderon, G. A., Iyer, L. K., and Rios, M. (2008). DISCUSSION It remains unclear what
mechanism(s) may underlie abnormal development of new born
granule cells after CPP exposure. BDNF, along with nerve growth
factor (NGF), neurotrophin-3 (NT3) and neurotrophin-4/5
(NT4/5), belongs to a family of closely related, small, secreted
proteins called neurotrophins. Mainly through acting on
tyrosine receptor kinases, BDNF affects neuronal proliferation,
differentiation, survival and morphological maintenance, as well
as dendrite outgrowth (Waterhouse and Xu, 2009). Additionally,
BDNF is known to be involved in numerous processes of
functional and structural synaptic plasticity (Zagrebelsky and
Korte, 2014). Evidence obtained from the hippocampal dentate
gyrus shows that global over-expression of BDNF promotes
growth of dendrites in dentate granule cells (Tolwani et al.,
2002), while deficits in BDNF signaling lead to abnormal
dendritic development of newborn granule cells (Bergami et al.,
2008; Chan et al., 2008; Wang et al., 2015). In addition to
dendritic morphology, it appears that following BDNF deficits,
the dendritic spines display more dramatic and specific changes
than dendrite morphology (Zagrebelsky and Korte, 2014). The features of dendritic abnormality after CPP treatment
are very similar to those caused by BDNF deficits, which
include decreased dendrite branching, length and complexity In conclusion, adult-born granule cells in the presence of
CPP, a widely used anti-cancer medication, display abnormal
dendritic morphologies and fewer dendritic spines, which
may
relate
to
chemotherapeutic
agents-induced
cognitive
dysfunction. ACKNOWLEDGMENTS This study was supported by a grant from the National Natural
Science Foundation of China (No: 31371183) to R-ZZ. The
authors would like to thank Prof. Yanxun Liu and Mrs. Qian Zhu
in the Shandong University School of Public Health for their help
in statistical analyses. AUTHOR CONTRIBUTIONS LW did most of animal experiments, behavioral studies and
imaging data processing. DG and QL did immunohistochemistry
and data analysis. FG, XS and XW did confocal imaging. FW helped in experimental design. R-ZZ designed the study and
wrote the article. REFERENCES doi: 10.1182/blood-2015-02-627414 Tolwani, R. J., Buckmaster, P. S., Varma, S., Cosgaya, J. M., Wu, Y., Suri, C., et al. (2002). BDNF overexpression increases dendrite complexity in hippocampal
dentate gyrus. Neuroscience 114, 795–805. doi: 10.1016/s0306-4522(02)0
0301-9 Janelsins, M. C., Kohli, S., Mohile, S. G., Usuki, K., Ahles, T. A., and Morrow, G. R. (2011). An
update
on
cancer-
and
chemotherapy-related
cognitive
dysfunction. Semin. Oncol. 38, 431–438. doi: 10.1053/j.seminoncol.2011. 03.014 Turrigiano, G. (2007). Homeostatic signaling: the positive side of negative
feedback. Curr. Opin. Neurobiol. 17, 318–324. doi: 10.1016/j.conb.2007. 04.004 Jim, H. S., Phillips, K. M., Chait, S., Faul, L. A., Popa, M. A., Lee, Y. H., et al. (2012). Meta-analysis of cognitive functioning in breast cancer survivors previously
treated with standard-dose chemotherapy. J. Clin. Oncol. 30, 3578–3587. doi: 10.1200/JCO.2011.39.5640 Tyler, W. J., and Pozzo-Miller, L. (2003). Miniature synaptic transmission and
BDNF modulate dendritic spine growth and form in rat CA1 neurones. J. Physiol. 553, 497–509. doi: 10.1113/jphysiol.2003.052639 Vardy, J., and Tannock, I. (2007). Cognitive function after chemotherapy in adults
with solid tumors. Crit. Rev. Oncol. Hematol. 63, 183–202. doi: 10.1016/j. critrevonc.2007.06.001 Kohler, S. J., Williams, N. I., Stanton, G. B., Cameron, J. L., and Greenough, W. T. (2011). Maturation time of new granule cells in the dentate gyrus of adult
macaque monkeys exceeds six months. Proc. Natl. Acad. Sci. U S A 108,
10326–10331. doi: 10.1073/pnas.1017099108 Wang, L., Chang, X., She, L., Xu, D., Huang, W., and Poo, M. M. (2015). Autocrine
action of BDNF on dendrite development of adult-born hippocampal neurons. J. Neurosci. 35, 8384–8393. doi: 10.1523/JNEUROSCI.4682-14.2015 Lindner, O. C., Phillips, B., McCabe, M. G., Mayes, A., Wearden, A., Varese, F.,
et al. (2014). A meta-analysis of cognitive impairment following adult cancer
chemotherapy. Neuropsychology 28, 726–740. doi: 10.1037/neu0000064 Waterhouse, E. G., and Xu, B. (2009). New insights into the role of brain-
derived neurotrophic factor in synaptic plasticity. Mol. Cell. Neurosci. 42,
81–89. doi: 10.1016/j.mcn.2009.06.009 Lyons, L., ElBeltagy, M., Bennett, G., and Wigmore, P. (2011). The effects of
cyclophosphamide on hippocampal cell proliferation and spatial working
memory in rat. PLoS One 6:e21445. doi: 10.1371/journal.pone.0021445 Wefel, J. S., Kesler, S. R., Noll, K. R., and Schagen, S. B. (2015). Clinical
characteristics, pathophysiology, and management of noncentral nervous
system cancer-related cognitive impairment in adults. CA Cancer J. Clin. 65,
123–138. doi: 10.3322/caac.21258 Matsuzaki, M., Ellis-Davies, G. C., Nemoto, T., Miyashita, Y., Iino, M., Kasai, H.,
et al. (2001). REFERENCES doi: 10.1016/j. neuroscience.2015.06.016 Sahay, A., Scobie, K. N., Hill, A. S., O’Carroll, C. M., Kheirbek, M. A.,
Burghardt, N. S., et al. (2011). Increasing adult hippocampal neurogenesis
is
sufficient
to
improve
pattern
separation. Nature
472,
466–470. doi: 10.1038/nature09817 ElBeltagy, M., Mustafa, S., Umka, J., Lyons, L., Salman, A., Dormon, K., et al. (2012). The effect of 5-fluorouracil on the long term survival and proliferation
of cells in the rat hippocampus. Brain Res. Bull. 88, 514–518. doi: 10.1016/j. brainresbull.2012.05.005 Seigers, R., Pourtau, L., Schagen, S. B., van Dam, F. S., Koolhaas, J. M.,
Konsman, J. P., et al. (2010). Inhibition of hippocampal cell proliferation by
methotrexate in rats is not potentiated by the presence of a tumor. Brain Res. Bull. 81, 472–476. doi: 10.1016/j.brainresbull.2009.10.006 Gao, F., Song, X., Zhu, D., Wang, X., Hao, A., Nadler, J. V., et al. (2015). Dendritic
morphology, synaptic transmission, and activity of mature granule cells born
following pilocarpine-induced status epilepticus in the rat. Front. Cell Neurosci. 9:e384. doi: 10.3389/fncel.2015.00384 Seigers, R., Schagen, S. B., Beerling, W., Boogerd, W., van Tellingen, O.,
van Dam, F. S., et al. (2008). Long-lasting suppression of hippocampal
cell proliferation and impaired cognitive performance by methotrexate
in the rat. Behav. Brain Res. 186, 168–175. doi: 10.1016/j.bbr.2007. 08.004 Grutzendler, J., Kasthuri, N., and Gan, W.-B. (2002). Long-term dendritic spine
stability in the adult cortex. Nature 420, 812–816. doi: 10.1038/nature01276 Hutchinson, A. D., Hosking, J. R., Kichenadasse, G., Mattiske, J. K., and Wilson, C. (2012). Objective and subjective cognitive impairment following chemotherapy
for cancer: a systematic review. Cancer Treat. Rev. 38, 926–934. doi: 10.1016/j. ctrv.2012.05.002 Seigers, R., Schagen, S. B., Coppens, C. M., van der Most, P. J., van Dam, F. S.,
Koolhaas, J. M., et al. (2009). Methotrexate decreases hippocampal cell
proliferation and induces memory deficits in rats. Behav. Brain Res. 201,
279–284. doi: 10.1016/j.bbr.2009.02.025 Irwin, M. L., McTiernan, A., Bernstein, L., Gilliland, F. D., Baumgartner, R.,
Baumgartner, K., et al. (2004). Physical activity levels among breast cancer
survivors. Med. Sci. Sports Exerc. 36, 1484–1491. Šišková, Z., Justus, D., Kaneko, H., Friedrichs, D., Henneberg, N., Beutel, T.,
et al. (2014). Dendritic structural degeneration is functionally linked to
cellular hyperexcitability in a mouse model of Alzheimer’s disease. Neuron 84,
1023–1033. doi: 10.1016/j.neuron.2014.10.024 Iyer, N. S., Balsamo, L. M., Bracken, M. B., and Kadan-Lottick, N. S. (2015). Chemotherapy-only treatment effects on long-term neurocognitive
functioning in childhood ALL survivors: a review and meta-analysis. Blood 126,
346–353. REFERENCES Depletion of central BDNF in mice impedes terminal differentiation of new
granule neurons in the adult hippocampus. Mol. Cell. Neurosci. 39, 372–383. doi: 10.1016/j.mcn.2008.07.017 Babus, L. W., Little, E. M., Keenoy, K. E., Minami, S. S., Chen, E., Song, J. M., et al. (2011). Decreased dendritic spine density and abnormal spine morphology
in Fyn knockout mice. Brain Res. 1415, 96–102. doi: 10.1016/j.brainres.2011. 07.059 Christie, L.-A., Acharya, M. M., Parihar, V. K., Nguyen, A., Martirosian, V.,
and Limoli, C. L. (2012). Impaired cognitive function and hippocampal June 2017 | Volume 11 | Article 171 Frontiers in Cellular Neuroscience | www.frontiersin.org 8 Wu et al. Cyclophosphamide and Dendritic Development neurogenesis following cancer chemotherapy. Clin. Cancer Res. 18, 1954–1965. doi: 10.1158/1078-0432.CCR-11-2000 Noguchi, J., Matsuzaki, M., Ellis-Davies, G. C., and Kasai, H. (2005). Spine-neck
geometry determines NMDA receptor-dependent Ca2+ signaling in dendrites. Neuron 46, 609–622. doi: 10.1016/j.neuron.2005.03.015 neurogenesis following cancer chemotherapy. Clin. Cancer Res. 18, 1954–1965. doi: 10.1158/1078-0432.CCR-11-2000 Christian, K. M., Song, H., and Ming, G. L. (2014). Functions and dysfunctions
of adult hippocampal neurogenesis. Annu. Rev. Neurosci. 37, 243–262. doi: 10.1146/annurev-neuro-071013-014134 Nokia, M. S., Anderson, M. L., and Shors, T. J. (2012). Chemotherapy disrupts
learning, neurogenesis and theta activity in the adult brain. Eur. J. Neurosci. 36,
3521–3530. doi: 10.1111/ejn.12007 Clelland, C. D., Choi, M., Romberg, C., Clemenson, G. D. Jr., Fragniere, A.,
Tyers, P., et al. (2009). A functional role for adult hippocampal neurogenesis in
spatial pattern separation. Science 325, 210–213. doi: 10.1126/science.1173215 Ono, M., Ogilvie, J. M., Wilson, J. S., Green, H. J., Chambers, S. K., Ownsworth, T.,
et al. (2015). A meta-analysis of cognitive impairment and decline associated
with adjuvant chemotherapy in women with breast cancer. Front. Oncol. 5:e59. doi: 10.3389/fonc.2015.00059 Cyclophosphamide
FDA
Packet
Insert. (2013). Reference
ID:
3304966. Available online at: https://www.accessdata.fda.gov/drugsatfda_docs/label/
2013/012141s090,012142s112lbl.pdf Opendak, M., and Gould, E. (2015). Adult neurogenesis: a substrate for
experience-dependent change. Trends Cogn. Sci. 19, 151–161. doi: 10.1016/j. tics.2015.01.001 Dickstein, D. L., Brautigam, H., Stockton, S. D. Jr., Schmeidler, J., and Hof, P. R. (2010). Changes in dendritic complexity and spine morphology in transgenic
mice expressing human wild-type tau. Brain Struct. Funct. 214, 161–179. doi: 10.1007/s00429-010-0245-1 Rugo, H. S., and Ahles, T. (2003). The impact of adjuvant therapy for breast cancer
on cognitive function: current evidence and directions for research. Semin. Oncol. 30, 749–762. doi: 10.1053/j.seminoncol.2003.09.008 Dietrich, J., Prust, M., and Kaiser, J. (2015). Chemotherapy, cognitive impairment
and hippocampal toxicity. Neuroscience 309, 224–232. Zhan, R.-Z., and Nadler, J. V. (2009). Enhanced tonic GABA current in
normotopic and hilar ectopic dentate granule cells after pilocarpine-induced
status epilepticus. J. Neurophysiol. 102, 670–681. doi: 10.1152/jn.00147.2009 Frontiers in Cellular Neuroscience | www.frontiersin.org REFERENCES Dendritic spine geometry is critical for AMPA receptor expression
in hippocampal CA1 pyramidal neurons. Nat. Neurosci. 4, 1086–1092. doi: 10.1038/nn736 Yang, M., Kim, J. S., Song, M. S., Kim, S. H., Kang, S. S., Bae, C. S., et al. (2010). Cyclophosphamide impairs hippocampus-dependent learning and
memory in adult mice: possible involvement of hippocampal neurogenesis in
chemotherapy-induced memory deficits. Neurobiol. Learn. Mem. 93, 487–494. doi: 10.1016/j.nlm.2010.01.006 Minisini, A., Atalay, G., Bottomley, A., Puglisi, F., Piccart, M., and Biganzoli, L. (2004). What is the effect of systemic anticancer treatment on cognitive
function? Lancet Oncol. 5, 273–282. doi: 10.1016/s1470-2045(04)01465-2 Mustafa, S., Walker, A., Bennett, G., and Wigmore, P. M. (2008). 5-Fluorouracil
chemotherapy affects spatial working memory and newborn neurons in the
adult rat hippocampus. Eur. J. Neurosci. 28, 323–330. doi: 10.1111/j.1460-9568. 2008.06325.x Yasumatsu, N., Matsuzaki, M., Miyazaki, T., Noguchi, J., and Kasai, H. (2008). Principles of long-term dynamics of dendritic spines. J. Neurosci. 28,
13592–13608. doi: 10.1523/JNEUROSCI.0603-08.2008 June 2017 | Volume 11 | Article 171 Frontiers in Cellular Neuroscience | www.frontiersin.org 9 Wu et al. Cyclophosphamide and Dendritic Development Cyclophosphamide and Dendritic Development Zagrebelsky, M., and Korte, M. (2014). Form follows function: BDNF and
its involvement in sculpting the function and structure of synapses. Neuropharmacology
76,
628–638. doi:
10.1016/j.neuropharm.2013. 05.029 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. Zhan, R.-Z., and Nadler, J. V. (2009). Enhanced tonic GABA current in
normotopic and hilar ectopic dentate granule cells after pilocarpine-induced
status epilepticus. J. Neurophysiol. 102, 670–681. doi: 10.1152/jn.00147.2009 Copyright © 2017 Wu, Guo, Liu, Gao, Wang, Song, Wang and Zhan. 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. p y
j
Zhao, C., Jou, J., Wolff, L. J., Sun, H., and Gage, F. H. (2014). Spine morphogenesis
in newborn granule cells is differentially regulated in the outer and
middle molecular layers. J. Comp. Neurol. 522, 2756–2766. doi: 10.1002/cne. 23581 June 2017 | Volume 11 | Article 171 Frontiers in Cellular Neuroscience | www.frontiersin.org 10
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КРИМІНАЛЬНА ВІДПОВІДАЛЬНІСТЬ ЗА ТЕРОРИСТИЧНУ ДІЯЛЬНІСТЬ В ІСПАНІЇ
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СТАНОВЛЕННЯ ТА РОЗВИТОК ПРАВОВОЇ ДЕРЖАВИ: ПРОБЛЕМИ ТЕОРІЇ ТА ПРАКТИКИ
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DOI https://doi.org/10.36059/978-966-397-287-9-116 DOI https://doi.org/10.36059/978-966-397-287-9-116 КРИМІНАЛЬНА ВІДПОВІДАЛЬНІСТЬ
ЗА ТЕРОРИСТИЧНУ ДІЯЛЬНІСТЬ В ІСПАНІЇ Попович Ольга Станіславівна
кандидат юридичних наук,
науковий співробітник відділу дослідження проблем кримінального права
Науково-дослідного інституту вивчення проблем злочинності
імені академіка В. В. Сташиса
Національної академії правових наук України
м. Харків, Україна Сучасний міжнародний тероризм є одним з нових факторів впливу на
світову політику, стан і розвиток міжнародної системи постбіполярного
часу. Терористичні акти дедалі частіше спрямовані на масові людські
жертви, спричиняють руйнування матеріальних і підрив духовних
цінностей, провокують війни, недовіру та ненависть між соціальними,
національними і релігійними групами, що сприяє виникненню і
поглибленню системних криз. Зарубіжний досвід боротьби з терористичними актами був предметом
наукового інтересу багатьох вчених. Іспанський досвід боротьби з
терористичною діяльністю є предметом наукових досліджень багатьох
українських і зарубіжних правознавців. Їх надбання становлять собою
цінне підґрунтя для набуття наукових знань у цьому напрямку. Кримінальну відповідальність за низку терористичних злочинів містить
книга ІІ «Злочини та покарання» в розділу XXII «Злочини проти публічного
порядку» (глава VII «Про терористичні організації і групи та терористичні
злочини») КК Королівства Іспанія [1]. Ця глава складається з 2-х частин
«Про терористичні організації і групи» (ст. 571) і «Про терористичні
злочини» (статті 572‒580). Особливо корисним для пізнання вважаємо ч. 1
ст. 573 КК Іспанії, де терористичним злочином визнано: (1) будь-який
тяжкий злочин проти Корони, життя, свободи, фізичної недоторканості,
моральної цілісності особи, статевої свободи й недоторканості, її власності;
(2) проти природних ресурсів, навколишнього природного середовища,
суспільного здоров’я; (3) злочинне діяння, що може спричинити ризики
катастрофи, пожежі; (4) злочини, що становить собою збройний напад,
володіння, зберігання і торгівлю зброї, боєприпасів і вибухових речовин,
передбачених цим Кодексом; (5) захоплення кораблів, літаків чи інших
транспортних засобів, якщо воно вчинено з такими цілями, як-от:
(а) повалення конституційного ладу, припинення або тяжка дестабілізація 432 діяльності економічних, політичних інститутів або соціальних структур
держави; (б) примушування публічної влади вчинити певну дію чи не
вчиняти її; (в) серйозне порушення громадського спокою; (г) серйозна
дестабілізація діяльності міжнародної організації; (д) провокація стану
страху серед населення або його частини. діяльності економічних, політичних інститутів або соціальних структур
держави; (б) примушування публічної влади вчинити певну дію чи не
вчиняти її; (в) серйозне порушення громадського спокою; (г) серйозна
дестабілізація діяльності міжнародної організації; (д) провокація стану
страху серед населення або його частини. р
у
р
Згідно з ч. 2 ст. 573 розглядуваного Кодексу терористичними злочинами
також вважаються інформаційні злочини, передбачені у статтях 197bis та
197 [2, с. 80, 81]. Згідно з ч. 2 ст. 573 розглядуваного Кодексу терористичними злочинами
також вважаються інформаційні злочини, передбачені у статтях 197bis та
197 [2, с. 80, 81]. КРИМІНАЛЬНА ВІДПОВІДАЛЬНІСТЬ
ЗА ТЕРОРИСТИЧНУ ДІЯЛЬНІСТЬ В ІСПАНІЇ 3 цієї ж статті за розроблення
біологічної або хімічної зброї, перевезення, володіння чи будь-яке
сприяння іншому поводженню з ядерними речовинами чи матеріалами, які
призначені
для
вироблення
радіоактивного
випромінювання,
або
відповідними пристроями. України. Частиной
2
ст. 574
Кодексу
підвищено
кримінальну
відповідальність у виді позбавлення волі на строк від десяти до двадцяти
років за ті ж дії, скоєні з метою тероризму, стосовно ядерної, хімічної,
радіоактивної або біологічної зброї, боєприпасів, речовин до неї або
пристроїв. Таке ж покарання передбачено у ч. 3 цієї ж статті за розроблення
біологічної або хімічної зброї, перевезення, володіння чи будь-яке
сприяння іншому поводженню з ядерними речовинами чи матеріалами, які
призначені
для
вироблення
радіоактивного
випромінювання,
або
відповідними пристроями. України. Частиной
2
ст. 574
Кодексу
підвищено
кримінальну
відповідальність у виді позбавлення волі на строк від десяти до двадцяти
років за ті ж дії, скоєні з метою тероризму, стосовно ядерної, хімічної,
радіоактивної або біологічної зброї, боєприпасів, речовин до неї або
пристроїв. Таке ж покарання передбачено у ч. 3 цієї ж статті за розроблення
біологічної або хімічної зброї, перевезення, володіння чи будь-яке
сприяння іншому поводженню з ядерними речовинами чи матеріалами, які
призначені
для
вироблення
радіоактивного
випромінювання,
або
відповідними пристроями. р
р
За ч. 1 ст. 575 КК Іспанії карається позбавленням волі на строк від двох
до п’яти років особа, яка пройшла бойове або військове навчання
(підготовку), а також навчання (підготовку) для розроблення біологічної
або
хімічної
зброї,
виготовлення
вогненебезпечних,
вибухових,
задушливих, запалювальних чи інших небезпечних речовин, скоєне з
метою вчинення терористичних злочинів. Згідно з частинами 2 і 3 цієї ж
статті таке ж покарання встановлене за різного роду підготовку до вчинення
злочинів, передбачених ч. 1 ст. 575 цього Кодексу, зокрема, за надання послуг
зв’язку (в мережі Інтернет тощо), за підготовку документів, якщо ці діяння
вчинені умисно і навіть на території інших держав. Стаття 576 передбачено
покарання у виді позбавлення волі на строк від п’яти до десяти років і штрафу
від трьох до п’яти його мінімальних розмірів за будь-яке пряме, а також
непряме, використання будь-яких товарів чи цінностей для сприяння
терористичній діяльності, тобто фінансування тероризму. Посилене покарання
може бути призначено, якщо товари чи цінності передано особі, яка є
відповідальною за вчинення терористичного злочину. За фінансування
терористичної діяльності у ст. 576 КК Іспанії передбачено кримінальну
відповідальність і юридичної особи у виді штрафу. Відповідно до ч. 1 ст. 577 КК Іспанії будь-яке співробітництво з
терористичною групою або організацією з метою вчинення терористичного
злочину карається позбавленням волі на строк від п’яти до десяти років. У ч. КРИМІНАЛЬНА ВІДПОВІДАЛЬНІСТЬ
ЗА ТЕРОРИСТИЧНУ ДІЯЛЬНІСТЬ В ІСПАНІЇ Статтею 573bis КК Іспанії встановлено покарання, що призначаються за
вчинення терористичних злочинів, передбачених у попередній нормі. Відповідно до § 1 – 4 ч. 2 ст. 573bis цього Кодексу скоєння терористичного
злочину карається позбавленням волі: Статтею 573bis КК Іспанії встановлено покарання, що призначаються за
вчинення терористичних злочинів, передбачених у попередній нормі. Відповідно до § 1 – 4 ч. 2 ст. 573bis цього Кодексу скоєння терористичного
злочину карається позбавленням волі: − на максимальний строк у випадку спричинення смерті хоча б одній
особі; – на строк від двадцяти до двадцяти п’яти років, якщо злочинні дії
супроводжувалися викраденням або незаконним позбавленням волі
людини, внаслідок яких особу не знайдено; – строком від п’ятнадцяти до двадцяти років, якщо злочинні дії
призвели до аборту або наслідків, передбачених ст. 149 (тілесні
ушкодження тяжкі), ст. 150 (тілесні ушкодження середньої тяжкості),
ст. 157 або 158 (пошкодження плода людини або його захворювання),
ст. 346 (вчинені загальнонебезпечним способом різного роду руйнування)
чи ст. 351 цього Кодексу (небезпечної для життя або здоров’я особи
пожежі); – строком від десяти до п’ятнадцяти років, якщо злочинні дії заподіяли
інше тілесне ушкодження або супроводжувалися насильством до особи чи
погрозами його заподіяти. У ст. 571 розглядуваного Кодексу наведено дефініцію терористичних
груп або організацій. Ними визнано угруповання, які характеризуються
визначеннями, наведеними у § 2 ч. 1 ст. 570bis та § 2 ч. 1 ст. 570ter і які
мають намір або мету вчинення терористичних злочинів. Згідно з ч. 1
ст. 572 цього Кодексу позбавленням волі від восьми до чотирнадцяти років,
забороною займати публічну посаду на строк від шести до чотирнадцяти
років караються діяння особи, яка засновує, організовує чи керує
терористичною групою чи організацією чи сприяє цьому. Частина 1 ст. 574 КК Іспанії містить кримінальну відповідальність у
виді позбавлення волі на строк від восьми до п’ятнадцяти років за
зберігання зброї, боєприпасів, володіння або зберігання вибухових,
запалювальних, легкозаймистих або задушливих речовин, пристроїв чи їх
компонентів, виготовлення, транспортування, продаж, або поставку
зазначених предметів у будь-якій формі, їх використання чи розміщення з
метою вчинення терористичних злочинів, перелічених у ч. 1 ст. 573 КК 433 України. Частиной
2
ст. 574
Кодексу
підвищено
кримінальну
відповідальність у виді позбавлення волі на строк від десяти до двадцяти
років за ті ж дії, скоєні з метою тероризму, стосовно ядерної, хімічної,
радіоактивної або біологічної зброї, боєприпасів, речовин до неї або
пристроїв. Таке ж покарання передбачено у ч. КРИМІНАЛЬНА ВІДПОВІДАЛЬНІСТЬ
ЗА ТЕРОРИСТИЧНУ ДІЯЛЬНІСТЬ В ІСПАНІЇ Київ, 30 верес. 2016 р.). Київ : Нац. акад. прокуратури України,
2016. 432 с. DOI https://doi.org/10.36059/978-966-397-287-9-117 КРИМІНАЛЬНА ВІДПОВІДАЛЬНІСТЬ
ЗА ТЕРОРИСТИЧНУ ДІЯЛЬНІСТЬ В ІСПАНІЇ 3 цієї ж норми визначено покарання у виді штрафу за вчинення цього
злочину з необережності. Згідно з ч. 1 ст. 578 позбавленням волі на строк
від одного до трьох років карається: (а) публічне виправдання або
звеличення терористичних злочинів, передбачених статтями 572 – 577
цього Кодексу Іспанії, осіб, які їх учинили; (б) зневага, дискредитація або
приниження жертв зазначених злочинів, а також членів їх сімей. Відповідно до ч. 1 ст. 579 поширення повідомлень і гасел з метою
підбурювання інших осіб до вчинення терористичних злочинів карається
штрафом. Крім того, у ст. 579bis Кодексу Іспанії передбачено можливість
призначення особам, засудженим за терористичні злочини, додаткових
покарань у виді позбавлення права займатися певними видами діяльності
[2, с. 80‒82]. Статтею 580 досліджуваного Кодексу спеціально закріплено 434 обов’язок визнання винесених раніше вироків іноземних судів за скоєння
терористичних злочинів з обтяжуючою обставиною у виді рецидиву
[1, с. 245]. Як відмічають фахівці-дослідники, проаналізовані вище норми
КК Іспанії забезпечують сувору кримінальну відповідальність за вчинення
злочинів, пов’язаних з тероризмом [49, с. 82]. Література:
1. Кримінальний кодекс Королівства Іспанія / під ред. В. Л. Менчин-
ського. Перекл. на укр. мову О. В. Лішевської. Київ : ОВК, 2017. 284 с. 2. Гуркевич В. Кримінальна відповідальність за терористичну
діяльність в Іспанії. Протидія терористичній діяльності: міжнародний
досвід і його актуальність для України : матеріали між нар. наук.-практ. конф. (м. Київ, 30 верес. 2016 р.). Київ : Нац. акад. прокуратури України,
2016. 432 с. Література:
1. Кримінальний кодекс Королівства Іспанія / під ред. В. Л. Менчин-
ського. Перекл. на укр. мову О. В. Лішевської. Київ : ОВК, 2017. 284 с. 2. Гуркевич В. Кримінальна відповідальність за терористичну
діяльність в Іспанії. Протидія терористичній діяльності: міжнародний
досвід і його актуальність для України : матеріали між нар наук практ Література:
1. Кримінальний кодекс Королівства Іспанія / під ред. В. Л. Менчин-
ського. Перекл. на укр. мову О. В. Лішевської. Київ : ОВК, 2017. 284 с. Література:
1. Кримінальний кодекс Королівства Іспанія / під ред. В. Л. Менчин-
ського. Перекл. на укр. мову О. В. Лішевської. Київ : ОВК, 2017. 284 с. 2. Гуркевич В. Кримінальна відповідальність за терористичну
діяльність в Іспанії. Протидія терористичній діяльності: міжнародний
досвід і його актуальність для України : матеріали між нар. наук.-практ. конф. (м. Київ, 30 верес. 2016 р.). Київ : Нац. акад. прокуратури України,
2016. 432 с. 2. Гуркевич В. Кримінальна відповідальність за терористичну
діяльність в Іспанії. Протидія терористичній діяльності: міжнародний
досвід і його актуальність для України : матеріали між нар. наук.-практ. конф. (м. Пустова Наталія Олександрівна у
др
аспірантка кафедри кримінального права і кримінології факультету № 1
Інституту з підготовки фахівців для підрозділів Національної поліції
Львівського державного університету внутрішніх справ
м. Львів, Україна Кримінальне право розвивається циклічно під впливом двох тенденції,
які споконвіку конкурують між собою. Перша – посилює кримінальну
відповідальність, яка виражається у розширенні її підстав шляхом
криміналізації, надмірної диференціації, а також у підвищенні суворості
покарання, скороченні можливостей для звільнення від кримінальної
відповідальності та покарання тощо. Друга – пом’якшує кримінальну
відповідальність, що полягає у декриміналізації та депеналізації,
пом’якшенні інших кримінально-правових наслідків вчинення злочину. Динамізм
кримінального
законодавства
залежить
від
низки
різноманітних чинників: політичних, соціальних, економічних, міжнародно-
правових. Неабиякий вплив також має наука кримінального права і практика
застосування приписів Кримінального кодексу України (далі – КК 435
|
https://openalex.org/W2077240908
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https://europepmc.org/articles/pmc3610620?pdf=render
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English
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The Role of the Arabidopsis Exosome in siRNA–Independent Silencing of Heterochromatic Loci
|
PLOS genetics
| 2,013
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cc-by
| 15,682
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Abstract The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: chekanovaj@umkc.edu * E-mail: chekanovaj@umkc.edu . These authors contributed equally to this work. . These authors contributed equally to this work. The exosome complex plays a central role in RNA metabolism
in eukaryotes. Evolutionarily conserved from archaea to humans,
the exosome is a stable complex of RNase-like and RNA binding
proteins that catalyzes 39 to 59 processing and decay of various
RNA substrates [15]. The current view of eukaryotic exosome
structure is based mostly on studies done in yeast and human. The
eukaryotic exosome has nuclear and cytoplasmic forms that share
ten components. The key structural feature is a nine-subunit
donut-shaped structure called the exosome ring. Six of the
subunits, RNase PH domain-containing proteins Rrp41, Rrp42,
Rrp43, Rrp45, Rrp46 and Mtr3, are organized into a hexameric
ring, capped on one side by a trimer of subunits that contain S1
and KH RNA binding domains (Rrp40, Rrp4 and Csl4) [16,17]. The 9-subunit ring structure has no catalytic activity in yeast and
human, due to amino acid replacements that disable binding of
RNA, phosphate ion, or catalysis [16,17]. The exosome active sites
are contributed by the tenth protein, Rrp44 (Dis3), which has
endonucleolytic and exonucleolytic activities and considered to be
the tenth subunit of the exosome core [18,19]. In addition to
Rrp44, the nuclear form of the eukaryotic exosome is associated
with a second active 39 to 59 exonuclease, Rrp6 [20,21]. Most
functions of the exosome are dependent on cofactors. One of the
notable complexes associated with the nuclear exosome is the Trf-
Air-Mtr4 polyadenylation (TRAMP) complex endowed with a
poly(A) polymerase activity that stimulates degradation [22–24]. Jun-Hye Shin., Hsiao-Lin V. Wang., Jinwon Lee, Brandon L. Dinwiddie, Dmitry A. Belostotsky,
Julia A. Chekanova* School of Biological Sciences, University of Missouri–Kansas City, Kansas City, Missouri, United States of America Abstract The exosome functions throughout eukaryotic RNA metabolism and has a prominent role in gene silencing in yeast. In
Arabidopsis, exosome regulates expression of a ‘‘hidden’’ transcriptome layer from centromeric, pericentromeric, and other
heterochromatic loci that are also controlled by small (sm)RNA-based de novo DNA methylation (RdDM). However, the
relationship between exosome and smRNAs in gene silencing in Arabidopsis remains unexplored. To investigate whether
exosome interacts with RdDM, we profiled Arabidopsis smRNAs by deep sequencing in exosome and RdDM mutants and
also analyzed RdDM-controlled loci. We found that exosome loss had a very minor effect on global smRNA populations,
suggesting that, in contrast to fission yeast, in Arabidopsis the exosome does not control the spurious entry of RNAs into
smRNA pathways. Exosome defects resulted in decreased histone H3K9 dimethylation at RdDM-controlled loci, without
affecting smRNAs or DNA methylation. Exosome also exhibits a strong genetic interaction with RNA Pol V, but not Pol IV,
and physically associates with transcripts produced from the scaffold RNAs generating region. We also show that two
Arabidopsis rrp6 homologues act in gene silencing. Our data suggest that Arabidopsis exosome may act in parallel with
RdDM in gene silencing, by epigenetic effects on chromatin structure, not through siRNAs or DNA methylation. Citation: Shin J-H, Wang H-LV, Lee J, Dinwiddie BL, Belostotsky DA, et al. (2013) The Role of the Arabidopsis Exosome in siRNA–Independent Silencing of
Heterochromatic Loci. PLoS Genet 9(3): e1003411. doi:10.1371/journal.pgen.1003411 Received December 19, 2012; Accepted February 11, 2013; Published March 28, 2013 pyright: 2013 Shin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, whic
e, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2013 Shin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
use, distribution, and reproduction in any medium, provided the original author and source are credited. et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
roduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by grants from USDA to DAB and JAC (grant #35301-18207), from MLSRB to JAC (grant #13230), NSF to DAB and JAC
(grant #0724168), and NIH to DAB and JAC (#GM073872). The Role of the Arabidopsis Exosome in siRNA–
Independent Silencing of Heterochromatic Loci Jun-Hye Shin., Hsiao-Lin V. Wang., Jinwon Lee, Brandon L. Dinwiddie, Dmitry A. Belostotsky
Julia A. Chekanova* Author Summary To maintain genomic stability and prevent expansion of
invasive genomic sequences such as transposable ele-
ments (TEs), eukaryotes have evolved defensive mecha-
nisms to control them. Here, we examine the role of the
Arabidopsis exosome complex in such mechanisms. Evo-
lutionarily
conserved
from
archaea
to
humans,
the
exosome is a stable complex of RNase-like and RNA
binding
proteins
that
plays
a
central
role
in
RNA
metabolism in eukaryotes. Depletion of the exosome
allows some repetitive sequences to escape from silencing. Most of these transcripts emanate from centromeric and
pericentromeric chromosomal regions and other hetero-
chromatic loci, and many derive from repetitive and
transposable elements. In plants, TEs are targeted for de
novo DNA methylation by smRNA–mediated pathways. However, we found that exosome depletion has only
minor effects on smRNA populations that are acting in the
main silencing mechanism in Arabidopsis, siRNAs–depen-
dent DNA methylation RdDM. Instead, exosome depletion
affects histone H3K9 dimethylation, an epigenetic mark
that affects chromatin structure and thus alters transcrip-
tion. Our data suggest that the exosome collaborates in
gene silencing, likely acting in a parallel pathway to other
mechanisms. We
also
propose
that
the
Arabidopsis
exosome may coordinate the transcriptional interplay of
different RNA polymerases to modulate repression of
some repetitive sequences. The model system in which siRNA-mediated silencing is the
best understood mechanistically is fission yeast. In S. pombe RNA
Pol II carries out the functions attributed to Pol IV and Pol V in
plants, therefore, it generates both siRNA precursors and scaffold
transcripts to which siRNAs bind at loci that are subject to siRNA-
mediated silencing. Exosome defects in S. pombe were reported to
result in the loss of transcriptional silencing from centromeric,
silent mating type, and telomeric loci [38–40]. In S. pombe, in the
absence of exosome-mediated degradation, abundant aberrant
RNA species enter the RNAi pathway and interfere with
heterochromatic silencing through competition for RNAi biogen-
esis machinery, resulting in the dramatic decrease in centromeric
siRNAs [38–40]. Recently, it was also shown that exosome plays
an important role in remodeling of facultative heterochromatin
[41]. Earlier work in plants also suggested that aberrant RNAs
could enter RNAi pathways unless they are degraded by the 59 to
39 pathway [42]. However, the role of the exosome complex in
smRNA metabolism in Arabidopsis has not been examined. It is
also not known whether the Arabidopsis exosome complex
interacts with the RdDM silencing pathway. Introduction High-throughput
analyses
have
revealed
that
eukaryotic
genomes are pervasively transcribed [1–4], and the majority of
the transcriptional activity takes place outside of protein-coding
genes, producing non-coding RNAs (ncRNAs) derived from
genome regions once thought to be transcriptionally silent,
including intergenic and heterochromatic regions [1–3,5]. Per-
vasive transcription constitutes a risk for the cell, as it can be
associated with expansion of TEs, loss of genomic stability and
defects in gene expression. However, recent studies have also
shown that ncRNAs themselves can have important regulatory
functions, including the establishment and maintenance of the
epigenetic architecture of eukaryotic genomes. In some cases,
long ncRNAs serve directly as molecular scaffolds for recruiting
chromatin modifiers [6,7], whereas in other cases ncRNAs are
processed by the RNAi machinery into short interfering siRNAs
that guide DNA methylation and chromatin modifications to
homologous regions of the genome [8,9]. Thus, RNA-mediated
heterochromatin formation requires an affected region to be
transcribed for transcriptional silencing to occur. Many of the
ncRNA transcripts earned the term ‘‘hidden’’ because they
remain invisible unless RNA degradation is prevented, for
example,
by
inactivation
of
the
degradation
machinery
[1,3,4,10–14], raising the important question of how these
ncRNAs are regulated. March 2013 | Volume 9 | Issue 3 | e1003411 1 PLOS Genetics | www.plosgenetics.org SmRNA–Independent Exosome Silencing biogenesis, RNA Pol IV transcripts are made double-stranded
by
RNA-DEPENDENT
RNA
POLYMERASE
2
(RDR2),
processed into 24 nt siRNA by DICER-LIKE 3 (DCL3), and
then incorporated into ARGONAUTE (AGO4 and possibly
AGO6) to form an AGO-siRNA complex [32–35]. The AGO-
siRNA complex and other RdDM effectors [31,35–37], assemble
on scaffold RNA to form a guiding complex that recruits DNA
methyltransferases and histone methyltransferases to direct the
silencing of specific genomic loci through a mechanism that is not
fully understood. Pol IV is thought to initiate RdDM pathway,
whereas Pol V and AGO4-associated siRNAs function down-
stream from Pol IV to promote DNA methylation by recruiting
the silencing complex to targeted loci. RNA Pol IV, Pol V and Pol
II activities in RdDM are functionally diversified and coordinated;
however, it is not yet clear how they are functionally integrated in
heterochromatin silencing. Author Summary The plant exosome might differ from yeast and human exosomes,
as its ring subunit Atrrp41p appears to retain an active site and
was also shown to have catalytic activity in vitro [1,25]. Our
previous genome-wide study using tiling microarrays to examine
exosome targets in Arabidopsis revealed that a large number of
exosome substrates correspond to ncRNAs originated from
promoters, 59UTRs, intergenic regions, repetitive elements and
TEs [1]. Many of these ncRNAs derive from centromeric and
pericentromeric regions and other heterochromatic loci known to
give rise to smRNAs that participate in silencing of these loci [26]. In Arabidopsis, the main and most-studied pathway for transcrip-
tional gene silencing of repetitive elements and transposons is the
siRNA-based silencing mechanism known as RNA-dependent
DNA methylation (RdDM) [9,27–29]. The effects of exosome
depletion on these ncRNAs and, potentially, on smRNAs are
unlikely to be attributable to indirect effects of exosome depletion
on the expression of RdDM pathway components, since no genes
acting in siRNA biogenesis, siRNA-mediated transcriptional gene
silencing (TGS), DNA methylation or demethylation, or histone
H3K9 modifications were found to be affected in these lines [1]. To answer these questions we employed next-generation
sequencing to profile populations of smRNAs in exosome-depleted
plants, and in mutants of RdDM pathway genes. Unexpectedly,
we found that loss of the exosome subunits had little effect on the
global populations of smRNAs and had no affect on the level of
DNA methylation in examined RdDM loci; rather, it resulted in a
reduction of histone H3K9 dimethylation. We propose that the
Arabidopsis exosome may coordinate the transcriptional interplay
of RNA polymerases Pol II, Pol V and Pol IV, to achieve the
appropriate level of transcriptional repression of heterochromatic
loci. Results RdDM induces de novo methylation of cytosines in all sequence
contexts at the region of siRNA–DNA or siRNA-RNA sequence
homology. This silencing pathway requires two plant-specific
RNA polymerases, Pol IV and Pol V, specializing in transcrip-
tional gene silencing (TGS) [28], although transcriptional activity
of Arabidopsis Pol II was also reported to be involved in siRNA-
directed gene silencing [30]. The mechanistic details of RNA-
dependent silencing are not fully understood and also appear to
vary from one genomic location to another, but the RdDM
pathway likely consists of three main steps: (i) siRNA production
from transcripts that are likely transcribed by RNA Pol IV [9], (ii)
synthesis of non-coding RNAs that could serve as scaffolds by
RNA Pol V and/or Pol II at some of the loci [30,31], and (iii)
assembly of AGO-siRNA effector complexes to recruit methyla-
tion machinery to complementary sequences [9]. In siRNA March 2013 | Volume 9 | Issue 3 | e1003411 Exosome depletion does not affect smRNA profiles Previously, we found that the majority of transcripts upregulated
in RRP4 and RRP41 exosome depletion mutants originate from
the promoters, repeats, intergenic, and siRNA generating regions
[1]. Most of these regions harbor repeats and TEs that are known
to be silenced by RdDM through siRNAs. Since microarray experiments allow estimation of only the
length of affected regions, but not the exact length of affected
transcripts, we set out to examine whether the exosome is involved
in down regulation of these regions through regulating either
quantity or quality of smRNAs. To characterize any changes in
smRNA populations that occur in response to exosome depletion,
we employed next-generation sequencing to deep sequence the
smRNA populations in depletion mutants of exosome subunits March 2013 | Volume 9 | Issue 3 | e1003411 2 PLOS Genetics | www.plosgenetics.org SmRNA–Independent Exosome Silencing and smRNAs between 15- and 32 nt in length were selected and
mapped to the Arabidopsis genome (TAIR version 9). RRP4 and RRP41. Null T-DNA insertion mutations in RRP4 and
RRP41 are lethal; therefore, we used inducible RNA-interference
(iRNAi) constructs to reduce RRP4 and RRP41. The seedlings of
RRP4 (rrp4-i) or RRP41 (rrp41-i) transgenic plants grown on
estradiol-containing medium to induce the RNAi constructs
subsequently exhibit a growth arrest ([1], Figure 1A). We selected
the earliest time-point of estradiol treatment corresponding to the
accumulation of underprocessed 5.8S rRNA species (the hallmark
of the exosome defect), but before growth retardation, to minimize
changes in gene expression that did not result directly from
exosome
depletion
[1]. Small
RNA
libraries
for
Illumina
sequencing were generated from the seedlings of rrp4-i and
rrp41-i iRNAi lines grown with and without estradiol (Table S1) We first examined the smRNAs from the iRNAi transgenes used
for inactivation of RRP4 or RRP41 [1]. As expected, these silencing
cassettes generate silencer sequences corresponding to RRP4 or
RRP41 (mapping to AT1G03360 and AT3G61620 loci). Profiling
silencer sequences by size and by first nucleotide revealed that the
majority of the silencer sequences are 21, 22 and 24 nt and start
with 59U or 59A (Figure S1), suggesting that they are preferentially
loaded into Ago1, Ago2 and Ago4 complexes [43] to silence their
target. Silencer sequences produced from iRNAi transgenes were
filtered out and libraries without silencer reads were termed FLR,
for filtered reads (Table S1). Each library was normalized either to Figure 1. Exosome depletion does not affect populations of
smRNAs corresponding to repeats and transposable
elements To further investigate whether the exosome participates in gene
silencing and interacts with the RdDM pathway, we examined the
transcription patterns of several specific loci regulated through
RdDM. solo LTR and AtSN1 are the heterochromatic loci for
which the role of RdDM players in their silencing and interactions
between them are best-understood [30,31,50–52]. Transcriptional
silencing of solo LTR and AtSN1 heterochromatic loci are
dependent on Pol IV and Pol V [30,31,50–52]. Based on previous
studies, both solo LTR and AtSN1 loci can be subdivided into
region A and an adjacent region B [30,31]. Region A represents
the siRNA-generating region likely transcribed by Pol IV, and
region B gives rise to the ncRNAs that are proposed to serve as a
scaffold for recruiting siRNA-mediated complexes that form
heterochromatin (Figure 4A). Pol V was proposed to produce
transcripts which serve as the scaffolds [31], although in case of
solo LTR, Pol II was also shown to be involved [30]. In Arabidopsis, repeats and TEs are silenced by siRNAs
through RdDM; therefore, we examined the effect of exosome loss
on 20–25 nt smRNAs corresponding specifically to TEs and
repeats. Surprisingly, we saw no changes in the groups of smRNAs
mapped to tandem repeats (TR), inverted repeats (IR), dispersed
repeats (DR) or the group of TEs in both exosome mutants
(Figure 2A and 2B). The diverse heterochromatic siRNAs
participating in TE silencing are mostly 24-mers and are Pol IV-
and/or Pol V-dependent [9]. Most siRNA production relies on Pol
IV, but there are also Pol V-dependent and Pol IV-independent
siRNA-generating loci [44,45]. Therefore, to examine whether the
exosome complex functionally overlaps with the components of
the RdDM pathway, we constructed lines containing rrp4-i or
rrp41-i iRNAi and mutations affecting Pol IV, Pol V, RDR2 and
DCL3, which are nrpd1, nrpe1, dcl3 and rdr2 respectively (allele
numbers provided in Methods). This approach also allowed us to
confirm that smRNAs observed in exosome depletion lines are
siRNAs produced by components of the RdDM pathway and not
short RNA degradation products accumulated in the absence of
functional exoribonucleolytic complex. We then used real-time RT–PCR to examine the levels of
transcript produced from region A, as a measure of the silencing
status of each locus. We found that exosome defects resulted in
accumulation of polyadenylated ncRNA produced from both
regions A and B of solo LTR (Figure 4B). Exosome depletion does not affect smRNA profiles Profiling repeat- and transposable element-generated
smRNAs by their size confirmed that the exosome defect did not
affect the group of 20–25 nt smRNAs even in Pol IV, Pol V,
RDR2 and DCL3 deficient genetic backgrounds. Typically,
siRNAs participating in RdDM are 24 nt long; therefore we
profiled smRNAs mapping to transposable elements by length, but
observed no change in abundance of 24 nt smRNAs (Figure 2E). Further analysis of the 24 nt smRNAs mapped specifically to the
different transposable element superfamilies led to the same
conclusion (Figure 2F and 2G). We therefore concluded that there
are
no
significant
changes
in
the
populations
of
siRNAs
corresponding to repeats and TE superfamilies in exosome
depletion mutants. We also did not observe any significant
differences in amounts of mature 21-mer miRNAs. The results of
our sequencing analysis were confirmed by Northern blot analysis
(Table S3, Figure 3, Figure S2). Together, these data suggest that
the Arabidopsis exosome complex is not involved in siRNA
metabolism on a global scale. Nevertheless, we can not exclude the
possibility that exosome might control a small number of smRNA
precursor transcripts at a few specific loci that would have been
missed in our experiments and with the data processing approach
we took while dissecting differences on genomic level. the total number of mapped non-redundant reads or to the total
number of non-redundant filtered reads (FLR), multiplied by 106
(RPM, reads per million). Both methods of normalization
produced similar results; therefore, only data normalized using
filtered reads (FLR) are presented graphically in this study. We then classified smRNAs based on their size, the nature of
their first nucleotide, and their genomic features. The majority of
functional smRNAs in A. thaliana range from 21 to 24 nt. Our
libraries were constructed using 15–32 nt smRNAs; therefore, we
were able to detect any effect exosome depletion might have on
smRNA metabolism. We found that exosome defect does not lead
to changes in smRNAs in the 15–19 nt and 26–32 nt categories
(data not shown). Importantly, the group of 20–25 nt smRNAs,
which contains the majority of functional smRNAs, was present in
similar proportions, although with minor variations, relative to the
number of total reads in the libraries of both of exosome depletion
mutants and in their corresponding non-induced lines, and
constituted about half of total smRNAs mapped to the genome
(Table S1, Figure 1B). Exosome depletion does not affect smRNA profiles Characterization of up-regulated loci and smRNA populations upon depletion of exosome subunits RRP4 and RRP41. (A)
Phenotypes of rrp41 iRNAi/nrpd1, rrp41 iRNAi/nrpe1, rrp41 iRNAi/dcl3, rrp41 iRNAi/rdr2 double mutants. RRP4 and RRP41 correspond to the iRNAi lines
grown without estradiol and rrp4-i and rrp41-i correspond to lines grown on estradiol-containing medium, to induce the RNAi-mediated knockdown
of RRP4 and RRP41, respectively. (B) 20–25 nt smRNAs sequences profiled by size in exosome depletion mutants rrp4-i and rrp41-i. (C) The relative
frequency of each 59 terminal nucleotide among populations 20–25 nt smRNAs in rrp4-i and rrp41-i mutants. (D) Genomic features of loci generating
20–25 nt small RNAs upon depletion of exosome subunits, according to TAIR9 annotation units. doi:10.1371/journal.pgen.1003411.g001 Figure 1. Characterization of up-regulated loci and smRNA populations upon depletion of exosome subunits RRP4 and RRP41. (A)
Phenotypes of rrp41 iRNAi/nrpd1, rrp41 iRNAi/nrpe1, rrp41 iRNAi/dcl3, rrp41 iRNAi/rdr2 double mutants. RRP4 and RRP41 correspond to the iRNAi lines
grown without estradiol and rrp4-i and rrp41-i correspond to lines grown on estradiol-containing medium, to induce the RNAi-mediated knockdown
of RRP4 and RRP41, respectively. (B) 20–25 nt smRNAs sequences profiled by size in exosome depletion mutants rrp4-i and rrp41-i. (C) The relative
frequency of each 59 terminal nucleotide among populations 20–25 nt smRNAs in rrp4-i and rrp41-i mutants. (D) Genomic features of loci generating
20–25 nt small RNAs upon depletion of exosome subunits, according to TAIR9 annotation units. doi:10.1371/journal.pgen.1003411.g001 March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org 3 SmRNA–Independent Exosome Silencing (Figure 2C) and the rrp41/rdr2 and rrp41/dcl3 double mutants
(Figure 2D). Similar to previous reports, we observed a significant
reduction in the amount of smRNAs corresponding to TEs, TRs
and IRs in nrpd1, nrpe1, rdr2, and dcl3 mutants [27,44,47,48]. Depletion of the exosome in nrpd1, nrpe1 and rdr2 mutants had no
effect on the amount of TE and repeat-associated smRNAs
produced in these mutants (Table S2, Figure 2C and 2D). Depletion of rrp41 in dcl3 led to a minor restoration of this defect in
all groups of repeats and TEs. In the absence of dcl3, other
Arabidopsis Dicer proteins are known to process dcl3 substrates [49];
therefore
this
minor
restoration
most
likely
resulted
from
compensatory effects of other DICER proteins (Table S2,
Figure 2D). Exosome depletion does not affect smRNA profiles Therefore, for simplicity we graphed only
data corresponding either to 20–25 nt smRNAs, or to smRNAs of
one specific length. In addition, the depletion of either RRP4 or RRP41, which are
both essential for exosome function, with slight variations, had no
effect on the smRNA size distribution (Figure 1B) or the
frequencies of their first nucleotide (Figure 1C). All together,
these results suggest that defects in exosome function do not lead to
accumulation of un-degraded smRNA fragments or to any
changes in the cleavage bias of Dicer proteins. Also, exosome
depletion did not change proportions of smRNAs mapped to
different classes of RNAs, such as mRNAs, tRNAs, rRNAs, and
snoRNAs (Figure 1D). Therefore, unlike the situation in S. pombe,
where exosome acts as a negative regulator of siRNA biogenesis,
Arabidopsis exosome does not act to prevent spurious RNAs from
entering RNAi pathway. Exosome depletion does not affect populations of
smRNAs corresponding to repeats and transposable
elements We then compared the
amplitudes of the region A derepression in the rrp41, with rrp41
iRNAi/nrpd1 and rrp41 iRNAi/nrpe1 double mutants relative to the
respective single mutants. As previously reported by others
[30,31], we observed solo LTR to be significantly derepressed in
Pol IV and Pol V single mutants (Figure 4C and 4F). Interestingly,
only the combination of exosome defect with mutation of Pol V, Pol IV, Pol V, RDR2 and DCL3 are not essential for viability
[27,29,46]. Combining mutations in nrpd1, nrpe1, dcl3 and rdr2 with
rrp41-i iRNAi knock-down line did not exacerbate the phenotypes
of single exosome depletion mutants (Figure 1A). We next analyzed the smRNAs corresponding to repeats and
TEs produced in the rrp41/nrpd1 and rrp41/nrpe1 double mutants March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org 4 SmRNA–Independent Exosome Silencing Figure 2. Characterization of 20–25 nt smRNAs corresponding to transposons and repeats in exosome and RdDM mutants
(TE = transposable element; TR = Tandem repeat; IR = Inverted repeat; DR = Dispersed repeat) (A) Results of depletion of exosome rrp4 subunit. (B
Results of depletion of rrp41 exosome subunit. (C) Results of depletion of rrp41 in nrpd1 and nrpe1 genetic backgrounds. (D) Results of depletion o
rrp41 in dcl3 and rdr2 mutants. (E) Characterization of smRNAs mapped to repeats and transposable elements in rrp4-i and rrp41-i libraries profiled
based on the reads length (F, G). Classification of 24 nt smRNAs corresponding to the different superfamilies of TEs in rrp4-i (F) and rrp41-i mutants (G
[76]. doi:10.1371/journal.pgen.1003411.g002 Figure 2. Characterization of 20–25 nt smRNAs corresponding to transposons and repeats in exosome and RdDM mutants
(TE = transposable element; TR = Tandem repeat; IR = Inverted repeat; DR = Dispersed repeat) (A) Results of depletion of exosome rrp4 subunit. (B Figure 2. Characterization of 20–25 nt smRNAs corresponding to transposons and repeats in exosome and RdDM mutants. (TE = transposable element; TR = Tandem repeat; IR = Inverted repeat; DR = Dispersed repeat) (A) Results of depletion of exosome rrp4 subunit. (B)
Results of depletion of rrp41 exosome subunit. (C) Results of depletion of rrp41 in nrpd1 and nrpe1 genetic backgrounds. (D) Results of depletion of
rrp41 in dcl3 and rdr2 mutants. (E) Characterization of smRNAs mapped to repeats and transposable elements in rrp4-i and rrp41-i libraries profiled
based on the reads length (F, G). Exosome depletion does not affect populations of
smRNAs corresponding to repeats and transposable
elements Classification of 24 nt smRNAs corresponding to the different superfamilies of TEs in rrp4-i (F) and rrp41-i mutants (G)
[76]. doi:10.1371/journal.pgen.1003411.g002 doi:10.1371/journal.pgen.1003411.g002 increased in abundance, we carried out strand-specific RT-PCR
for the A and B regions. but not with mutation of Pol IV, resulted in the synergistic increase
of region A transcript (Figure 4C). Reverse transcription with oligo
dT primers does not discriminate between transcripts originating
from either DNA strand; thus an elevated level of polyadenylated
transcript could result from transcription from either one of the
DNA strands. Therefore, to find out which of the transcripts Following standard nomenclature, the top transcript (also called
top strand RNA) corresponds to the transcript identical to the
sequence of the DNA top strand (and therefore produced from the
bottom DNA strand), and the bottom transcript is identical to the March 2013 | Volume 9 | Issue 3 | e1003411 March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org 5 SmRNA–Independent Exosome Silencing Figure 3. Expression of miRNAs in exosome mutants. miRNA families, miR-159a, miR-167a, miR-173 and variations in sequence length in each
family. smRNAs mapped to matching mature miR-159, miR-167miR-173, and miR-167 sequences [94](miRBase release 18) were plotted versus the
sum of their normalized reads per million (rpm) from smRNA libraries constructed from RRP4, rrp4-i, RRP41, rrp41-i, RRP41/nrpd1, rrp41 iRNAi/nrpd1,
RRP4 iRNAi/nrpe1 and rrp41 iRNAi/nrpd1 mutants. Detection of miRNAs by Northern Blot analysis demonstrates that mature miRNA levels are not
affected by exosome depletion, and confirms the results of bioinformatic analysis. Total RNA stained with ethidium bromide was used as a loading
control. doi:10.1371/journal.pgen.1003411.g003 Figure 3. Expression of miRNAs in exosome mutants. miRNA families, miR-159a, miR-167a, miR-173 and variations in sequence length in each
family. smRNAs mapped to matching mature miR-159, miR-167miR-173, and miR-167 sequences [94](miRBase release 18) were plotted versus the
sum of their normalized reads per million (rpm) from smRNA libraries constructed from RRP4, rrp4-i, RRP41, rrp41-i, RRP41/nrpd1, rrp41 iRNAi/nrpd1,
RRP4 iRNAi/nrpe1 and rrp41 iRNAi/nrpd1 mutants. Detection of miRNAs by Northern Blot analysis demonstrates that mature miRNA levels are not
affected by exosome depletion, and confirms the results of bioinformatic analysis. Total RNA stained with ethidium bromide was used as a loading
control. d i 10 1371/j
l
1003411 003 doi:10.1371/journal.pgen.1003411.g003 sequence of DNA bottom strand. The scaffold RNAs were
reported to correspond to region B top strand [30,31]. Exosome depletion does not affect populations of
smRNAs corresponding to repeats and transposable
elements Pol V mutants had no additive or synergistic effect on the level of
region A top transcript. Surprisingly, the level of expression of
region A top transcript was even somewhat decreased in rrp41
iRNAi/nrpd1 and rrp41 iRNAi/nrpe1, compared to nrpd1 and nrpe1
single mutants, opposite to the pattern we observed for the bottom
strand (Figure 4E). Production of scaffold transcripts is central in
silencing of the locus and it was reported that even in the presence
of functional Pol IV and siRNAs, silencing of solo LTR fails when
scaffold RNAs are not produced [30,31]. Similar to previous results [30,31], we observed region A top
and bottom transcripts to be significantly derepressed in Pol IV
and Pol V single mutants (Figure 4D and 4E), and depletion of
RRP41 lead to increased accumulation of the region A top and
bottom transcripts (inserts in Figure 4D and 4E). Interestingly, we
found that the bottom transcript was synergistically derepressed in
rrp41 iRNAi/nrpe1 double mutants relative to nrpe1 and rrp41 iRNAi
single mutants, while no change was observed in rrp41 iRNAi/nrpd1
double mutants (Figure 4D). Despite the fact that the exosome
defect equally affected the levels of both top and bottom region A
transcripts, combining the exosome defect with either Pol IV or We therefore examined the scaffold-producing region B and
found that the exosome also affects the amount of region B top
transcript, but there is no synergistic increase of this transcript in
rrp41 iRNAi/nrpe1 double mutants (Figure 4F and 4G). When we March 2013 | Volume 9 | Issue 3 | e1003411 March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org 6 SmRNA–Independent Exosome Silencing Figure 4. Effect of exosome subunits depletion on expression of ncRNA transcripts from RdDM-regulated loci. (A) Diagrams of solo
LTR and AtSN1 loci, based on analysis of transcription units by Wierzbicki et al. (2008). Region A corresponds to the siRNA-producing region of solo
LTR, region B corresponds to the adjacent to solo LTR region that produces scaffold RNA, and red lines mark regions amplified by RT-PCR and qPCR. The dotted line corresponds to the region of scaffold RNAs hypothesized to be complementary to the siRNAs produced from region A. (B) Depletion
PLOS Genetics | www.plosgenetics.org
7
March 2013 | Volume 9 | Issue 3 | e1003411 Figure 4. Effect of exosome subunits depletion on expression of ncRNA transcripts from RdDM-regulated loci. RRP41 depletion does not affect de novo DNA
methylation in solo LTR and AtSN1 loci The solo LTR, AtSN1 and IGN5 loci are silenced primarily by
RdDM, through siRNA mediated de novo methylation of DNA
[9,30,31]. We reasoned that if the exosome is involved in
controlling the amount of RNA expressed from these loci in a
siRNA-dependent manner, then the exosome defect might affect
the amount of siRNAs generated from these regions. To address
this question, we first compared solo LTR and AtSN1-specific
smRNAs. We found that production of smRNAs from the siRNA-
generating A regions was not altered in rrp4-i or rrp41-i mutants
relative to WT (Figure 5A and 5B), similar to the results of the
global smRNA analysis we described above. The increased
amount of smRNAs observed in dcl3 mutants is because in the
absence of DCL3, the other Dicer proteins process DCL3
substrates [49]. In order to make sure that the smRNAs produced
from one strand of region A are not masking the smRNAs
produced from the opposite strand in exosome depletion mutants,
we also analyzed these smRNA populations in a strand-specific
manner. However, the patterns of strand-specific siRNAs were
very similar to the patterns we observed previously and siRNAs
were not affected by exosome depletion (Figure 5C and 5D). We
examined an additional region controlled by RdDM, the IGN5
locus [31], and found that IGN5-specific smRNAs are also not
affected in exosome mutants, similar to solo LTR and AtSN1 loci
(Figure S3C). This implies that the increase in accumulation of
transcripts in exosome-depleted plants was not a result of siRNA
defect. To verify this directly, we examined the patterns of DNA
methylation in these regions by using methylation sensitive
restriction enzymes (Figure 5E). The DNA of the solo LTR
region was examined by two different assays (Figure 5E and 5F). We found that, consistent with the results of the region-specific
siRNA analysis, de novo DNA methylation was not changed in
rrp41-i plants (Figure 5A–5D). Taken together, these results
indicate that an increase in transcript accumulation is not caused
by the loss of de novo methylation and the region is still silenced by
RdDM. It also suggests that in the examined loci, the exosome
complex functions independently of RdDM. Interestingly, the
increased amount of transcripts accumulated in these regions does
not contribute to increased smRNA amounts in the exosome-
depleted plants. This was observed regardless of whether these
transcripts originated from siRNA-generating regions, or adjacent
regions. Exosome depletion does not affect populations of
smRNAs corresponding to repeats and transposable
elements (A) Diagrams of solo
LTR and AtSN1 loci, based on analysis of transcription units by Wierzbicki et al. (2008). Region A corresponds to the siRNA-producing region of solo
LTR, region B corresponds to the adjacent to solo LTR region that produces scaffold RNA, and red lines mark regions amplified by RT-PCR and qPCR. The dotted line corresponds to the region of scaffold RNAs hypothesized to be complementary to the siRNAs produced from region A. (B) Depletion March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org PLOS Genetics | www.plosgenetics.org 7 SmRNA–Independent Exosome Silencing SmRNA–Independent Exosome Silencing of exosome subunits RRP4 and RRP41 leads to an increase in noncoding transcripts generated from siRNA-producing region A and scaffold RNA-
producing region B of solo LTR. RT was primed with oligo(dT). (C, D, E) Expression of region A of solo LTR in exosome depleted plants and various
mutants. (C) Combining depletion of RRP41 with mutation in Pol V leads to a synergistic increase in accumulation of transcripts from region A. Strand-specific RT-PCR analysis revealed that both top and bottom transcripts of region A are affected by depletion of RRP41 subunit, but only
bottom transcript is synergistically affected in rrp41-i/nrpe1 double mutants (D), while the amount of top transcript is decreased in both rrp41-i/nrpd1
and rrp41-i/nrpe1 double mutants (E). (F, G) Expression of region B of solo LTR in exosome depleted plants and various mutants. (F) Depletion of rrp41
leads to increased amounts of transcript produced from both strands of region B. (G) RT-PCR analysis of solo LTR top transcript. (H, I) Depletion of
exosome subunit RRP41 leads to increase in ncRNA transcripts generated from the AtSN1 region. (H) Combining depletion of RRP41 with mutation in
Pol V leads to synergistic increase in accumulation of polyadenylated transcript from region A. (I) Amount of the region A bottom strand of AtSN1 is
synergistically increased in rrp41-i/nrpe1 double mutants. doi:10.1371/journal.pgen.1003411.g004 A transcript in iRNAi/nrpe1 mutants (Figure 4B, 4C, 4G and 4H)
does not produce any increase in the amount of siRNAs
(Figure 5A–5D). A transcript in iRNAi/nrpe1 mutants (Figure 4B, 4C, 4G and 4H)
does not produce any increase in the amount of siRNAs
(Figure 5A–5D). examined AtSN1, we observed a very similar synergistic increase
in the level of the siRNA-producing region A of bottom strand
transcript of AtSN1 in rrp41 iRNAi/nrpe1 mutants (Figure 4H and
4I). H3K9me2 levels are affected in exosome-depleted plants H3K9me2 levels are affected in exosome depleted plants
DNA methylation and histone modification are two major
epigenetic marks regulating gene expression and chromatin state
in plants. Monomethylated histone H3 lysine 27 (H3K27me1) and
dimethylated histone H3 lysine 9 (H3K9me2) are hallmarks of
heterochromatin, and silencing of solo LTR, AtSN1 and IGN5
loci also involves histone modifications [30,31]. Although de novo
methylation does not directly affect the level of H3K9me2, it does
affect the level of H3K27me1 [31], suggesting that in addition to
histone modification pathways, which are dependent on RdDM,
other, RdDM-independent, pathways also contribute to transcrip-
tional silencing of these regions. We therefore used chromatin
immunoprecipitation (ChIP) to examine whether the exosome is
involved in regulation of histone modifications in these loci. Exosome depletion does not affect populations of
smRNAs corresponding to repeats and transposable
elements Together, these results suggest that the exosome participates in
controlling the amount of top transcripts emanating from the
scaffold-producing region B of solo LTR, and thus may contribute
to the repression of region A through regulating the level of region
B transcripts. RRP41 depletion does not affect de novo DNA
methylation in solo LTR and AtSN1 loci Indeed, even several thousand-fold upregulation of region Similar to the results reported by others [30,31], we observed a
significant decrease in the level of H3K9me2 in the solo LTR
locus in nrpd1 and nrpe1 mutants, which affect Pol IV and Pol V,
respectively. We found that RRP41 depletion also led to a
decrease in H3K9me2 but less than observed in nrpd1 and nrpe1
mutants (Figure 6A). The decrease in level of this repressive
histone modification also correlated with a mild increase in RNA
Pol II occupancy in the solo LTR region, as would be expected
with a release of transcriptional block (Figure 6B). The rrp41
iRNAi/nrpe1 double mutant did not exhibit any additive or
synergistic effect on the loss of H3K9me2 relative to respective
single mutants. When we examined AtSN1, we found that the level of
H3K9me2
was
mildly
decreased
in
all
mutants
tested
(Figure 6A). For AtSN1, it was previously suggested that RNA
Pol III is the main RNA polymerase transcribing the region when
the region is in a derepressed state [31], although RNA Pol II was
also reported to be associated with this region [30]. We found that
RNA Pol II occupancy in AtSN1 was very low but it increased
significantly in rrp41 iRNAi/nrpe1 double mutants (Figure 6B), in
accordance with the synergistic increase of the transcript level we
observed (Figure 4H and 4I). Depletion of another exosome subunit, RRP4, caused a similar
loss of H3K9me2 at solo LTR and AtSN1 loci (Figure 6C). We
then chose several additional regions, termed REG3 and REG4
(Figure S3A), that are mildly upregulated in exosome mutants
according to our previous microarray analysis [1], and examined
them using ChIP. REG3 harbors a MuDR transposon, and REG4
is situated in a tandem repeat area. Neither of these loci produces
smRNAs or is controlled by DNA methylation (Figure 6E and data
not shown). We found that the H3K9me2 in these loci was
similarly affected by exosome depletion (Figure 6C), while the level
of H3K27 methylation in these regions didn’t show any difference
(Figure 6D). Exosome associates with transcripts produced from a
scaffold-generating area adjacent to solo LTR g
g
j
We then examined exosome association with ncRNA loci. Detection of some protein–nascent mRNA interactions by ChIP
were reported previously for proteins working on RNA, but the
results of our attempts to localize tagged exosome subunits at solo
LTR locus have proven inconclusive. Transcripts from region A
are normally below the level of detection in wild-type plants, but
transcription from the region B adjacent to solo LTR has been
previously documented in wild-type plants [1,30,31]. In order to
confirm that the exosome directly associates with these transcripts,
we conducted RNA immunoprecipitation (RIP) using plants
carrying a transgene expressing RRP41-TAP, and examined the
ncRNAs associated with the exosome by RT-PCR. No region A
transcripts were detected in immunoprecipitates, but we found
that region B transcripts were co-precipitated with exosome
(Figure 7A). These data suggest that in wild-type plants, exosome
physically associates with polyadenylated transcripts produced
from region B of solo LTR. We found that, similar to depletion of the core subunits RRP4
and RRP41, rrp6l1-2 and rrp6l2-2 mutants exhibited increased
accumulation of transcripts produced from region A. As would be
expected based on cytoplasmic localization of RRP6L3 protein, no
effect was observed in rrp6l3-1 mutants. To our surprise, we
observed a dramatic derepression of region A in rrp6l1-2/rrp6l2-2
double mutants, suggesting that both RRP6L1 and RRP6L2
proteins are involved in the silencing of this region and might have
a redundant function in this process. We also examined the status of solo LTR DNA methylation in
rrp6l1-2, rrp6l2-2, and rrp6l1-2/rrp6l2-2 double mutants. We found
that methylation was not affected in these mutants regardless of
the extent of derepression of the region (Figure 7D), consistent
with the results obtained using rrp4-i and rrp41-i depletion mutants. Taken together, these results indicate that the observed increase in
transcript accumulation is not caused by the loss of de novo
methylation and the region is still methylated by RdDM. This
further confirms that the exosome complex functions indepen-
dently of the RdDM pathway. In contrast to solo LTR, we did not detect a physical association
of exosome with AtSN1 region B transcript (Figure 7A). This
implies that exosome depletion may not directly affect the
silencing of AtSN1. However, we observed that exosome depletion
resulted in accumulation of transcript in the AtSN1 locus and we
detected a synergistic derepression of the locus in rrp41/nrpe1
mutants, similar to solo LTR locus (Figure 4H and 4I). RRP41 depletion does not affect de novo DNA
methylation in solo LTR and AtSN1 loci These results suggest that the exosome may
participate in maintaining chromatin structure in these regions March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org 8 SmRNA–Independent Exosome Silencing March 2013 | Volume 9 | Issue 3 | e1003411 March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org PLOS Genetics | www.plosgenetics.org SmRNA–Independent Exosome Silencing Figure 5. smRNA accumulation and DNA methylation in solo LTR and AtSN1 loci is unaltered upon exosome depletion. (A, B) 20–
25 nt smRNAs produced from region A of solo LTR (A) and region A of AtSN1 (B) in rrp4-i, rrp41-i exosome depletion lines and RdDM mutants. All
locus-specific datasets of 20–25 nt smRNAs are plotted versus the sum of their normalized reads per million (rpm). (C, D) Strand-specific analysis of
smRNAs generated at regions A and B of AtSN1 (C) and solo LTR (D) loci in different mutants. (E, F) DNA methylation analysis of AtSN1 and solo LTR
loci by digestion of purified DNA with the methylation-sensitive endonucleases HaeIII for AtSN1 (E), AluI for solo LTR (E), and McrBC for solo LTR (D),
followed by PCR. doi:10 1371/journal pgen 1003411 g005 y
doi:10.1371/journal.pgen.1003411.g005 as well, and does so by specifically affecting the level of H3K9me2
in addition to controlling the level of transcripts. our preparations. In addition, RRP6L2 was later shown to have at
least some commonalities with core exosome substrates [54]. We
therefore examined whether the Arabidopsis RRP6-like proteins
control the amount of ncRNA at the solo LTR locus. To
determine this, we used T-DNA insertion alleles in RRP6L1,
RRP6L2 and RRP6L3. We isolated the rrp6l1-2 allele from the
University of Wisconsin BASTA population (Ws ecotype), and the
alleles of the rrp6l2-2 and rrp6l3-1 are SALK alleles (Col-0
ecotype). To control for effects of ecotype, we compared the
amount of region A transcript in rrp6l3-1, rrp6l2-2, rrp6l1-2/rrp6l2-
2 mutants to Col-0 wild type plants, and rrp6l1-2, rrp6l1-2/rrp6l2-2
mutants to Ws ecotype plants (Figure 7B and 7C). The exosome and smRNA metabolism The exosome functions in virtually all aspects of RNA
metabolism and it appears to also have a prominent role in
transcriptional gene silencing in different species [1,10,38–41,55–
59]. This study examined the role of the exosome complex in
metabolism of smRNAs and explored the possible relationship
between the exosome and the RdDM pathway in gene silencing in
Arabidopsis. Our results showed that exosome-mediated silencing did not
produce global changes in smRNA profiles, nor in DNA
methylation at specific loci. However, we did find effects on
histone methylation, indicating that the exosome may regulate
chromatin structure, thereby playing an important role in
maintenance of gene silencing on a much broader scale than the
RdDM pathway. It is clear from our results using suppression of
key exosome components that plants have an exosome-dependent
pathway that relies on ncRNAs to target heterochromatin. Exosome associates with transcripts produced from a
scaffold-generating area adjacent to solo LTR Most likely
the regulation of AtSN1 is more complex because an additional
RNA polymerase, RNA Pol III, is involved. AtSN1 is transcribed
mostly by RNA Pol III [31,53], suggesting that the double
deficiency in exosome and Pol V may increase both Pol II and Pol
III access to the locus. We also observed the increased Pol II
association with AtSN1 in rrp41/nrpe1 mutants by ChIP assay
using anti-Pol II (Figure 6B), which is consistent with the results of
qRT-PCR. Therefore, it is also possible that the loss of exosome
function may lead to the alteration of chromatin structure in
regions adjacent to AtSN1 and thus affect the stability of silencing
in AtSN1 indirectly. Nevertheless, these results are similar to the
interplay between exosome and Pol V observed for solo LTR. March 2013 | Volume 9 | Issue 3 | e1003411 RRP6 is involved in controlling levels of ncRNA from the
solo LTR locus The 9-subunit exosome complex is catalytically inactive in yeast
and human. Instead, active sites are contributed by Rrp44 (Dis3)
and by the subunit Rrp6, which is substoichiometric, nuclear-
specific, and not essential for viability. Degradation of S. cerevisiae
nuclear ncRNAs depends on polyadenylation by the TRAMP
complex and involves Rrp6, the subunit that is also responsible for
elimination of heterochromatic RNAs in S. pombe [20,22–24,39–
41]. In Arabidopsis there are three RRP6-like proteins – nuclear
localized RRP6L1 and RRP6L2, and cytoplasmic RRP6L3; these
were suggested to be functional homologues of RRP6 [54]. None
of the RRP6-like proteins co-purified with the exosome complex in
our proteomic studies [1], but may have been underrepresented in Our finding that the increase in ncRNA transcribed from
heterochromatic loci in exosome-depleted plants did not lead to an
increase in levels of smRNA indicates that exosome function in
Arabidopsis differs from that in fission yeast. In fission yeast,
exosome defects have a dramatic effect on siRNAs leading to
redistribution of the spectrum of Ago1-associated siRNAs, from March 2013 | Volume 9 | Issue 3 | e1003411 March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org 10 SmRNA–Independent Exosome Silencing Figure 6. The effect of the exosome subunits depletion on the levels of H3K9me2 in different loci. (A, B) The levels of H3K9me2, and Pol
II occupancy at solo LTR and AtSN1 examined by ChIP in RRP41, rrp41-i, RRP41/nrpd1, rrp41-i/nrpd1, RRP41/nrpe1, and nrpd1/nrpe mutants using
ib di
i
H K
(A)
d RNA P l II (B)
i
l
(C) Eff
f RRP
d
l
i
l
l
f H K
i
d b ChIP
l
LTR Figure 6. The effect of the exosome subunits depletion on the levels of H3K9me2 in different loci. (A, B) The levels of H3K9me2, and Pol
II occupancy at solo LTR and AtSN1 examined by ChIP in RRP41, rrp41-i, RRP41/nrpd1, rrp41-i/nrpd1, RRP41/nrpe1, and nrpd1/nrpe mutants using
antibodies against H3K9me2 (A), and RNA Pol II (B), respectively. (C) Effect of RRP4 depletion on levels of H3K9me2 examined by ChIP at solo LTR,
IGN5, REG 3, and REG 4 (C), and on levels of H3K27me1 at solo LTR, AtSN1, and IGN5 loci (D). No Ab, ChIP with no antibody, is used as a negative
control. RRP6 is involved in controlling levels of ncRNA from the
solo LTR locus (D) Analysis of DNA methylation in solo LTR by McrBC treatment in Col-0, Ws,
rrp6l1-2, rrp6l2-2, double mutant rrp6l1-2/rrp6l2-2, rrp6l3-1mutant plants. RRP41/nrpd1 mutant DNA is used as a control. doi:10.1371/journal.pgen.1003411.g007 reported [63–71]. One of the DNA methylation-independent gene
silencing pathways is mediated by MOM1 (Morpheus’ molecule 1)
protein [63,65], which predominantly silences transposons and
loci harboring sequences related to gypsy-like transposons. Activa-
tion of transcription in mom1 mutants occurs with no change in
DNA methylation, histone modifications or chromatin condensa-
tion, and the investigation of the relationship between RdDM and
MOM1 revealed a very complex interplay between these two
pathways [63,69,72–74]. However, a reduction in H3K9 dimethy-
lation was reported in some loci in mom1 mutants and it was
suggested that MOM1 may transduce RdDM signals to repressive
histone modifications by an unknown mechanism [75]. mostly repeat-associated to those derived predominantly from
exosome substrates such as rRNA and tRNA [39], indicative of
exosome acting as a negative regulator of siRNA biogenesis. Our
data indicate that the Arabidopsis exosome most likely lost this
function during evolution, meaning that exosome substrates do not
compete with siRNA precursors for siRNA biogenesis machinery
and spurious transcripts do not enter RNAi pathways in plants. Additionally, it suggests that perhaps only very few of the ncRNA
transcripts controlled by the exosome could be bona fide siRNA
precursors. One of the reasons for this could be the fact that plants
evolved two plant-specific RNA polymerases, Pol IV and Pol V,
which specialize in siRNA-mediated TGS. Pol IV is required for
biogenesis of the majority of 24-nt siRNAs and is supported by Pol
V, which is responsible for production of a subset of siRNAs
[31,44,45,60]. It is also plausible that there might be other
unknown plant-specific ribonucleases that specialize in controlling
stability of siRNAs or the amount of siRNA precursors generated
by Pol IV and/or Pol V in plants. We also cannot rule out the
possibility that some of the transcripts controlled by the exosome
in a small subset of loci are legitimate siRNA precursors; this
definitely warrants further in-depth investigation. Also, a recent study of MORC family ATPases revealed that
mutation of AtMORC1 or AtMORC6 caused derepression of DNA
methylated genes and TEs without any loss of DNA methylation,
change in histone methylation or alteration of siRNA levels [71]. These
proteins
are
involved
in
alteration
of
chromosome
superstructure and are likely to act downstream of DNA
methylation. RRP6 is involved in controlling levels of ncRNA from the
solo LTR locus (E) Analysis of DNA methylation in REG 3 and REG 4 regions by McrBC treatment in RRP41, rrp41-i, RRP41/nrpd1, rrp41-i/nrpd1, RRP41/nrpe1,
and nrpd1/nrpe1 mutant plants. REG 3 and REG 4 are not methylated in wild type plants and no changes were observed in mutants. The error bars in
ChIP experiments represent the standard error of the mean (SE) and correspond to the difference between 2 biological replicates. doi:10.1371/journal.pgen.1003411.g006 March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org 11 SmRNA–Independent Exosome Silencing Figure 7. Exosome associates with transcripts produced from the region adjacent to the solo LTR scaffold-generating area. (A). RT-
PCR of RNA-immunoprecipitation using plants carrying a functional RRP41-TAP transgene and empty-TAP transgene to examine the association of
exosome with noncoding transcripts produced at siRNA and scaffold RNA producing loci. Region B transcripts were co-precipitated with exosome,
while no region A transcripts were detected in immunoprecipitates. Transgenic plants are in Ws ecotype. (B, C) Two homologous rrp6 catalytic
subunits of exosome are involved in controlling the amount of ncRNA emanating from the solo LTR locus. (C) Expression pattern of region A of solo
LTR locus in rrp6l3-1, rrp6l2-2, 6l1-2, and the double mutant rrp6l1-2/rrp6l2-2 compared relative to the RNA expression in Col-0 ecotype wild-type (B),
compared relative to the RNA expression in Ws ecotype wild-type (C). (D) Analysis of DNA methylation in solo LTR by McrBC treatment in Col-0, Ws,
rrp6l1-2, rrp6l2-2, double mutant rrp6l1-2/rrp6l2-2, rrp6l3-1mutant plants. RRP41/nrpd1 mutant DNA is used as a control. doi:10.1371/journal.pgen.1003411.g007 Figure 7. Exosome associates with transcripts produced from the region adjacent to the solo LTR scaffold-generating area. (A). RT-
PCR of RNA-immunoprecipitation using plants carrying a functional RRP41-TAP transgene and empty-TAP transgene to examine the association of
exosome with noncoding transcripts produced at siRNA and scaffold RNA producing loci. Region B transcripts were co-precipitated with exosome,
while no region A transcripts were detected in immunoprecipitates. Transgenic plants are in Ws ecotype. (B, C) Two homologous rrp6 catalytic
subunits of exosome are involved in controlling the amount of ncRNA emanating from the solo LTR locus. (C) Expression pattern of region A of solo
LTR locus in rrp6l3-1, rrp6l2-2, 6l1-2, and the double mutant rrp6l1-2/rrp6l2-2 compared relative to the RNA expression in Col-0 ecotype wild-type (B),
compared relative to the RNA expression in Ws ecotype wild-type (C). RRP6 is involved in controlling levels of ncRNA from the
solo LTR locus These results indicate that there are multiple parallel
pathways for DNA methylation-independent gene silencing in
Arabidopsis. The exosome-mediated silencing we observed here
bears some similarities to the silencing observed for MOM1 and
MORC; for example, they show effects on repetitive sequences
and an absence of effects on siRNAs, although there are notable
differences as well. Here we show that, similar to MOM1 and
MORC mechanisms, exosome-dependent gene silencing also
affects repetitive sequences and acts independent of RdDM,
although our results are limited in scope. Characterization of the Exosome and DNA methylation-independent gene
silencing How can exosome function in gene silencing in
Arabidopsis? There are multiple mechanisms by which the exosome can be
envisioned to participate in gene silencing in Arabidopsis. Heterochromatin assembly is used by all eukaryotes in gene
silencing. In addition to repressive histone modifications employed
by all organisms, humans and plants widely use DNA methylation
as well, and ncRNAs play a central role in the control of
chromatin structure in all organisms. While ncRNA-mediated
silencing proceeds through multiple mechanisms some of which
are organism-specific, the end result appears to be the same
repressive histone modifications. For example, budding yeast,
which lacks RNAi machinery, employs strategies that include, but
not limited to, the use of antisense, cryptic or read-through
transcripts, as well as transcripts originating from divergent
promoters to guide histone modifications. Fission yeast is more
similar to higher eukaryotes and uses all of the above strategies in
addition to utilizing RNAi as well. However, DNA methylation is
not used by budding and fission yeast. Plants, on the other hand,
evolved very sophisticated epigenetic mechanisms that include the
use of both RNAi-dependent and RNAi-independent pathways to
guide DNA methylation and histone modifications for gene
silencing [9,31–33,44,45,47,61,68,70,75,77,78]. Exosome com-
plex proved to be amazingly versatile in impacting gene silencing
in budding and fission yeasts. In fission yeast, the organism which
takes full advantage of RNAi machinery to regulate its gene
expression, the exosome is involved in silencing of both facultative
and constitutive heterochromatin by acting in several different
pathways through smRNAs, produced in either an RNAi-
dependent or RNAi-independent manner [38,39,79,80]. It was
also found to act through surveillance of RNA quantity and quality
as
well
as
by
collaborating
with
termination
machinery
[40,41,57,80,81], similarly to the manner exosome participates
in gene silencing in bakers yeast, which lacks RNAi machinery
[55,58,59]. Our results showed that the exosome depletion produced no
effect on siRNAs and DNA methylation of solo LTR, AtSN1 and
IGN5 loci, arguing that the exosome complex functions indepen-
dently of RdDM. However, our findings also indicated that the
exosome is involved in the silencing of these loci and does interact
with
the
RdDM
pathway,
possibly
through
its
functional
interaction with RNA Pol V. The converging transcripts we
observed in the rrp41-i and rrp4-i mutants in solo LTR and AtSN1
suggest that the exosome is involved in regulation of either
processing or level of RNA from these loci (Figure 4A–4I, and
model Figure 8). Exosome and DNA methylation-independent gene
silencing siRNA-dependent RdDM is thought to be the main pathway for
transcriptional gene silencing of repetitive elements and transpo-
sons in plants [27,28,31,61,62], although existence of other DNA
methylation-independent gene silencing pathways have also been March 2013 | Volume 9 | Issue 3 | e1003411 March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org 12 SmRNA–Independent Exosome Silencing relationship between these pathways remains an interesting topic
for future study. subunit AtRrp44a [J. Lee and J. Chekanova unpublished data] is
involved in this process, and whether components of the TRAMP
complex also participate. The different silencing pathways likely have different functions,
depending on the genomic region, the nature of the regulated
sequences, and the precision and dynamics of silencing required. For example, methylated sequences can affect the expression of
nearby genes. The expression of nearby genes is negatively
correlated with the density of methylated, but not unmethylated
TEs. Methylated TEs are preferentially removed from gene-dense
regions over time and TE families that have a higher proportion of
methylated insertions are distributed farther from genes [76],
arguing that positional effects and the surrounding landscape most
likely contributes to the choice of silencing mechanisms and the
interplay between them. p
p
p
We also observed that the exosome physically associates with
the polyadenylated ncRNA transcripts from scaffold producing
regions. We could not reliably crosslink the exosome to the DNA
of the target locus by ChIP (data not shown), although this could
simply reflect the difficulty of reliably crosslinking proteins to DNA
through RNA, or it could mean that the exosome binds to the
transcripts after they are released from the locus and that
exosome-mediated regulation of the transcripts may be important
for maintenance of chromatin structure around the locus. H3K9
dimethylation was reported to be disturbed and lost when isolated
Arabidopsis nuclei were treated with RNase A [82], meaning that
histone modification may be affected by RNA level and/or RNA
in close proximity to the target loci. In fission yeast, the mutation
of Cid14, one of the subunits of the TRAMP complex, results in
accumulation of aberrant heterochromatic RNA close to the target
loci and leads to a mild decrease in H3K9 methylation. Exosome and DNA methylation-independent gene
silencing It was
recently shown that decrease of H3K9 methylation in yeast is the
result of HP1 protein (Heterochromatin Protein1), which binds to
H3K9me2 heterochromatin and propagates H3K9me2 spreading,
being titrated by an excess of heterochromatic RNA [83]. In our
study, we also observed a combination of the transcripts
accumulation in exosome mutants relative to WT with a weak
decrease in H3K9me2 levels in solo LTR (Figure 6A). Taken
together, these data could suggest that a similar mechanism to
regulate the stability of chromatin structure might operate in
plants. However, LHP1 (Like-HP1), the closest Arabidopsis
homolog of yeast HP1, has specificity for H3K27me3 [84], not
H3K9me2, and the rrp41 iRNAi/nrpe1 double mutant did not
exhibit any additive or synergistic effect on the loss of H3K9me2
relative to respective single mutants as well, suggesting that the loss
of H3K9me2 observed in the exosome mutants is unlikely to result
from an unknown functional homolog of Arabidopsis HP1 simply
titrating an excess of ncRNA off chromatin, as reported in fission
yeast. How can exosome function in gene silencing in
Arabidopsis? We found that production of smRNAs from the
siRNA-generating A regions was totally abolished in rrp41/nrpd1
double mutant (Figure 5A–5D), ruling out a possibility for these
transcripts to serve as a double stranded precursors for RNA Pol
IV-independent siRNAs. We also found that the exosome
physically associates with the polyadenylated transcripts produced
from the scaffold region (region B) and exhibits synergistic
derepression of the locus (region A) when combined with a Pol
V mutant, while there was no change in the derepression in rrp41/
nrpd1 double mutants (Figure 4B, 4C, 4H and 4I). Based on these
results, we speculate that RNA polymerase V may function in gene
silencing of these loci in two ways, the first acting in the DNA-
methylation-dependent RdDM pathway, and the second acting
independently of a DNA-methylation. Indeed, RdDM- indepen-
dent roles of Pol V in silencing of 5S rDNA [31,85] and several
other loci [82] were previously reported. A recent genome-wide
study of Pol V-associated loci also hints at the possibility of Pol V In Arabidopsis, silencing of repetitive elements involves siRNA-
dependent DNA methylation guided by homologous siRNAs [9]. Repressive histone modifications always appear to accompany
DNA methylation, however, the mechanistic link between them is
not yet fully understood. In budding and fission yeasts, degrada-
tion of nuclear ncRNAs depends on polyadenylation by the
TRAMP complex and involves Rrp6. We also found that
mutations in two RRP6-like proteins AtRRP6 L1and AtRRP6
L2 led to significant dereperession of solo LTR (Figure 7B, 7C and
7D) and occurred in a DNA methylation-independent manner as
in rrp4 and rrp41 (Figure 7F). These results suggest that Atrrp6s
may be true nuclear catalytic subunits of Arabidopsis exosome, or
may also work independently of core exosome. It will be
interesting to examine whether another putative exosome catalytic March 2013 | Volume 9 | Issue 3 | e1003411 13 PLOS Genetics | www.plosgenetics.org SmRNA–Independent Exosome Silencing Figure 8. Model for the role of the exosome complex in gene silencing at solo LTR in Arabidopsis. The process of silencing of the solo LTR
locus is substantially more complex than portrayed here, but for simplicity only the factors examined in this study are represented. No modifications
to the prevailing views on the roles of Pol IV, Pol V, Pol II, RDR2, DCL3, and AGO4 are proposed [9,28,30,31,60]. A. How can exosome function in gene silencing in
Arabidopsis? Either Pol II transcripts or the process of transcription from region
B recruits Pol V (blue), complexed with AGO4 and siRNA, to the scaffold-producing region B. Due to the sequence complementarity between siRNAs,
which are derived from region A only, and the portion of the scaffold transcripts that partially overlaps with region A, AGO4/siRNA RISC localizes to
region A and recruits other components of the silencing machinery. Both Pol II and Pol V were implicated in producing region B scaffold transcripts
[30,31]. Exosome is not involved in siRNA metabolism and does not contribute to DNA methylation. Exosome participates in controlling the amount
of top transcripts emanating from the scaffold-producing region B of solo LTR, and thus may contribute to the repression of region A through
regulating the level of region B transcripts. The exosome associates with transcripts emanating from the scaffold-producing region and plays a role in
locus silencing through maintaining or establishing chromatin structure. B. More than one silencing pathway controls the solo LTR locus. The
exosome associates with transcripts emanating from the adjacent scaffold-producing region, and plays a role in locus silencing through maintaining
or establishing chromatin structure by affecting histone methylation (H3K9), in parallel to the RdDM pathway, which affects siRNAs and DNA
methylation (‘‘M’’ in red hexagons). doi:10 1371/journal pgen 1003411 g008 Figure 8. Model for the role of the exosome complex in gene silencing at solo LTR in Arabidopsis. The pr y
g
doi:10.1371/journal.pgen.1003411.g008 transcripts, one of which is controlled by the exosome, and the
exosome functions by regulating the Pol II transcripts that are
distinct from the transcripts that are used in RdDM pathway. This
possibility would be very interesting to examine, particularly in
light of the yeast exosome involvement in gene silencing through
regulation of cryptic transcripts, transcripts originating from
divergent promoters and read-through transcripts [4,55,58,59]. How the Arabidopsis exosome complex and the exosome
controlled ncRNAs facilitate recruitment of chromatin modifiers
in order to enforce silencing through repressive histone modifica-
tions remains an interesting topic of future studies. We suggest that
the exosome may coordinate the transcriptional interplay of RNA
polymerases Pol II and Pol V to achieve the right level of
transcriptional repression of heterochromatic loci (Figure 8). having unknown functions in addition to the function it plays in
the RdDM pathway [45]. How can exosome function in gene silencing in
Arabidopsis? The exosome complex is not
involved in the regulation of quality or quantity of siRNAs produced from region A. RNA Pol II (green) generates transcripts from region B of solo LTR. It is also possible that Pol II transcribes both A and B regions in opposite directions. Either Pol II transcripts or the process of transcription from region
B recruits Pol V (blue), complexed with AGO4 and siRNA, to the scaffold-producing region B. Due to the sequence complementarity between siRNAs,
which are derived from region A only, and the portion of the scaffold transcripts that partially overlaps with region A, AGO4/siRNA RISC localizes to
region A and recruits other components of the silencing machinery. Both Pol II and Pol V were implicated in producing region B scaffold transcripts
[30,31]. Exosome is not involved in siRNA metabolism and does not contribute to DNA methylation. Exosome participates in controlling the amount
of top transcripts emanating from the scaffold-producing region B of solo LTR, and thus may contribute to the repression of region A through
regulating the level of region B transcripts. The exosome associates with transcripts emanating from the scaffold-producing region and plays a role in
locus silencing through maintaining or establishing chromatin structure. B. More than one silencing pathway controls the solo LTR locus. The
exosome associates with transcripts emanating from the adjacent scaffold-producing region, and plays a role in locus silencing through maintaining
or establishing chromatin structure by affecting histone methylation (H3K9), in parallel to the RdDM pathway, which affects siRNAs and DNA
methylation (‘‘M’’ in red hexagons). doi:10 1371/journal pgen 1003411 g008 Figure 8. Model for the role of the exosome complex in gene silencing at solo LTR in Arabidopsis. The process of silencing of the solo LTR
locus is substantially more complex than portrayed here, but for simplicity only the factors examined in this study are represented. No modifications
to the prevailing views on the roles of Pol IV, Pol V, Pol II, RDR2, DCL3, and AGO4 are proposed [9,28,30,31,60]. A. The exosome complex is not
involved in the regulation of quality or quantity of siRNAs produced from region A. RNA Pol II (green) generates transcripts from region B of solo LTR. It is also possible that Pol II transcribes both A and B regions in opposite directions. March 2013 | Volume 9 | Issue 3 | e1003411 Plant materials and growth conditions g
iRNAi lines of exosome subunits RRP4 and RRP41, RNA Pol
IV
(SALK_128428.20.10,
nrpd1a-3,
nrpd1-3),
RNA
Pol
V
(SALK_029919, nrpd1b-11, nrpe1-11), RDR2 ( SAIL_1277808,
rdr2-1), and DCL3 ( SALK_005512.38.70.x0, dcl3-1) mutants were
described previously [1,27,33,86]. rrp41 iRNAi/nrpd1-3, rrp41
iRNAi/nrpe1-11, rrp4 iRNAi/nrpd1-3,and rrp4 iRNAi/nrpe1-11 dou-
ble mutants were obtained by crossing of rrp41 iRNAi and rrp4
iRNAi with nrpd1/nrpe1-11 line. rrp41 iRNAi/dcl3-1, rrp41 iRNAi/
rdr2-1 double mutants were obtained by crossing. The small RNA reads with 20 to 25 nt length were calculated
and plotted versus the sum of their normalized reads per million
(rpm). The relative frequencies of each 59 terminal nucleotide of
the small RNAs were calculated (Tables S1, S2 ) and represented
graphically. Repetitive genomic features were classified using TAIR9
Tandem Repeat Finder (version 4.04) [95] and Inverted Repeat
Finder (version 3.05) [96]. Annotation of dispersed repeats was
done with Repeat Masker (version 3-3-0) [97]. The
alleles
of
the
rrp6l2-2
and
rrp6l3-1
correspond
to
SALK_011429
and
SALK_122492
lines,
respectively. The
rrp6l1-2 allele was isolated from the University of Wisconsin
BASTA population. The ecotype background is Col-0 for all Salk
alleles and Ws for University of Wisconsin alleles. To induce
iRNAi, seedlings were germinated and grown for 7 days on K6
MS plates with 8 mM 17b-estradiol, as described before [1]. For analysis of locus-specific expression of smRNAs (solo LTR,
AtSN1, IGN5, REG3, and REG4), the expressed normalized
reads per million (rpm) were calculated for respective genomic
locus and locus-specific datasets were plotted for comparisons. Analysis of DNA methylation Genomic DNA was isolated from 7-day-old seedlings using a
DNeasy kit (QIAGEN). The methylation analysis using DNA
sensitive
methylation
enzymes
was
followed
as
described
[27,31,77]. SmRNA–Independent Exosome Silencing AT1G03360 and AT3G61620 loci), respectively. Therefore,
silencer sequences produced from iRNAi transgenes were filtered
out from each library and libraries were analyzed separately to
ensure accurate interpretations. The remaining smRNA reads,
termed FLR for filtered reads, were used for further analysis. ases to modulate repression of heterochromatic sequences. The
mechanisms that link this RNA metabolic complex, the epigenetic
modification of histone methylation, and heterochromatic silenc-
ing in plants remain to be elucidated. Our results indicate that
there is no one-size-fits-all pathway or mechanism that exclusively
governs silencing of all loci; rather, different loci and different
players in RdDM interact with different pathways and are silenced
by different, likely overlapping mechanisms. The positional effects
and the surrounding landscape most likely also play important
roles in the choice of silencing mechanisms and the interplay
between them. This may reflect the crucial importance of silencing
in developmental gene regulation and in maintenance of genomic
stability by suppression of invasive sequences. Each library was normalized either to the total number of
mapped non-redundant reads or to the total number of non-
redundant filtered reads (FLR), multiplied by 106 (rpm, reads per
million). Both methods of normalizations were compared and
found to produce results which lead to identical interpretations,
therefore, only data analyzed using filtered reads are presented in
this study. Classification of small RNAs was performed by BEDTools
(v2.10.0) [93] and in-house UNIX shell programming using the
following databases: TAIR9 annotations for protein coding and
non-coding features (tRNA, rRNA, ncNRA, miRNA, snRNA,
snoRNA, and transposable elements [76]), miRBase (release 18)
[94] or mature miRNA annotations. Some smRNAs match more
than one annotation category; therefore the sum of the numbers is
bigger than the total input number. Bioinformatic analysis of small RNAs Data processing was done using available tools and custom in-
house
UNIX
shell
programming
[43,75,87–90]. The
raw
sequences in Illumina GAIIx and demultiplexed HiSeq 2000
sequencing reads were trimmed removing adapter using ‘‘fas-
tx_clipper’’ in the FASTX-Toolkit (version 0.0.13) [91] and
smRNAs with lengths between 15- and 32-nt were selected and
mapped to the Arabidopsis genomic sequences (TAIR9 version)
using BOWTIE (version 0.12.7) [92]. Reads that failed to perfectly
map to the nuclear genome with no mismatches, and reads present
in fewer than two counts were discarded. All Arabidopsis lines used
in this study carried iRNAi cassette transgenes used for inactivation
of either RRP4 or RRP41 exosome subunit genes [1]. These
silencing cassettes generate a number of 21-, 22- and 24-nt silencer
sequences corresponding to RRP4 or RRP41 genes (mapping to RNA analysis Total RNA was isolated from 7-day-old seedlings using the
mirVana
miRNA
isolation
kit
(Ambion)
according
to
the
manufacturer’s protocol. The total RNA sample was used for
sequencing library construction using the Small RNA sample Prep
v1.5 kit and TruSeq Small RNA Sample Prep kit (Illumina, San
Diego, CA) according to the manufacturer’s instructions. The
smRNA libraries were sequenced using the Illumina Genetic
Analyzer II (by DNA Core Facility, University of Missouri) and
Illumina HiSeq 2000 (by Biotechnology Center, University of
Wisconsin) according to the manufacturer’s instructions. HiSeq
2000 sequencing reads were demultiplexed using Casava v 1.8 (by
Bioinformatic Resource Center, University of Wisconsin) before
further bioinformatic analysis Total RNA was isolated from 7-day-old seedlings using the
Trizol method. For RT-qPCR, 1–4 mg of total RNA digested with
DNase I (Fermentas) was reverse transcribed 1 hour either at 50uC
(for oligo-dT primer) or 55uC (for specific primers) using 60–100
units SuperScript III Reverse Transcriptase (Invitrogen). Tran-
scripts were quantified by RT-qPCR using the comparative
threshold cycle method (DDCt, primers listed in Table S4), using
Actin2 (At3g18780) as endogenous reference. Polyacrylamide
Northern Blot analyses were performed as described [25]. How can exosome function in gene silencing in
Arabidopsis? Alternative possibility is that Pol II produces two distinct pools of In summary, our data suggest that the exosome likely acts in a
parallel pathway to RdDM pathways in gene silencing, possibly
affecting the transcriptional interplay of different RNA polymer- March 2013 | Volume 9 | Issue 3 | e1003411 March 2013 | Volume 9 | Issue 3 | e1003411 14 PLOS Genetics | www.plosgenetics.org SmRNA–Independent Exosome Silencing How can exosome function in gene silencing in
Arabidopsis? The DNA-methylation-independent
function of Pol V may then be in addition to its function in
RdDM, and may operate in parallel to the exosome pathway. If
this is the case, the depletion of both rrp41 and nrpd1 may not lead
to synergistic derepression because it would be compensated by
the RdDM-independent function of Pol V. However, deficiencies
in exosome and Pol V would result in synergistic desilencing due to
the loss of three different pathways. Both Pol II and Pol V were
reported to be responsible for the transcription of scaffold RNA
and be required for silencing [30,31], although it is not known how
their activities are functionally integrated. It is also not known how
Pol V initiation sites are chosen, but they appear to be promoter
independent [31]. Perhaps transcription by Pol II helps maintain
open chromatin architecture at this site, and together with the
resulting noncoding RNAs facilitates Pol V transcription initiation. Alternative possibility is that Pol II produces two distinct pools of having unknown functions in addition to the function it plays in
the RdDM pathway [45]. The DNA-methylation-independent
function of Pol V may then be in addition to its function in
RdDM, and may operate in parallel to the exosome pathway. If
this is the case, the depletion of both rrp41 and nrpd1 may not lead
to synergistic derepression because it would be compensated by
the RdDM-independent function of Pol V. However, deficiencies
in exosome and Pol V would result in synergistic desilencing due to
the loss of three different pathways. Both Pol II and Pol V were
reported to be responsible for the transcription of scaffold RNA
and be required for silencing [30,31], although it is not known how
their activities are functionally integrated. It is also not known how
Pol V initiation sites are chosen, but they appear to be promoter
independent [31]. Perhaps transcription by Pol II helps maintain
open chromatin architecture at this site, and together with the
resulting noncoding RNAs facilitates Pol V transcription initiation. Author Contributions Conceived the experiments: JAC DAB. Designed the experiments: JAC J-
HS. Performed the experiments: J-HS. Analyzed the data: JAC J-HS H-
LVW. Contributed reagents/materials: JL. Wrote the paper: JAC. Bioinformatic pipeline development: H-LVW JAC. Bioinformatic analysis:
H-LVW. Contributed to bioinformatics tool development/writing: BLD
JHS H-LVW. Conceived the experiments: JAC DAB. Designed the experiments: JAC J-
HS. Performed the experiments: J-HS. Analyzed the data: JAC J-HS H-
LVW. Contributed reagents/materials: JL. Wrote the paper: JAC. Bioinformatic pipeline development: H-LVW JAC. Bioinformatic analysis:
H-LVW. Contributed to bioinformatics tool development/writing: BLD
JHS H-LVW. Figure S3
Effects of exosome deletion, RdDM, and other
mutants. (A) Diagrams of IGN5, REG 3 and REG 4 genomic loci,
based on analysis of transcription units by Wierzbicki et al. (2008)
[1,31]. Region A corresponds to siRNA producing region, region Supporting Information Table S2
Summary of smRNA sequence reads in the libraries of
RRP41/nrpd1,
rrp41-i/nrpd1,
RRP41/nrpe1,
rrp41-i/nrpe1,
RRP41/rdr2, rrp41-i/rdr2, RRP41/dcl3 and rrp41-i/dcl3 plants. (XLS) Figure S1
iRNAi silencer sequences produced by rrp4-i and
rrp41-i cassettes in response to estradiol treatment. (A, B) 20–25 nt
smRNAs corresponding RRP4 in rrp4-i (A) and corresponding and
to RRP4 in rrp41-i (B) depletion mutants profiled based on the
length of the reads. (C, D) 20–25 nt smRNAs produced from in
rrp4-i (C) and rrp4-i (D) depletion mutants profiled based on both
their length and the terminal 59 nucleotide. The major silencer
sequences are 59U and 59A smRNA species. (TIF) Table S3
Expression profiling of known mature miRNAs in the
libraries of RRP4, rrp4-i, RRP41, and rrp41-i mutant plants. (XLS) Table S4
Oligonucleotides used in this study. (XLS) Figure S2
miRNA families, miR-158a, miR-158b, miR-860,
miR-823, miR-841, miR-5561 and variations in sequence length. miRNA families miR-158a, miR-158b, miR-860, miR-823, miR-
841, and miR-5561 and variations in sequence length in each
family. smRNAs mapped to matching mature miR-158, miR-860,
miR-823, miR-841, and miR-5561 sequences [94](miRBase
release 18) were plotted versus the sum of their normalized reads
per million (rpm) from smRNA libraries constructed from RRP4,
rrp4-i, RRP41, rrp41-i, RRP41/nrpd1, rrp41 iRNAi/nrpd1, RRP4
iRNAi/nrpe1 and rrp41 iRNAi/nrpd1 mutants. (TIF) Acknowledgments We thank Aaron Stevens for IT assistance, 3D artist Thal Syhabout for
preparing the digital artwork for our model, and Xuemei Chen and Steven
Jacobsen for RdDM mutants. This paper is dedicated to the memory of
Dima Belostotsky, an eminent scientist and a valued colleague whose
untimely death in March 2009 is a big loss for the field of RNA biology. (TIF) Table S1
Summary of smRNA sequence reads in the libraries of
RRP4, rrp4-i, RRP41, and rrp41-i plants. (XLS) References 1. Chekanova JA, Gregory BD, Reverdatto SV, Chen H, Kumar R, et al. (2007)
Genome-Wide High-Resolution Mapping of Exosome Substrates Reveals
Hidden Features in the Arabidopsis Transcriptome. Cell 131: 1340–1353. 11. Wyers F, Rougemaille M, Badis G, Rousselle J-C, Dufour M-E, et al. (2005)
Cryptic pol II transcripts are degraded by a nuclear quality control pathway
involving a new poly(A) polymerase. Cell 121: 725–737. 12. Kapranov P, Cheng J, Dike S, Nix DA, Duttagupta R, et al. (2007) RNA maps
reveal new RNA classes and a possible function for pervasive transcription. Science 316: 1484–1488. 2. Kapranov P, Willingham AT, Gingeras TR (2007) Genome-wide transcription
and the implications for genomic organization. Nature Reviews Genetics 8: 413–
423. 13. Seila AC, Calabrese JM, Levine SS, Yeo GW, Rahl PB, et al. (2008) Divergent
transcription from active promoters. Science 322: 1849–1851. 3. Wilhelm BT, Marguerat S, Watt S, Schubert F, Wood V, et al. (2008) Dynamic
repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution. Nature 453: 1239–1243. 14. Preker P, Nielsen J, Kammler S, Lykke-Andersen S, Christensen MS, et al. (2008) RNA exosome depletion reveals transcription upstream of active human
promoters. Science 322: 1851–1854. 4. Neil H, Malabat C, d’Aubenton-Carafa Y, Xu Z, Steinmetz LM, et al. (2009)
Widespread bidirectional promoters are the major source of cryptic transcripts in
yeast. Nature 457: 1038–1042. 15. Mitchell P, Petfalski E, Shevchenko A, Mann M, Tollervey D (1997) The
Exosome: A Conserved Eukaryotic RNA Processing Complex Containing
Multiple 39R59 Exoribonucleases. Cell 91: 457–466. 5. Jacquier A (2009) Applications of next-generation sequencing: The complex
eukaryotic transcriptome: unexpected pervasive transcription and novel small
RNAs. Nature Reviews Genetics 10: 833–844. p
16. Bonneau F, Basquin J, Ebert J, Lorentzen E, Conti E (2009) The Yeast Exosome
Functions as a Macromolecular Cage to Channel RNA Substrates for
Degradation. Cell 139: 547–559. 6. Guttman M, Rinn JL (2012) Modular regulatory principles of large non-coding
RNAs. Nature 482: 339–346. 17. Liu Q, Greimann JC, Lima CD (2006) Reconstitution, Activities, and Structure
of the Eukaryotic RNA Exosome. Cell 127: 1223–1237. 7. Wang KC, Yang YW, Liu B, Sanyal A, Corces-Zimmerman R, et al. (2011) A
long noncoding RNA maintains active chromatin to coordinate homeotic gene
expression. Nature 472: 120–124. 18. Dziembowski A, Lorentzen E, Conti E, Se´raphin B (2007) A single subunit,
Dis3, is essentially responsible for yeast exosome core activity. Nat Struct Mol
Biol 14: 15–22. 8. SmRNA–Independent Exosome Silencing B corresponds to scaffold producing region in both loci, red lines
mark regions amplified in RT-PCR and qPCR. (B) 20–25 nt
smRNAs produced from region A of IGN5 in rrp4-i, rrp41-i
exosome depletion lines and RdDM mutants. All locus-specific
datasets of 20–25 nt smRNAs are plotted versus the sum of their
normalized reads per million (rpm). (C, D) RT-PCR analysis of
RRP6L1 and RRP6L mRNA expression in rrp6L1 and rrp6L2
insertion mutants. Chromatin immunoprecipitation (ChIP) assays y
ChIP was performed as described [98]. One gram of 7-day-old
seedlings was used for each experiment. All ChIP experiments
were reproduced at least twice on each of the two or more
biological replicates. The normalization was done relative to input
using [99]. Anti-RNA Pol II (ab817) and anti-H3K9me2 (ab1220)
were obtained from Abcam, and anti-H3K27me1 antibody from
Upstate. An equal amount of chromatin not treated with antibody
was used as the mock antibody control. The ChIPed DNA was
purified using PCR purification kit (Fermentas) before being used
for qPCR. The primer sets used for the PCR are listed in Table
S4. March 2013 | Volume 9 | Issue 3 | e1003411 15 PLOS Genetics | www.plosgenetics.org RNA immunoprecipitation (RIP) RNA immunoprecipitation (RIP) RIP assays were performed by adapting an existing protocol
[100]. Transgenic plants expressing TAP-tagged RRP41 at
physiological levels [1] were used in the experiment. Two grams
of 2-week-old seedlings were collected and fixed with 1%
formaldehyde. For RRP41-RNA complex purification, the chro-
matin solution was incubated overnight with prewashed IgG
Sepharose 6 Fast Flow (GE Healthcare) at 4uC. Immunoprecip-
itated RNA was purified with phenol: chloroform and cDNA
synthesis was performed using SuperScript III reverse transcrip-
tase (Invitrogen) and random hexamers (Promega). The primer
sets used for the PCR are listed in Table S4. SmRNA–Independent Exosome Silencing (2005) Plant Nuclear
RNA Polymerase IV Mediates siRNA and DNA Methylation-Dependent
Heterochromatin Formation. Cell 120: 613–622. 28. Haag JR, Pikaard CS (2011) Multisubunit RNA polymerases IV and V:
purveyors of non-coding RNA for plant gene silencing. Nature Reviews
Molecular Cell Biology 12: 483–492. 55. Vasiljeva L, Kim M, Terzi N, Soares LM, Buratowski S (2008) Transcription
Termination and RNA Degradation Contribute to Silencing of RNA
Polymerase II Transcription within Heterochromatin. Molecular Cell 29:
313–323. 29. Chan SWL, Zilberman D, Xie Z, Johansen LK, Carrington JC, et al. (2004)
RNA silencing genes control de novo DNA methylation. Science 303: 1336. 56. Reyes-Turcu FE, Grewal SI (2012) Different means, same end—heterochro-
matin formation by RNAi and RNAi-independent RNA processing factors in
fission yeast. Current Opinion in Genetics & Development 22: 156–163. 30. Zheng B, Wang Z, Li S, Yu B, Liu JY, et al. (2009) Intergenic transcription by
RNA Polymerase II coordinates Pol IV and Pol V in siRNA-directed
transcriptional gene silencing in Arabidopsis. Genes & Development 23:
2850–2860. fission yeast. Current Opinion in Genetics & Development 22: 156 57. Wang S-W, Stevenson AL, Kearsey SE, Watt S, Ba¨hler J (2008) Global role for
polyadenylation-assisted nuclear RNA degradation in posttranscriptional gene
silencing. Molecular and Cellular Biology 28: 656–665. 31. Wierzbicki AT, Haag JR, Pikaard CS (2008) Noncoding Transcription by RNA
Polymerase Pol IVb/Pol V Mediates Transcriptional Silencing of Overlapping
and Adjacent Genes. Cell 135: 635–648. g
gy
58. Camblong J, Iglesias N, Fickentscher C, Dieppois G, Stutz F (2007) Antisense
RNA stabilization induces transcriptional gene silencing via histone deacetyla-
tion in S. cerevisiae. Cell 131: 706–717. 32. Zilberman D, Cao X, Jacobsen SE (2003) ARGONAUTE4 Control of Locus-
Specific siRNA Accumulation and DNA and Histone Methylation. Science 299:
716–719. 59. Houseley J, Rubbi L, Grunstein M, Tollervey D, Vogelauer M (2008) A ncRNA
modulates histone modification and mRNA induction in the yeast GAL gene
cluster. Molecular Cell 32: 685–695. 33. Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, et al. (2004)
Genetic and Functional Diversification of Small RNA Pathways in Plants. PLoS
Biol 2: e104. doi:10.1371/journal.pbio.0020104.sg002. 60. Mosher RA, Schwach F, Studholme D, Baulcombe DC (2008) PolIVb influences
RNA-directed DNA methylation independently of its role in siRNA biogenesis. PNAS 105: 3145–3150. 34. Zheng X, Zhu J, Kapoor A, Zhu J-K (2007) Role of Arabidopsis AGO6 in
siRNA accumulation, DNA methylation and transcriptional gene silencing. The
EMBO Journal 26: 1691–1701. 61. SmRNA–Independent Exosome Silencing Herr AJ, Molnar A, Jones A, Baulcombe DC (2006) Defective RNA processing
enhances RNA silencing and influences flowering of Arabidopsis. PNAS 103:
14994–15001. J
35. Gao Z, Liu H-L, Daxinger L, Pontes O, He X, et al. (2010) An RNA polymerase
II- and AGO4-associated protein acts in RNA-directed DNA methylation. Nature 465: 106–109. 62. Wierzbicki AT, Ream TS, Haag JR, Pikaard CS (2009) RNA polymerase V
transcription guides ARGONAUTE4 to chromatin. Nat Genet 41: 630–634. 36. Kanno T, Bucher E, Daxinger L, Huettel B, Bo¨hmdorfer G, et al. (2008) A
structural-maintenance-of-chromosomes hinge domain–containing protein is
required for RNA-directed DNA methylation. Nat Genet 40: 670–675. 63. Amedeo P, Habu Y, Afsar K, Scheld OM, Paszkowski J (2000) Disruption of the
plant gene wyw releases transcriptional silencing of methylated genes. Nature
405: 203–206. 37. Kanno T, Bucher E, Daxinger L, Huettel B, Kreil DP, et al. (2010) RNA-
directed DNA methylation and plant development require an IWR1-type
transcription factor. EMBO reports 11: 65–71. 64. Tariq M, Habu Y, Paszkowski J (2002) Depletion of MOM1 in non-dividing
cells of Arabidopsis plants releases transcriptional gene silencing. EMBO reports 3:
951–955. p
p
38. Bu¨hler M, Haas W, Gygi SP, Moazed D (2007) RNAi-Dependent and -
Independent RNA Turnover Mechanisms Contribute to Heterochromatic Gene
Silencing. Cell 129: 707–721. 65. Vaillant I, Schubert I, Tourmente S, Mathieu O (2006) MOM1 mediates DNA-
methylation-independent silencing of repetitive sequences in Arabidopsis. EMBO reports 7: 1273–1278. 39. Bu¨hler M, Spies N, Bartel DP, Moazed D (2008) TRAMP-mediated RNA
surveillance prevents spurious entry of RNAs into the Schizosaccharomyces
pombe siRNA pathway. Nat Struct Mol Biol 15: 1015–1023. 66. Shin Takeda, Zerihun Tadele, Hofmann I, Aline V . Probst, Angelis KJ, et al. (2004) BRU1, a novel link between responses to DNA damage and epigenetic
gene silencing in Arabidopsis. Genes & Development 18: 782–793. 40. Reyes-Turcu FE, Zhang K, Zofall M, Chen E, Grewal SIS (2011) Defects in
RNA quality control factors reveal RNAi-independent nucleation of hetero-
chromatin. Nat Struct Mol Biol 18: 1132–1138. 67. Elmayan T, Proux F, Vaucheret H (2005) Arabidopsis RPA2: A Genetic Link
among Transcriptional Gene Silencing, DNA Repair, and DNA Replication. Current Biology 15: 1919–1925. 41. Zofall M, Yamanaka S, Reyes-Turcu FE, Zhang K, Rubin C, et al. (2012) RNA
Elimination Machinery Targeting Meiotic mRNAs Promotes Facultative
Heterochromatin Formation. Science 335: 96–100. 68. SmRNA–Independent Exosome Silencing 21. Schneider C, Kudla G, Wlotzka W, Tuck A, Tollervey D (2012) Transcriptome-
wide Analysis of Exosome Targets. Molecular Cell: 1–12. 48. Pontier D, Yahubyan G, Vega D, Bulski A, Saez-Vasquez J, et al. (2005)
Reinforcement of silencing at transposons and highly repeated sequences
requires the concerted action of two distinct RNA polymerases IV in
Arabidopsis. Genes & Development 19: 2030–2040. 22. LaCava J, Houseley J, Saveanu C, Petfalski E, Thompson E, et al. (2005) RNA
degradation by the exosome is promoted by a nuclear polyadenylation complex. Cell 121: 713–724. 49. Gasciolli V, Mallory AC, Bartel DP, Vaucheret H (2005) Partially Redundant
Functions of Arabidopsis DICER-like Enzymes and a Role for DCL4 in
Producing trans-Acting siRNAs. Current Biology 15: 1494–1500. 23. Callahan KP, Butler JS (2010) TRAMP complex enhances RNA degradation by
the nuclear exosome component Rrp6. Journal of Biological Chemistry 285:
3540–3547. 50. Huettel B, Kanno T, Daxinger L, Aufsatz W, Matzke AJ, et al. (2006)
Endogenous targets of RNA-directed DNA methylation and Pol IV in
Arabidopsis. The EMBO Journal 25: 1–9. 24. Schmidt K, Xu Z, Mathews DH, Butler JS (2012) Air proteins control
differential TRAMP substrate specificity for nuclear RNA surveillance. RNA 18:
1934–1945. p
J
51. Herr AJ, Jensen MB, Dalmay T, Baulcombe DC (2005) RNA Polymerase IV
Directs Silencing of Endogenous DNA. Science 308: 118–120. 25. Chekanova JA (2000) Poly(A) Tail-dependent Exonuclease AtRrp41p from
Arabidopsis thaliana Rescues 5.8 S rRNA Processing and mRNA Decay Defects
of the Yeast ski6 Mutant and Is Found in an Exosome-sized Complex in Plant
and Yeast Cells. Journal of Biological Chemistry 275: 33158–33166. 52. Kanno T, Huettel B, Mette MF, Aufsatz W, Jaligot E, et al. (2005) Atypical
RNA polymerase subunits required for RNA-directed DNA methylation. Nat
Genet 37: 761–765. 53. Myouga F, Tsuchimoto S, Noma K, Ohtsubo H, Ohtsubo E (2001)
Identification and structural analysis of SINE elements in the Arabidopsis
thaliana genome. Genes and Genetics systems 76: 169–179. 26. Lister R, O’Malley RC, Tonti-Filippini J, Gregory BD, Berry CC, et al. (2008)
Highly Integrated Single-Base Resolution Maps of the Epigenome in
Arabidopsis. Cell 133: 523–536. 54. Lange H, Holec S, Cognat VE, Pieuchot L, Le Ret M, et al. (2008) Degradation
of a Polyadenylated rRNA Maturation By-Product Involves One of the Three
RRP6-Like Proteins in Arabidopsis thaliana. Molecular and Cellular Biology 28:
3038–3044. 27. Onodera Y, Haag JR, Ream T, Nunes PC, Pontes O, et al. SmRNA–Independent Exosome Silencing Baurle I, Smith L, Baulcombe DC, Dean C (2007) Widespread Role for the
Flowering-Time Regulators FCA and FPA in RNA-Mediated Chromatin
Silencing. Science 318: 109–112. 42. Gazzani S, Lawrenson T, Woodward C, Headon D, Sablowski R (2004) A Link
Between mRNA Turnover and RNA Interference in Arabidopsis. Science 306:
1046–1048. 69. Yokthongwattana C, Bucher E, aikovski MC, Vaillant I, Nicolet JEL, et al. (2009) MOM1 and Pol-IV/V interactions regulate the intensity and specificity of
transcriptional gene silencing. The EMBO Journal 29: 1–12. 43. Mi S, Cai T, Hu Y, Chen Y, Hodges E, et al. (2008) Sorting of Small RNAs into
Arabidopsis Argonaute Complexes Is Directed by the 59 Terminal Nucleotide. Cell 133: 116–127. 70. Liu F, Bakht S, Dean C (2012) Cotranscriptional Role for Arabidopsis DICER-
LIKE 4 in Transcription Termination. Science 335: 1621–1623. LIKE 4 in Transcription Termination. Science 335: 1621–162 71. Moissiard G, Cokus SJ, Cary J, Feng S, Billi AC, et al. (2012) MORC Family
ATPases Required for Heterochromatin Condensation and Gene Silencing. Science 336: 1448–1451. 44. Lee T-F, Gurazada SGR, Zhai J, Li S, Simon SA, et al. (2012) RNA polymerase
V-dependent small RNAs in Arabidopsis originate from small, intergenic loci
including most SINE repeats. Epigenetics 7: 781–795. 72. Habu Y, Mathieu O, Tariq M, Probst AV, Smathajitt C, et al. (2006) Epigenetic
regulation of transcription in intermediate heterochromatin. EMBO reports 7:
1279–1284. 45. Wierzbicki AT, Cocklin R, Mayampurath A, Lister R, Rowley MJ, et al. (2012)
Spatial and functional relationships among Pol V-associated loci, Pol IV-
dependent siRNAs, and cytosine methylation in the Arabidopsis epigenome. Genes & Development 26: 1825–1836. 73. Steimer A, Amedeo P, Afsar
Karin, Fransz P, Scheid OM, et al. (2000)
Endogenous Targets of Transcriptional Gene Silencing in Arabidopsis. The
plant Cell 12: 1165–1178. 46. Haag JR, Pontes O, Pikaard CS (2009) Metal A and metal B sites of nuclear
RNA polymerases Pol IV and Pol V are required for siRNA-dependent DNA
methylation and gene silencing. PLoS ONE 4: e4110. doi:10.1371/journal.-
pone.0004110. p
74. Probst AV, Fransz PF, Paszkowski J, Scheid OM (2003) Two means of
transcriptional reactivation within heterochromatin. THe plant journal 33: 743–
749. 47. Kristin D . Kasschau, Noah Fahlgren, Elisabeth J . Chapman, Christopher M . Sullivan, Jason S . Cumbie, et al. (2007) Genome-Wide Profiling and Analysis of
Arabidopsis siRNAs. PLoS Biol 5: 1–15. 75. References Moazed D (2009) Small RNAs in transcriptional gene silencing and genome
defence. Nature 457: 413–420. 9. Matzke M, Kanno T, Daxinger L, Huettel B, Matzke AJ (2009) RNA-mediated
chromatin-based silencing in plants. Current Opinion in Cell Biology 21: 367–376. 19. Lebreton A, Tomecki R, Dziembowski A, Se´raphin B (2008) Endonucleolytic
RNA cleavage by a eukaryotic exosome. Nature 456: 993–996. 10. Belostotsky D (2009) Exosome complex and pervasive transcription in eukaryotic
genomes. Current Opinion in Cell Biology 21: 352–358. doi:10.1016/
j.ceb.2009.04.011. 20. Briggs MW, Burkard KTD, Butler JS (1998) Rrp6p, the Yeast Homologue of the
Human PM-Scl 100-kDa Autoantigen, Is Essential for Efficient 5.8 S rRNA 39
End Formation. Journal of Biological Chemistry 273: 13255–13263. 10. Belostotsky D (2009) Exosome complex and pervasive transcription in eukaryotic
genomes. Current Opinion in Cell Biology 21: 352–358. doi:10.1016/
j.ceb.2009.04.011. March 2013 | Volume 9 | Issue 3 | e1003411 March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org 16 SmRNA–Independent Exosome Silencing 76. Hollister JD, Gaut BS (2009) Epigenetic silencing of transposable elements: A
trade-off between reduced transposition and deleterious effects on neighboring
gene expression. Genome Research 19: 1419–1428. 88. Fischer SEJ, Montgomery TA, Zhang C, Fahlgren N, Breen PC, et al. (2011)
The ERI-6/7 Helicase Acts at the First Stage of an siRNA Amplification
Pathway That Targets Recent Gene Duplications. PLoS Genet 7: e1002369. doi:10.1371/journal.pgen.1002369.g005. 77. Jullien PE, Kinoshita T, Ohad N, Berger F (2006) Maintenance of DNA
Methylation during the Arabidopsis Life Cycle Is Essential for Parental
Imprinting. The plant Cell 18: 1360–1372. 89. Zheng Q, Ryvkin P, Li F, Dragomir I, Valladares O, et al. (2010) Genome-Wide
Double-Stranded RNA Sequencing Reveals the Functional Significance of Base-
Paired RNAs in Arabidopsis. PLoS Genet 6: e1001141. doi:10.1371/journal. pgen.1001141.g007. 78. Hamilton A, Voinnet O, Chappell L, Baulcombe D (2002) Two classes of short
interfering RNA in RNA silencing. The EMBO Journal 21: 4671–4679. 90. Olson AJ, Brennecke J, Aravin AA, Hannon GJ, Sachidanandam R (2008)
Analysis of large-scale sequencing of small RNAs. Pacific Symposium on
Biocomputing 13: 126–136. g
g
79. Halic M, Moazed D (2010) Dicer-Independent Primal RNAs Trigger RNAi and
Heterochromatin Formation. Cell 140: 504–516. 80. Zhang K, Fischer T, Porter RL, Dhakshnamoorthy J, Zofall M, et al. (2011)
Clr4/Suv39 and RNA Quality Control Factors Cooperate to Trigger RNAi and
Suppress Antisense RNA. Science 331: 1624–1627. 91. Blankenberg D, Gordon A, Kuster Von G, Coraor N, Taylor J, et al. (2010)
Manipulation of FASTQ data with Galaxy. Bioinformatics 26: 1783–1785. 92. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-
efficient alignment of short DNA sequences to the human genome. Genome Biol
10: R25. pp
81. Bernard P, Drogat J, Dheur S, Longo G, Javerzat JP (2010) Splicing Factor
Spf30 Assists Exosome-Mediated Gene Silencing in Fission Yeast. Molecular
and Cellular Biology 30: 1145–1157. 93. Aaron R . Quinlan, Hal IM (2010) BEDTools: a flexible suite of utilities for
comparing genomic features. Bioinformatics 26: 841–842. 82. Pontes O, Costa-Nunes P, Vithayathil P, Pikaard CS (2009) RNA Polymerase V
Functions in Arabidopsis Interphase Heterochromatin Organization Indepen-
dently of the 24-nt siRNA-Directed DNA Methylation Pathway. Molecular
Plant 2: 700–710. p
g g
94. Kozomara A, Griffiths-Jones S (2010) miRBase: integrating microRNA
annotation and deep-sequencing data. Nucleic Acids Research 39: D152–D157. p
q
g
95. Benson G (1998) Tandem repeats finder: a program to analyze DNA sequences. SmRNA–Independent Exosome Silencing Numa H, Kim J-M, Matsui A, Kurihara Y, Morosawa T, et al. (2009)
Transduction of RNA-directed DNA methylation signals to repressive histone
marks in Arabidopsis thaliana. The EMBO Journal 29: 352–362. March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org March 2013 | Volume 9 | Issue 3 | e1003411 17 SmRNA–Independent Exosome Silencing Nucleic Acids Research 27: 573–580. 83. Keller C, Adaixo R, Stunnenberg R, Woolcock KJ, Hiller S, et al. (2012)
HP1Swi6 Mediates the Recognition and Destruction of Heterochromatic RNA
Transcripts. Molecular Cell 47: 215–227. 96. Warburton PE, Giordano J, Cheung F, Gelfand Y, Benson G (2004) Inverted
Repeat Structure of the Human Genome: The X-Chromosome Contains a
Preponderance of Large, Highly Homologous Inverted Repeats That Contain
Testes Genes. Genome Research 14: 1861–1869. 84. Zhang X, Germann S, Blus BJ, Khorasanizadeh S, Gaudin V, et al. (2007) The
Arabidopsis LHP1 protein colocalizes with histone H3 Lys27 trimethylation. Nat
Struct Mol Biol 14: 869–871. Testes Genes. Genome Research 14: 1861–1869. 97. Smit A, Hubley R, Green P (1996) RepeatMasker Open-3.0. Available: http://
www.repeatmasker.org. 85. Douet J, Tutois S, Tourmente S (2009) A Pol V–Mediated Silencing,
Independent of RNA–Directed DNA Methylation, Applies to 5S rDNA. PLoS
Genet 5: e1000690. doi:10.1371/journal.pgen.1000690.t001. 98. Morohashi K, Xie Z, Grotewold E (2009) Gene-specific and genome-wide ChIP
approaches to study plant transcriptional networks. Methods Mol Biol 553: 3–
12. 86. Pontes O, Li CF, Nunes PC, Haag J, Ream T, et al. (2006) The Arabidopsis
Chromatin-Modifying Nuclear siRNA Pathway Involves a Nucleolar RNA
Processing Center. Cell 126: 79–92. 99. Haring M, Offermann S, Danker T, Horst I, Peterhansel C, et al. (2007)
Chromatin immunoprecipitation: optimization, quantitative analysis and data
normalization. Plant Methods 3: 11. 87. Wang H, Zhang X, Liu J, Kiba T, Woo J, et al. (2011) Deep sequencing of small
RNAs specifically associated with Arabidopsis AGO1 and AGO4 uncovers new
AGO functions. THe plant journal 67: 292–304. 100. Terzi LC, Simpson GG (2009) Arabidopsis RNA immunoprecipitation. THe
plant journal 59: 163–168. March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org 18
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English
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The role of interferon regulatory factor 8 for retinal tissue homeostasis and development of choroidal neovascularisation
|
Journal of neuroinflammation
| 2,021
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cc-by
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Zhang et al. Journal of Neuroinflammation (2021) 18:215
https://doi.org/10.1186/s12974-021-02230-y Zhang et al. Journal of Neuroinflammation (2021) 18:215
https://doi.org/10.1186/s12974-021-02230-y (2021) 18:215 Zhang et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-021-02230-y The role of interferon regulatory factor 8
for retinal tissue homeostasis and
development of choroidal
neovascularisation Peipei Zhang1, Anja Schlecht1,2, Julian Wolf1, Stefaniya Boneva1, Yannik Laich1, Jana Koch1, Franziska Ludwig1,
Myriam Boeck1, Adrian Thien1, Carmen Härdtner3,4, Katrin Kierdorf5,6,7, Hansjürgen Agostini1, Günther Schlunck1,
Marco Prinz5,7,8, Ingo Hilgendorf3,4, Peter Wieghofer5,9*† and Clemens Lange1*† Abstract Background: Microglia cells represent the resident innate immune cells of the retina and are important for retinal
development and tissue homeostasis. However, dysfunctional microglia can have a negative impact on the
structural and functional integrity of the retina under native and pathological conditions. Methods: In this study, we examined interferon-regulatory factor 8 (Irf8)–deficient mice to determine the
transcriptional profile, morphology, and temporospatial distribution of microglia lacking Irf8 and to explore the
effects on retinal development, tissue homeostasis, and formation of choroidal neovascularisation (CNV). Results: Our study shows that Irf8-deficient MG exhibit a considerable loss of microglial signature genes
accompanied by a severely altered MG morphology. An in-depth characterisation by fundus photography,
fluorescein angiography, optical coherence tomography and electroretinography revealed no major retinal
abnormalities during steady state. However, in the laser-induced CNV model, Irf8-deficient microglia showed an
increased activity of biological processes critical for inflammation and cell adhesion and a reduced MG cell density
near the lesions, which was associated with significantly increased CNV lesion size. Conclusions: Our results suggest that loss of Irf8 in microglia has negligible effects on retinal homeostasis in the
steady state. However, under pathological conditions, Irf8 is crucial for the transformation of resident microglia into
a reactive phenotype and thus for the suppression of retinal inflammation and CNV formation. Keywords: Irf8, Interferon regulatory factor 8, Retinal microglia, Choroidal neovascularisation, RNA sequencing * Correspondence: peter.wieghofer@medizin.uni-leipzig.de;
clemens.lange@uniklinik-freiburg.de †Peter Wieghofer and Clemens Lange both contributed equally to the work. 5Medical Faculty, Institute of Neuropathology, University Hospital, University
of Freiburg, Freiburg im Breisgau, Germany
1Medical Faculty, Eye Center, University Hospital, University of Freiburg,
Freiburg im Breisgau, Germany
Full list of author information is available at the end of the article * Correspondence: peter.wieghofer@medizin.uni-leipzig.de;
clemens.lange@uniklinik-freiburg.de
†Peter Wieghofer and Clemens Lange both contributed equally to the work.
5Medical Faculty, Institute of Neuropathology, University Hospital, University
of Freiburg, Freiburg im Breisgau, Germany
1Medical Faculty, Eye Center, University Hospital, University of Freiburg,
Freiburg im Breisgau, Germany
Full list of author information is available at the end of the article Background flow cytometry, immunohistochemistry and RNA se-
quencing (RNA-seq). We found that Irf8-deficient MG
exhibited functionally relevant alterations in gene ex-
pression patterns that were associated with a significant
disruption of microglial development, normal postnatal
retinal vascular and functional development, and in-
creased CNV lesion size in the adult situation. flow cytometry, immunohistochemistry and RNA se-
quencing (RNA-seq). We found that Irf8-deficient MG
exhibited functionally relevant alterations in gene ex-
pression patterns that were associated with a significant
disruption of microglial development, normal postnatal
retinal vascular and functional development, and in-
creased CNV lesion size in the adult situation. g
Retinal microglia cells (rMG) constitute the resident
myeloid cell population in the neuroretina and are crit-
ical for retinal development, tissue homeostasis and re-
sponse to cell damage. During development, rMG are in
intimate contact to endothelial tip cells and contribute
to postnatal vascular development [16] as well as neur-
onal survival by modulating programmed cell death and
trophic influences [56]. During adulthood, rMG interact
closely with synapses to maintain synaptic structure and
electroretinal function and continuously scan the local
environment for danger signals associated with injury or
pathogens [56]. In response to tissue damage or infec-
tion, rMG rapidly attain an activated phenotype, migrate
towards the site of injury and contribute to phagocytosis,
inflammation and pathological events [4, 52, 59]. As
such, activated microglia cells have been found in the
subretinal space of patients with age-related macular de-
generation (AMD) and in particular at sites of choroidal
neovascularisation (CNV) in neovascular AMD [11, 20]
which is a common cause of irreversible blindness in the
elderly [10, 60]. Studies on the role of rMG in the devel-
opment of CNV, however, revealed conflicting results,
and both detrimental and protective roles of MG for the
progression of CNV have been discussed in the past [2,
14, 36, 49, 51]. Methods
Mice All animal experiments were authorized by the local ani-
mal care and use committee under the respective EU,
national, federal and institutional regulations for animal
experiments (ethical protocol numbers G14/89, G20/13). Mice were bred on a C57BL/6J background and devoid
of the Crb1 mutation. Cx3cr1GFP/GFP mice were crossed
with C57BL/6J mice to generate Cx3cr1GFP/+ (Irf8 WT)
mice. Irf8-/- mice were crossed with Irf8-/-Cx3cr1GFP/GFP
mice to obtain Cx3cr1GFP/+:Irf8-/- (Irf8 KO) mice [24,
26]. Phage artificial chromosome-transgenic Irf8-VENUS
reporter mice were used to trace the expression of IRF8
[53]. CAG::mRFP1 mice were purchased from the Jack-
son Laboratory (Bar Harbor, ME). Laser-induced choroidal neovascularisation (CNV) Laser-induced choroidal neovascularisation (CNV)
The laser-induced CNV model was used as previously
described [15, 31, 50]. In brief, mice were anaesthetized
by intraperitoneal administration of ketamine hydro-
chloride (100 mg/kg, Pharmacia & Upjohn, Erlangen,
Germany) and xylazine (6 mg/kg, Bayer Vital GmbH, Le-
verkusen, Germany). Pupillary dilatation was achieved by
applying 0.5% tropicamide (Bausch + Lomb, Berlin,
Germany) and 5% phenylephrine hydrochloride (URSA-
PHARM Arzneimittel GmbH, Saarbrücken, Germany). After covering the cornea with a coverslip coated with
dexpanthenol eye gel (50 mg/g, Bausch + Lomb, Berlin,
Germany), three to six laser spots (488 nm, 150 mW,
100 μm and 100 ms) were applied to each eye using the
VISULAS 532s Laser System (Carl Zeiss, Jena, Germany)
in combination with ZEISS Laser Slit Lamp 532s (Carl
Zeiss, Jena, Germany). Only laser spots with visible for-
mation of vaporisation bubbles were included in this
study. The aim of this study was to determine the function of
IRF8 in retinal microglia in the healthy as well as per-
turbed retina. Specifically, we aimed to investigate
whether IRF8 is involved in microglial cell homeostasis,
neuroretinal function and pathological CNV formation. The latter is of particular interest, as PU.1 and CSF1R
signalling acting upstream and downstream of IRF8, re-
spectively, are critical for postnatal angiogenesis and for-
mation of pathological neovascularisation in the eye [16,
54]. For this purpose, we analysed Irf8 reporter and
knockout mice by in vivo imaging, functional studies, © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://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. Page 2 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Page 2 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Page 2 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Genotyping Transgenic mice
were genotyped
according to
the
primers and programs shown in supplemental table 1. The interferon regulatory factor (IRF) family of tran-
scription factors consists of nine members that are in-
volved in hematopoietic differentiation, oncogenesis,
Toll-like and purinergic receptor signalling and expres-
sion of interferons and interferon-inducible genes [39,
57]. In particular, Irf8 plays a pivotal role in the regula-
tion of lineage commitment and MG cell maturation
during brain development [27, 47]. Besides its essential
role during development, Irf8 is crucial for the function
of resident myeloid cells in the adult steady state. As
such, the deletion of Irf8 in mice leads to a disturbed
homeostasis of resident tissue macrophages in the liver,
the kidney and brain including microglia and other
CNS-associated macrophages [19, 25, 48, 58]. The role
of Irf8 in regulating rMG gene expression and its influ-
ence on retinal development and neuroretinal function,
however, are currently unknown. Bone marrow transplantation Stainings
were imaged using the Nano Zoomer S60 digital slide
scanner
(Hamamatsu,
Herrsching
am
Ammersee,
Germany) and analysed with NDP viewer software (Ha-
mamatsu, Herrsching am Ammersee, Germany) or with
a confocal laser scanning microscope (Zeiss LSM 510 or
Leica TCS SP8 or Olympus FV1000), Zen software (Carl
Zeiss, Jena, Germany), LAS X software (Leica, Nussloch,
Germany) or Fluoview FV1000 (Olympus, Tokyo, Japan). For a detailed list of antibodies used, see supplementary
table 2. Meanwhile, bone marrow cells (BMCs) were collected
from the tibias and femurs of CAG-mRFP1 mice and re-
suspended in phosphate-buffered saline (PBS). The re-
cipient mice were intravenously injected with 3 × 106
BMCs via the tail vein. Nine weeks after bone marrow
transplantation, the efficiency of reconstitution was
assessed by flow cytometry which will be explained
below. Three-dimensional reconstruction of retinal microglia Three-dimensional reconstruction of retinal microglia
Imaging for 3D reconstruction was performed using a
Zeiss LSM 510 confocal laser scanning microscope with
a 20× objective, 3× zoom and 1024 × 1024 pixel reso-
lution. The interval thickness of the z-stacks was set to
1.0 μm. The morphology of retinal microglia in the inner
plexiform layer (IPL) and outer plexiform layer (OPL)
was determined by a three-dimensional reconstruction
using the filament mode of IMARIS software (Bitplane,
Zurich, Switzerland). Three cells per layer and mouse
were reconstructed and analysed. Bone marrow transplantation Bone marrow transplantation experiments were carried
out as previously described [22]. In brief, a total of 12 re-
cipient control and 14 Irf8 knockout mice were head-
shielded and lethally irradiated (RS2000 irradiator, Rad
Source, Kanas, USA) in two independent experiments. Page 3 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation (2021) 18:215 to embedding in Tissue-Tek O.C.T. compound (Sakura,
Aplphen
aan
den
Rijn,
The
Netherlands). Seven
micrometre-thick cryosections were cut using a cryostat
(Leica CM1950, Leica, Nussloch, Germany). Following
blocking
in
Ultra
V
block
for
10
min
at
room
temperature, the sections were incubated with primary
antibodies against ßIII tubulin (1:500, ab18207, Abcam,
Cambridge, UK), collagen type IV (1:1000, ab6586,
Abcam, Cambridge, UK), Ceh-10 homeo domain con-
taining homolog (CHX10, 1:200, ab16141, Abcam, Cam-
bridge, UK), GFP (1:500, 600-101-215, ROCKLAND,
Limerick, PA, USA), glial fibrillary acidic protein (GFAP,
1:500, 087A1005RE, Fremont, CA, USA) or Iba1 (1:500)
for 60 min with corresponding Alexa Fluor® 568-
conjugated secondary antibodies (1:500, Life technolo-
gies, Eugene, OR, USA). Nuclei were counterstained
with
4,6-diamidino-2-phenylindole
(DAPI). Stainings
were imaged using the Nano Zoomer S60 digital slide
scanner
(Hamamatsu,
Herrsching
am
Ammersee,
Germany) and analysed with NDP viewer software (Ha-
mamatsu, Herrsching am Ammersee, Germany) or with
a confocal laser scanning microscope (Zeiss LSM 510 or
Leica TCS SP8 or Olympus FV1000), Zen software (Carl
Zeiss, Jena, Germany), LAS X software (Leica, Nussloch,
Germany) or Fluoview FV1000 (Olympus, Tokyo, Japan). For a detailed list of antibodies used, see supplementary
table 2. to embedding in Tissue-Tek O.C.T. compound (Sakura,
Aplphen
aan
den
Rijn,
The
Netherlands). Seven
micrometre-thick cryosections were cut using a cryostat
(Leica CM1950, Leica, Nussloch, Germany). Following
blocking
in
Ultra
V
block
for
10
min
at
room
temperature, the sections were incubated with primary
antibodies against ßIII tubulin (1:500, ab18207, Abcam,
Cambridge, UK), collagen type IV (1:1000, ab6586,
Abcam, Cambridge, UK), Ceh-10 homeo domain con-
taining homolog (CHX10, 1:200, ab16141, Abcam, Cam-
bridge, UK), GFP (1:500, 600-101-215, ROCKLAND,
Limerick, PA, USA), glial fibrillary acidic protein (GFAP,
1:500, 087A1005RE, Fremont, CA, USA) or Iba1 (1:500)
for 60 min with corresponding Alexa Fluor® 568-
conjugated secondary antibodies (1:500, Life technolo-
gies, Eugene, OR, USA). Nuclei were counterstained
with
4,6-diamidino-2-phenylindole
(DAPI). Immunohistochemistry and imaging Immunohistochemistry and imaging
After intracardiac perfusion with PBS and 4% parafor-
maldehyde (PFA), eyes were fixated in 4% PFA for 45
min at room temperature and processed to RPE-
choroidal-scleral and retinal flat mounts. After incuba-
tion in PBST/BSA blocking buffer overnight, the flat
mounts were incubated with primary antibodies against
collagen type IV (1:500, AB769, Merck Millipore, Darm-
stadt, Germany), Iba1 (1:500, #019-19741, Wako, Neuss,
Germany) or alpha smooth muscle actin (SMA, 1:500,
ab5694, Abcam, Cambridge, UK) for two nights at 4 °C,
followed by incubation with Alexa Fluor® 568 or 647-
conjugated secondary antibodies overnight at 4 °C (1:
500, Life technologies, Eugene, OR, USA). Eyes of Irf8-
VENUS mice were fixated in 4% PFA for 1 h and incu-
bated in 10%, 20% and 30% sucrose for 24 h each prior In vivo characterisation and analysis Fundus morphology, retinal structure and physiological
function were investigated using fundus photography,
fundus fluorescein angiography (FFA), optical coherence
tomography (OCT) and electroretinography (ERG) as
previously described [32]. Fundus photography, FFA and
OCT were performed using a Micron III retinal micro-
scope (Phoenix Technology Group, Pleasanton, CA,
USA) and the StreamPix software (Norpix Inc., Mon-
treal, Canada). For FFA, 10% sodium fluorescein (Alcon,
Freiburg, Germany) was diluted to a concentration of 50
μL/mL in 0.9% sodium chloride for injection (VWR,
Leuven, Belgium) and administered intraperitoneally (2
μL/g). Ninety seconds after dye injection, the angiograms
were recorded. For quantification of CNV size, hyper-
fluorescent areas in early-phase angiograms were mea-
sured in pixels using ImageJ. Image-guided OCT was
performed using the OCT2 scan head. In OCT images,
the thickness of the inner nuclear layer (INL) (200 pixels
from the optic nerve head) was measured using ImageJ
(https://imagej.nih.gov/ij/). For ERG, mice were dark-
adapted overnight and anaesthetized by intraperitoneal
injection of ketamine hydrochloride (66.8 mg/kg) and
xylazine (12.76 mg/kg). ERG signals were amplified, re-
corded and analysed automatically using Ganzfeld Q450
(Roland-Consult, Brandenburg, Germany) with the inte-
grated software developed by Prof. Dr. rer. nat. Michael
Bach (Eye Center, University of Freiburg, Germany). RNA sequencing First-strand cDNA was generated using SMARTer Ultra
Low Input RNA Kit for Sequencing v4 (Clontech La-
boratories, Inc., Mountain View, CA, USA). Double-
standed cDNA was amplified with LD PCR and purified
with AMPure XP beads. Library preparation was con-
structed conforming to the Illumina Nextera XT Sample
Preparation Guide (Illumina, San Diego, CA, USA). In
brief, 150 pg of input cDNA was tagmented via Nextera
XT transposome. The products were purified and ampli-
fied with a limited-cycle PCR program to construct se-
quencing libraries. The libraries were quantified with the
KAPA SYBR FAST ABI Prism Library Quantification Kit
(Kapa
Biosystems,
Wobum,
MA,
USA). Equimolar
amounts
of
each
library
were
pooled
for
cluster Statistical analysis Statistical analysis was performed using GraphPad Prism
v6 (La Jolla, USA) as follows: an unpaired t test was ap-
plied if the normality was given by the Kolmogorov-
Smirnov test. Otherwise, the Mann–Whitney U test was
used. Difference with significance was defined as p < 0.05. Protein analysis
P
i Protein was extracted from the choroid and the retinae using
RIPA buffer (#R2078, Sigma Aldrich) containing protease in-
hibitor (cOmplete, Mini; edta-FREE Protease inhibitor Cock-
tail,
Roche
Diagnostics,
Manheim,
Germany)
and
phosphatase inhibitors (PhosSTOP, Roche Diagnostics, Man-
heim, Germany), respectively, for preservation. Total protein
concentration for each sample was measured with the Pier-
ceTM Bicinchoninic Acid Protein Assay Kit (Thermo Fisher
Scienticis, Inc., Rockland, IL, USA). RNA extraction RNA extraction, RNA library preparation and RNA se-
quencing were performed in collaboration with the Gen-
omics Core Facility "KFB-Center of Excellence for
Fluorescent
Bioanalytics"
(University
of
Regensburg,
Germany). RNA extraction was performed according to
manufacturer’s instructions using the RNeasy Plus Mini
Kit (QIAGEN, Hilden, Germany). After pelleting the
sample by centrifugation, the RNA stabilisation reagent
was removed and replaced by RLT Plus buffer for lysing
retinal microglia. Genomic DNA was removed select-
ively and efficiently by using gDNA Eliminator spin col-
umns for RNA purification. After adding Ethanol to the
flow-through, the sample was applied to an RNeasy
MinElute spin column to collect RNA. Finally, after
washing the column, total purified RNA was eluted in
RNase-free water. The quality and integrity of total RNA
was assessed with a Agilent 2100 Bioanalyser in combin-
ation with the RNA 6000 Pico LabChip Kit (Agilent,
Palo Alto, CA, USA). Fluorescence-activated cell sorting Fluorescence-activated cell sorting
Following transcardial perfusion with 1× PBS and enu-
cleation, eyes were dissected in ice-cold 1× PBS to iso-
late
the
retinae
of
Irf8+/+Cx3cr1GFP/+
or
Irf8-/-Cx3cr1GFP/+. For the lasered mice, the central parts
(70%) of the retinae were used for FACS while the per-
ipheral parts were omitted. After tissue homogenisation
and filtration through a 50-μm cell strainer (Sysmex,
Goerlitz, Germany), dead cell exclusion was performed
by incubation with fixable viability dye 780 (1:1000, 65-
0865-14, eBioscience, Waltham, MA, USA). Anti-CD16/
CD32 (Fc) receptor (1:200, 553142, BD Biosciences, Hei-
delberg, Germany) was used to avoid unspecific binding. Following
staining
with
anti-CD45
(1:200,
103133, Page 4 of 18 Page 4 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation BioLegend, San Diego, CA, USA), anti-CD11b (1:200,
17-0112-83, eBioscience, Waltham, MA, USA), anti-
Ly6C
(1:200,
560593,
BD
Bioscience,
Heidelberg,
Germany) and anti-Ly6G (1:200, 560601, BD Biosci-
ences, Heidelberg, Germany) for 20 min at 4 °C, retinal
microglia
characterised
as
CD45lowCD11b+Cx3cr1GF-
P/+Ly6C-Ly6G- were analysed and sorted into RNA sta-
bilisation reagent (QIAGEN, Hilden, Germany) using a
MoFlo Astrios EQ High Speed Cell Sorter (Beckman
Coulter, Munich, Germany). For RNA sequencing, an
average number of 10,000 retinal microglia per sample
was obtained from pooling 3 to 4 mice. Flow cytometric
purification of wild-type retinal and brain microglia to-
gether with bone marrow–derived monocytes was de-
scribed before [59]. Data are available under the GSE
accession number GSE160845. For a detailed list of anti-
bodies used, see supplementary table 2. generation on the cBot using the Illumina TruSeq SR
Cluster Kit v3. The sequencing run was performed on a
HiSeq1000 instrument with TruSeq SBS Kit v3 accord-
ing to the Illumina HiSeq 1000 System User Guide. Illu-
mina image analysis and base calling were recorded in
library base call format (.bcl) and further converted to
Fastq files via the CASAVA1.8.2 software. RNA sequencing data analysis q
g
y
Quality control and transcriptome profiling including
reads mapping, annotation, quantification and normal-
isation were performed by GenXPro (GenXPro, Frank-
furt, Germany). Briefly, FastQC was performed to assess
sequencing quality. After removing reads containing
adapter sequences and duplicate reads via cutadapt soft-
ware (GitHub, San Francisco, CA, USA) and FastUniq,
the filtered reads were mapped to the mouse genome
from ENSEMBL (https://www.ensembl.org/Mus_
musculus/Info/Index) using bowtie2. The transcripts
were functionally annotated with gene transfer format
file version 90, quantified using HTSeq and normalised
as transcripts per kilobase million (TPM) via DESeq2. Differential gene expression analysis with threshold
(log2 fold change greater than 1.5 or less than −1.5, p <
0.05, TPM ≥100 in at least one of the two compared
groups) was performed using DESeq2. Data was visua-
lised using RStudio (v1.2.1335) and R (v3.5.3). Volcano
plots were created using the ggplot2 package, and Gene
Ontology (GO) analysis was performed using R with the
clusterProfiler 3.10.1 package [62]. IRF8 is the most abundantly expressed member of the IRF
family in retinal microglia The interferon regulatory factor (IRF) family is critical
for
the
development,
maturation
and
function
of Page 5 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation CD45loCD11b+ rMG in the steady state (Fig. 1C). In line
with this finding, immunofluorescence analysis of retinal
flat mounts and cryosections confirmed that IRF8 is
mostly expressed in IBA1-positive microglial cells in the
steady state (Fig. 1D and Supplementary Figure 1C). Since expression of Irf family members may change in
response to stress and inflammation, we next assessed
the expression levels of all IRF family members (1–9)
and of common MG signature genes, such as Tmem119
and
P2ry12,
in
retinal
MG
in
2–3
months
old
Cx3cr1GFP/+ mice in the steady state and upon MG acti-
vation in the laser-induced CNV model. While the ex-
pression of the signature genes P2ry12 and Tmem119
decreased in retinal MG after laser injury, activation
markers such as Cd74 were increased, as reported before
[59]. However, most members of the IRF family were
expressed at the same level in the rMG in the context of myeloid cells [40]. Using flow cytometry and RNA se-
quencing, we first determined the expression levels of
different IRF family members in adult retinal microglia
(rMG), brain microglia (bMG) and bone marrow (BM)
monocytes (Fig. 1A, B). In general, genes belonging to
the IRF family were expressed at different levels in rMG,
bMG and BM monocytes (Fig. 1B). Compared with
other IRF family members, Irf5 and Irf8 exhibited the
highest expression levels in rMG, bMG and BM mono-
cytes. In the retina, Irf8 emerged as the most prominent
IRF member in rMG. Interestingly, the expression of Irf8
in rMG exceeded the expression in bMG and BM mono-
cytes indicating a distinct function of Irf8 for rMG. To
validate Irf8 expression in adult rMG, we next analysed
Irf8-VENUS reporter mice by flow cytometry and immu-
nohistochemistry. Flow cytometry analysis revealed a
strong
VENUS
expression
(98.7%
±
0.5%)
in Fig. 1 Irf8 is predominantly expressed in retinal microglia (MG) and blood monocytes. A Flow chart of retinal MG RNA sequencing. B Differential
expression of IRF family members in retinal MG (rMG), brain MG (bMG) and bone marrow (BM) monocytes. The gene expression profile was
generated from four CD45loCD11b+Ly6C-Ly6G- rMG samples, four CD45loCD11b+Ly6C-Ly6G- bMG samples and four
CD45+CD11b+SscloCD115+Ly6C+ bone marrow (BM) monocytes. morphology, distribution and transcriptional activity of
retinal MG in the steady state To assess the role of IRF8 in distribution and cell morph-
ology of rMG in the adult situation, we next examined ret-
inal flat mounts from 8-week-old Irf8 knockout (KO)
mice by immunofluorescence microscopy (Fig. 2A,B). Irf8
KO mice revealed a strikingly altered rMG distribution
and morphology compared with wild-type (WT) animals,
characterised by slightly reduced MG numbers in the
inner plexiform layer (IPL, Irf8 WT: 104 ± 7 cells/mm2,
Irf8 KO: 88 ± 5 cells/mm2, p = 0.09) and highly significant
decrease of cell numbers in the outer plexiform layer
(OPL, Irf8 WT: 116 ± 6 cells/mm2; Irf8 KO: 43 ± 1 cells/
mm2, p < 0.0001, Fig. 2B,C). Quantitative morphometric
analysis using IMARIS revealed a severely altered morph-
ology of retinal microglia in Irf8 KO mice, including sig-
nificantly shorter length of dendrites (IPL: Irf8 KO: 356.5
± 14.3 μm, Irf8 WT 1043.0 ± 59.4 μm, p = 0.009; OPL:
Irf8 KO: 297.7 ± 35.6 μm, Irf8 WT: 788.8 ± 26.1 μm, p =
0.01), and reduced number of dendrite segments (IPL: Irf8
KO: 62 ± 4, Irf8 WT: 167 ± 14, p = 0.009; OPL: Irf8 KO:
44 ± 6, Irf8 WT: 139 ± 6, p = 0.01), branch points (IPL:
Irf8 KO: 30 ± 2, Irf8 WT: 82 ± 7, , p = 0.009; OPL: Irf8
KO: 21 ± 3, Irf8 WT: 68 ± 3, p = 0.01) and terminal points
(IPL: Irf8 KO: 33 ± 2, Irf8 WT: 86 ± 7, p = 0.009; OPL:
Irf8 KO: 23 ± 3, Irf8 WT: 71 ± 3, p = 0.01, Fig. 2D,E) com-
pared with controls. Having established a profoundly al-
tered rMG distribution and phenotype in the adult
situation, we next explored rMG cell numbers at earlier
stages of postnatal development. Interestingly, reduced
microglia cell numbers were already present in the neuro-
blast layer at postnatal day 1 (P1) and later in the OPL at
P7 but only transiently in the IPL at P7 that could be com-
pensated until adulthood (Suppl. Figure 2A, B). These
findings suggest an impaired MG distribution specifically
in the deeper layers of the retina which is already present
shortly after birth and persists into adulthood. morphology, distribution and transcriptional activity of
retinal MG in the steady state Taken together these results strongly suggest that
IRF8 plays a critical role in maintaining the distribution,
morphology and homeostasis of retinal microglia and
has a substantial impact on the transcriptional activity of
rMG in the steady state. IRF8 is the most abundantly expressed member of the IRF
family in retinal microglia In ac-
cordance with the RNA-seq data, we found a strong im-
munoreactivity for TMEM119 and P2RY12 in Irf8-
competent rMG cells, which was almost absent in Irf8-
deficient rMG. Conversely, we found an increased im-
munoreactivity for the mannose receptor (CD206, Mrc1)
in Irf8-deficient rMG, consistent with and further sup-
porting the RNA-seq results (Fig. 3D). In addition, flow
cytometry analysis of homeostatic CD45+CD11b+ rMG
confirmed our RNA-seq results showing a reduced but
still detectable protein expression of CX3CR1 and CD64
in Irf8-deficient rMG compared with controls with a
trend towards lower MERTK and higher F4/80 expres-
sion as reported before in bMG [44] (Fig. 3E). laser-induced inflammation compared with controls. Only Irf2 and Irf5 were slightly downregulated in retinal
MG, whereas Irf7 was modestly upregulated. Notably,
the expression of Irf8 in retinal MG remained stable in
the laser-induced CNV model compared with controls
(data not shown). IRF8 is the most abundantly expressed member of the IRF
family in retinal microglia C Flow cytometry analysis of Irf8-VENUS expression in CD45loCD11b+ retinal MG
(98.7% ± 0.5, green solid line) in comparison with a negative control (grey dotted line). D Immunohistochemistry of the retinal flat mounts (upper
panel) and cryosections (lower panel) from Irf8-VENUS mice reveal that all IBA1+ (red) rMG express Irf8-VENUS (green) Fig. 1 Irf8 is predominantly expressed in retinal microglia (MG) and blood monocytes. A Flow chart of retinal MG RNA sequencing. B Differential
expression of IRF family members in retinal MG (rMG), brain MG (bMG) and bone marrow (BM) monocytes. The gene expression profile was
generated from four CD45loCD11b+Ly6C-Ly6G- rMG samples, four CD45loCD11b+Ly6C-Ly6G- bMG samples and four
CD45+CD11b+SscloCD115+Ly6C+ bone marrow (BM) monocytes C Flow cytometry analysis of Irf8-VENUS expression in CD45loCD11b+ retinal MG Fig. 1 Irf8 is predominantly expressed in retinal microglia (MG) and blood monocytes. A Flow chart of retinal MG RNA sequencing. B Differential
expression of IRF family members in retinal MG (rMG), brain MG (bMG) and bone marrow (BM) monocytes. The gene expression profile was
generated from four CD45loCD11b+Ly6C-Ly6G- rMG samples, four CD45loCD11b+Ly6C-Ly6G- bMG samples and four
CD45+CD11b+SscloCD115+Ly6C+ bone marrow (BM) monocytes. C Flow cytometry analysis of Irf8-VENUS expression in CD45loCD11b+ retinal MG
(98.7% ± 0.5, green solid line) in comparison with a negative control (grey dotted line). D Immunohistochemistry of the retinal flat mounts (upper
panel) and cryosections (lower panel) from Irf8-VENUS mice reveal that all IBA1+ (red) rMG express Irf8-VENUS (green) Page 6 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation (2021) 18:215 processes such as “cell migration” (GO:0016477, p.adj. <
1.3 × 10-5), “cell motility” (GO:0048870, p.adj. < 4.8 ×
10-5), “localization of cells” (GO:0051674, p.adj. < 4.8 ×
10-5), “regulation of cell proliferation” (GO:0032944,
p.adj. < 3.5 × 10-3) and “cell adhesion” (GO:0007155,
p.adj. < 7.7 × 10-4) suggesting a reduced migratory po-
tential of retinal microglia in the Irf8 KO mice compared
with controls (Fig. 3B). Among the downregulated
DEGs, we found numerous microglia signature genes es-
sential for microglia homeostasis, including the spalt like
transcription factor 1 (Sall1, log2FC = −10.69, −log10p
= 24.57), allograft inflammatory factor 1 (Aif1 or Iba1,
log2FC = −2.73, −log10p = 35.04), purinergic receptor
P2Y, G-Protein couple 12 (P2ry12, log2FC = −2.50, −
log10p = 45.06) and transmembrane 119 (Tmem119,
log2FC = −2.13, −log10p = 24.57) (Fig. 3A,C). IRF8 is not required for the development of the retinal
structure, vasculature and function Having
established
the
quantitative
and
qualitative
changes in Irf8-deficient rMG, we next investigated
whether Irf8-deficiency influences retinal structure and
electroretinal function during steady state. To this end,
we examined the retina of adult Irf8-deficient and con-
trol mice using color fundus photography (CF), fluores-
cein angiography (FA), optical coherence tomography
(OCT), electroretinography (ERG) and immunohisto-
chemical staining of retinal flat mounts for retinal ves-
sels (Suppl. Figure 3). To explore the associated transcriptional changes in
Irf8-deficient rMG, we next performed RNA sequencing
(RNA-seq) of FACS-sorted rMG from adult Irf8 KO
mice and controls. We determined 277 differentially
expressed genes (DEGs) of which 142 were significantly
upregulated, and 135 genes were downregulated in Irf8
KO microglia compared with microglia of control mice
(Fig. 3A). Gene ontology (GO) cluster analysis revealed
that most of these downregulated genes contribute to In general, Irf8 KO mice showed regular retinal struc-
ture, vasculature and function compared with controls. Qualitative assessment of retinal structure and vessels
on CF and FA images of Irf8-deficient and control mice
revealed no gross abnormalities, particularly no vascular
dye leakage as an indicator of disturbed vascular archi-
tecture or spontaneous neovascularisation (Suppl. Figure Zhang et al. Journal of Neuroinflammation (2021) 18:215
Page 7 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Page 7 of 18 Fig. 2 Irf8 deficiency alters retinal MG distribution, morphology and transcriptional profile. A Breeding scheme. B Irf8 KO mice demonstrate
reduced density of Cx3cr1-GFP+ (green) retinal MG. Quantitative analysis are shown in (C). Pictures are representative for n = 5 mice in each
group. C Quantification in microglial numbers in the inner plexiform (IPL) and outer plexiform layer (OPL). n = 5 mice were analysed per group. Data are shown as mean ± SEM. D 3D reconstruction of retinal MG by IMARIS reveals that Irf8 deficiency alters morphology of retinal MG,
resulting in shorter total length of dendrites, less dendrite segments and attenuated branching. E Quantitative analysis of retinal morphology. Four to six cells were reconstructed per mouse per IPL and OPL separately. Statistics were performed with the mean values per mouse (n = 6–7
in the Irf8 WT and n = 3–4 in the Irf8 KO group). Data are shown as mean ± SEM Fig. 2 Irf8 deficiency alters retinal MG distribution, morphology and transcriptional profile. A Breeding scheme. B Irf8 KO mice demonstrate
reduced density of Cx3cr1-GFP+ (green) retinal MG. IRF8 is not required for the development of the retinal
structure, vasculature and function The mannose receptor CD206
(encoded by Mrc1) is absent under homeostatic conditions but detectable under Irf8-deficient conditions. E Myeloid expression levels shown as transcripts per
million (TPM) and analogue surface marker expression, as determined by flow cytometry, of Irf8 WT (blue) and Irf8 KO (red) mice, expressed as mean
fluorescence intensity (MFI) (left). Representative histograms are shown (right) including fluorescence minus one controls (grey line). Six mice per group were
analysed for CX3CR1, CD64 and MERTK, three mice per group for F4/80. Data are shown as mean ± SEM controls (Suppl. Figure 3D). Specifically, no significant
difference was detected between Irf8 KO mice and con-
trols with respect to scotopic a-waves emanating from
rods, scotopic b-waves corresponding to depolarisation
of bipolar cells, and photopic b-waves arising from
cones. Immunohistochemical staining of retinal flat
mounts revealed a regular retinal vasculature in Irf8 KO
mice, including equal numbers of arteries labelled by
smooth muscle actin (SMA, Irf8 WT 6.2 ± 0.4, Irf8 KO:
5.9 ± 0.3 per animal) and major vessels stained with
Isolectin-B4 (IB4, Irf8 WT: 11.3 ± 0.5, Irf8 KO: 11.0 ±
0,5 per retinal per animal, Suppl. Figure 3E). Addition-
ally, branch points in the central superficial vascular
plexus (Irf8 WT: 17.0 ± 2.7, Irf8 KO 22.1 ± 2.4 per ani-
mal), central deep plexus (Irf8 WT: 8.7 ± 4.7, Irf8 KO:
74.0 ± 3.4 per animal), peripheral superficial plexus (Irf8
WT: 29.0 ± 3.5, Irf8 KO: 28.1 ± 2.0 per animal) and per-
ipheral deep plexus (Irf8 WT: 59.0 ± 5.1, Irf8 KO: 63.9 ±
5.7 per animal) were similar between both groups
(Suppl. Figure 3F). revealed more than 2-fold larger CNV lesions in Irf8 KO
mice (8603 ± 1309 pixels per animal) compared with con-
trols (3697 ± 425 pixels per animal, p < 0.005). Measure-
ment of collagen type IV-labelled CNV area on RPE/
choroidal flat mounts confirmed significantly enlarged
CNV lesions in Irf8 KO mice (52143 ± 7670 μm2) com-
pared with Irf8 WT (25203 ± 4156 μm2, p < 0.005, Fig. 4B). As expected, microscopic evaluation of RPE/choroidal
flat mounts revealed that activated amoeboid Cx3cr1-
GFP+ cells accumulate at CNV lesions in Irf8 KO mice as
well as in controls. IRF8 is not required for the development of the retinal
structure, vasculature and function Quantitative analysis are shown in (C). Pictures are representative for n = 5 mice in each
group. C Quantification in microglial numbers in the inner plexiform (IPL) and outer plexiform layer (OPL). n = 5 mice were analysed per group. Data are shown as mean ± SEM. D 3D reconstruction of retinal MG by IMARIS reveals that Irf8 deficiency alters morphology of retinal MG,
resulting in shorter total length of dendrites, less dendrite segments and attenuated branching. E Quantitative analysis of retinal morphology. Four to six cells were reconstructed per mouse per IPL and OPL separately. Statistics were performed with the mean values per mouse (n = 6–7
in the Irf8 WT and n = 3–4 in the Irf8 KO group). Data are shown as mean ± SEM 3A,B). In OCT images, the thickness of the inner nuclear
layer (INL) and the outer nuclear layer (ONL) contain-
ing the photoreceptors (PR) was similar in Irf8-deficient
mice (INL: 15.2 ± 0.5 μm, ONL: 69.8 ± 1.0 μm) compared with control animals (INL: 15.8 ± 0.4 μm,
ONL: 72.3 ± 1.0 μm, Suppl. Figure 3C). ERG measure-
ments demonstrated similar dark-adapted scotopic and
light-adapted photopic responses in Irf8 KO mice and Zhang et al. Journal of Neuroinflammation (2021) 18:215 Page 8 of 18 Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page.) Page 9 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 (See figure on previous page.)
Fig. 3 Irf8 deficiency leads to expression loss of homeostatic signature genes. A Volcano plot of differentially expressed genes in Irf8 KO retinal MG (n = 3)
compared with control (n = 5). Significantly up- and downregulated genes are shown in red and blue, respectively. The top significantly up- and
downregulated genes are labelled. B The top 5 downregulated GO clusters in Irf8 KO retinal MG. Significance is represented as p.adjust, the size of each data
circle indicates the number of genes involved in each enriched GO term. C Representative signature genes found to be highly expressed in competent retinal
MG are significantly downregulated in the Irf8 KO mice. D Immunohistochemistry of retinal flat mounts demonstrate a strong immunoreactivity for P2RY12 and
TMEM119 shown as colour-coded signal intensity in Irf8-competent retinal MG that is reduced or absent in Irf8 KO mice. IRF8 is not required for the development of the retinal
structure, vasculature and function The number of Cx3cr1-GFP+ cells
around CNV lesions, however, was significantly decreased
in Irf8 KO mice (34 ± 7 cells per lesion per animal) com-
pared with control animals (84.6 ± 8.8 cells per lesion per
animal, p < 0.001, Fig. 4C). In addition, the number of
rMG significantly increased in the IPL above CNV areas
in controls, whereas no such increase was observed in Irf8
KO mice, further pointing to a defect in rMG migration
(Suppl. Figure 4). Taken together, Irf8-deficient mice revealed reduced
MG cell numbers suggesting an impaired MG migratory
behaviour under physiological and pathological condi-
tions which was associated with increased CNV lesion
size in the laser-CNV model. Overall, these data show that Irf8 is not essential for
the development and maintenance of homeostatic retinal
structure, vascular network and function. This is particu-
larly surprising given the significant changes in rMG cell
numbers in the Irf8 KO mice during development and in
the adult. Thus, the impaired retinal MG cell morph-
ology and expression profile in otherwise unremarkable
retinal homeostasis in Irf8-deficient mice provide a
unique opportunity to investigate the role of retinal MG
in the development of CNV. Retinal microglia rather than infiltrating monocytes
account for the larger CNV lesions in the Irf8 KO mice Since Cx3cr1 is expressed in retinal microglia and infil-
trating monocyte-derived macrophages from the blood
[26], the observed Cx3cr1GFP/+ positive cells around
CNV lesions could belong to both cell populations. However, Irf8-deficient mice are characterized by a low
number of peripheral monocytes, which suggests that
very few monocytes from the blood infiltrated the CNV
lesion (Terry et al, 2015 PMID: 25277331). To investi-
gate the influence of peripheral monocytes in our model,
we next performed bone marrow transplantation experi-
ments with bone marrow from CAG-RFP reporter
animals to restore the peripheral monocyte pool in Irf8-
deficient mice with Irf8 potent monocytes (Fig. 5A). Following head-shielded bone marrow transplantation,
we observed a successful reconstitution of RFP+ Irf8-po-
tent
peripheral
monocytes
in
Irf8-deficient
animals Irf8 deficiency aggravates CNV formation Retinal MG change their phenotype and transcriptional
profile after tissue injury and modulate the development
of pathological CNV, which represents a hallmark of
neovascular AMD [59]. To investigate the role of IRF8 in
microglial cell activation after tissue injury and formation
of CNV, we next studied Irf8-deficient and control mice in
the laser-induced CNV model. Both Irf8 KO and Irf8 WT
mice developed typical laser-induced CNV 7 days after
laser photocoagulation visible, as hyperfluorescent lesions
with clear demarcation in FA images (Fig. 4A). Quantifica-
tion
of
hyperfluorescent
CNV
areas
in
angiograms Zhang et al. Journal of Neuroinflammation (2021) 18:215
Page 10 of 18 Page 10 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Fig. 4 Irf8-deficiency aggravates CNV formation. A Fundus fluorescein angiography at day 7 following laser treatment demonstrates enlarged
CNV lesions in Irf8 KO mice (n = 11) compared with Irf8 WT (n = 13). Data are presented as mean ± SEM. B 3D reconstruction of collagen type IV-
labelled CNV lesions using IMARIS. IHC of RPE-choroid-scleral flat mounts confirms that Irf8 deficiency (n = 11) increases CNV severity in
comparison with control mice (n = 12). Data are presented as mean ± SEM. C Less Cx3cr1GFP/+ (green) cells around CNV lesions (red) at day 7
after laser treatment were observed in the Irf8 KO mice (n = 6) compared with control mice (n = 7). Data are presented as mean ± SEM Fig. 4 Irf8-deficiency aggravates CNV formation. A Fundus fluorescein angiography at day 7 following laser treatment demonstrates enlarged
CNV lesions in Irf8 KO mice (n = 11) compared with Irf8 WT (n = 13). Data are presented as mean ± SEM. B 3D reconstruction of collagen type IV-
labelled CNV lesions using IMARIS IHC of RPE choroid scleral flat mounts confirms that Irf8 deficiency (n = 11) increases CNV severity in Fig. 4 Irf8-deficiency aggravates CNV formation. A Fundus fluorescein angiography at day 7 following laser treatment demonstrates enlarged
CNV lesions in Irf8 KO mice (n = 11) compared with Irf8 WT (n = 13). Data are presented as mean ± SEM. B 3D reconstruction of collagen type IV-
labelled CNV lesions using IMARIS. IHC of RPE-choroid-scleral flat mounts confirms that Irf8 deficiency (n = 11) increases CNV severity in
comparison with control mice (n = 12). Data are presented as mean ± SEM. Irf8 deficiency aggravates CNV formation C Less Cx3cr1GFP/+ (green) cells around CNV lesions (red) at day 7
after laser treatment were observed in the Irf8 KO mice (n = 6) compared with control mice (n = 7). Data are presented as mean ± SEM mice (Mildner et al, 2007 PMID: 18026096). Interest-
ingly, the reconstituted Irf8-deficient animals still dem-
onstrated
increased
CNV
lesion
size
that
was
approximately twice as large compared with reconsti-
tuted Irf8 WT animals (Irf8 KO 82,009 ± 16,242 μm2;
Irf8 WT 53,386 ± 4793 μm2, p = 0.06) which was associ-
ated with slightly reduced numbers of RFP-GFP+ micro-
glia at sites of CNV (Irf8 KO 74 ± 16.5; Irf8 WT 197.3 ±
12.1, p = 0.14) (Fig. 5C,D). The numbers of reconstituted
RFP+GFP- monocyte-derived macrophages, in contrast,
were around twofold increased in Irf8-deficient mice compared with controls by using flow cytometry (Irf8
KO:
95.78
±
0.59%
(RFP+Ly6Chi),
97.42
±
0.14%
(RFP+Ly6Clo), Irf8 WT: 31.19 ± 4.85% (RFP+Ly6Chi),
40.24 ± 5.5% (RFP+Ly6Clo), Fig. 5B). The observed
higher recombination efficiency of Ly6Chi and Ly6Clo
monocytes in Irf8 KO is likely due to the initially low
abundance of these cells and their respective progenitors
in Irf8 KO mice. Of note, due to the head shielding, the
recipients’ bone marrow in the skull is still active and
not substituted by the donor cells that could explain the
comparably lower recombination efficiency in Irf8 WT Zhang et al. Journal of Neuroinflammation (2021) 18:215 Page 11 of 18 (2021) 18:215 Fig. 5 Bone marrow chimera experiments: reconstituted Irf8-deficient mice contain similar monocyte numbers as controls and reveal increased
CNV lesion size. A Experimental setup. After head-shielded irradiation, bone marrow of CAG-RFP mice was transplanted intravenously to irradiated
control (Irf8+/+:Cx3cr1GFP/+) and Irf8-deficient mice (Irf8-/-:Cx3cr1GFP/+) at the age of 8 weeks, respectively. Nine weeks after transplantation, flow
cytometry was performed to check the reconstitution of blood cells. Ten weeks after transplantation, all mice underwent laser treatment to
induce CNV. Analysis was performed at day 7 after laser induction. B After bone marrow transplantation, reconstitution of the blood cells was
analyzed. Following head-shielded bone marrow transplantation, we observed a successful reconstitution of RFP+ peripheral
CD45+CD11b+CD115+SScloLy6Chi/lo monocytes in Irf8-deficient animals compared with controls by using flow cytometry. Irf8 KO (n = 6) and Irf8
WT (n = 8) mice were used per group. Data are presented as mean ± SEM. Irf8 deficiency aggravates CNV formation C,D Following bone marrow transplantation, Irf8-deficient mice exhibit
a 2-fold increase of laser-induced CNV compared with control mice, while the number of Cx3cr1GFP/+ GFP-positive and RFP-negative microglia at
sites of CNV was similar in both groups. The number of reconstituted RFP-positive and GFP-negative blood-derived monocytes were increased in
Irf8-deficient mice compared with controls. Irf8 KO (N = 12) and Irf8 WT (n = 14) mice were used per group. Data are presented as mean ± SEM Fig. 5 Bone marrow chimera experiments: reconstituted Irf8-deficient mice contain similar monocyte numbers as controls and reveal increased
CNV lesion size. A Experimental setup. After head-shielded irradiation, bone marrow of CAG-RFP mice was transplanted intravenously to irradiated
control (Irf8+/+:Cx3cr1GFP/+) and Irf8-deficient mice (Irf8-/-:Cx3cr1GFP/+) at the age of 8 weeks, respectively. Nine weeks after transplantation, flow
cytometry was performed to check the reconstitution of blood cells. Ten weeks after transplantation, all mice underwent laser treatment to
induce CNV. Analysis was performed at day 7 after laser induction. B After bone marrow transplantation, reconstitution of the blood cells was
analyzed. Following head-shielded bone marrow transplantation, we observed a successful reconstitution of RFP+ peripheral
CD45+CD11b+CD115+SScloLy6Chi/lo monocytes in Irf8-deficient animals compared with controls by using flow cytometry. Irf8 KO (n = 6) and Irf8
WT (n = 8) mice were used per group. Data are presented as mean ± SEM. C,D Following bone marrow transplantation, Irf8-deficient mice exhibit
GFP/+ Fig. 5 Bone marrow chimera experiments: reconstituted Irf8-deficient mice contain similar monocyte numbers as controls and reveal increased
CNV lesion size. A Experimental setup. After head-shielded irradiation, bone marrow of CAG-RFP mice was transplanted intravenously to irradiated
control (Irf8+/+:Cx3cr1GFP/+) and Irf8-deficient mice (Irf8-/-:Cx3cr1GFP/+) at the age of 8 weeks, respectively. Nine weeks after transplantation, flow
cytometry was performed to check the reconstitution of blood cells. Ten weeks after transplantation, all mice underwent laser treatment to
induce CNV. Analysis was performed at day 7 after laser induction. B After bone marrow transplantation, reconstitution of the blood cells was
analyzed. Following head-shielded bone marrow transplantation, we observed a successful reconstitution of RFP+ peripheral
CD45+CD11b+CD115+SScloLy6Chi/lo monocytes in Irf8-deficient animals compared with controls by using flow cytometry. Irf8 KO (n = 6) and Irf8
WT (n = 8) mice were used per group. Data are presented as mean ± SEM. Irf8 deficiency aggravates CNV formation C,D Following bone marrow transplantation, Irf8-deficient mice exhibit
a 2-fold increase of laser-induced CNV compared with control mice, while the number of Cx3cr1GFP/+ GFP-positive and RFP-negative microglia at
sites of CNV was similar in both groups. The number of reconstituted RFP-positive and GFP-negative blood-derived monocytes were increased in
Irf8-deficient mice compared with controls. Irf8 KO (N = 12) and Irf8 WT (n = 14) mice were used per group. Data are presented as mean ± SEM Fig. 5 Bone marrow chimera experiments: reconstituted Irf8-deficient mice contain similar monocyte numbers as controls and reveal increased
CNV lesion size. A Experimental setup. After head-shielded irradiation, bone marrow of CAG-RFP mice was transplanted intravenously to irradiated
control (Irf8+/+:Cx3cr1GFP/+) and Irf8-deficient mice (Irf8-/-:Cx3cr1GFP/+) at the age of 8 weeks, respectively. Nine weeks after transplantation, flow
cytometry was performed to check the reconstitution of blood cells. Ten weeks after transplantation, all mice underwent laser treatment to
induce CNV. Analysis was performed at day 7 after laser induction. B After bone marrow transplantation, reconstitution of the blood cells was
analyzed. Following head-shielded bone marrow transplantation, we observed a successful reconstitution of RFP+ peripheral
CD45+CD11b+CD115+SScloLy6Chi/lo monocytes in Irf8-deficient animals compared with controls by using flow cytometry. Irf8 KO (n = 6) and Irf8
WT (n = 8) mice were used per group. Data are presented as mean ± SEM. C,D Following bone marrow transplantation, Irf8-deficient mice exhibit
a 2-fold increase of laser-induced CNV compared with control mice, while the number of Cx3cr1GFP/+ GFP-positive and RFP-negative microglia at
sites of CNV was similar in both groups. The number of reconstituted RFP-positive and GFP-negative blood-derived monocytes were increased in
Irf8-deficient mice compared with controls. Irf8 KO (N = 12) and Irf8 WT (n = 14) mice were used per group. Data are presented as mean ± SEM Zhang et al. Journal of Neuroinflammation (2021) 18:215
Page 12 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Page 12 of 18 Fig. 6 Transcriptional profiling of Irf8 KO mice during CNV formation. A Volcano plot of differentially expressed genes in Irf8 KO retinal MG (n = 4)
compared with control (n = 5) after laser photocoagulation. Significantly up- and downregulated genes are shown in red and blue, respectively. The top significantly up- and downregulated genes are labelled. B The top 5 upregulated GO clusters in retinal MG of lasered Irf8 KO mice. Irf8 deficiency aggravates CNV formation Significance is represented as p.adjust, the size of each data circle indicates the number of genes involved in each enriched GO term. C
Differential expression of purinergic receptors in rMG from Irf8 KO compared with Irf8 WT mice. D Cnet-plot links Fibronectin (Fn1) to the GO
terms inflammatory response, angiogenesis and cell adhesion. Genes written in bold belong to the top differentially upregulated genes (see A). E
Expression of Fn1 is significantly increased on transcriptional level in Irf8-deficient microglia, shown as transcripts per million (Irf8 KO N = 4, Irf8 WT
n = 5). Data are presented as mean ± SEM. F Expression of FN1 is significantly increased on protein level in Irf8-deficient microglia, measured by
ELISA. N = 5 mice per group. Data are presented as mean ± SEM. G Expression of Fn1 can be traced back to myeloid Cx3cr1-GFP+ cells at the
boarder of the CNV lesion at day 7 following laser. H Fibronectin and Collagen IV are strongly associated in the CNV lesions in both Irf8 WT and
Irf8 KO mice Fig. 6 Transcriptional profiling of Irf8 KO mice during CNV formation. A Volcano plot of differentially expressed genes in Irf8 KO retinal MG (n = 4)
compared with control (n = 5) after laser photocoagulation. Significantly up- and downregulated genes are shown in red and blue, respectively. The top significantly up- and downregulated genes are labelled. B The top 5 upregulated GO clusters in retinal MG of lasered Irf8 KO mice. Significance is represented as p.adjust, the size of each data circle indicates the number of genes involved in each enriched GO term. C
Differential expression of purinergic receptors in rMG from Irf8 KO compared with Irf8 WT mice. D Cnet-plot links Fibronectin (Fn1) to the GO
terms inflammatory response, angiogenesis and cell adhesion. Genes written in bold belong to the top differentially upregulated genes (see A). E
Expression of Fn1 is significantly increased on transcriptional level in Irf8-deficient microglia, shown as transcripts per million (Irf8 KO N = 4, Irf8 WT
n = 5). Data are presented as mean ± SEM. F Expression of FN1 is significantly increased on protein level in Irf8-deficient microglia, measured by
ELISA. N = 5 mice per group. Data are presented as mean ± SEM. G Expression of Fn1 can be traced back to myeloid Cx3cr1-GFP+ cells at the
boarder of the CNV lesion at day 7 following laser. Discussion The interferon regulatory factor 8 (IRF8) is an essential
transcription factor for the development, maturation and
homeostasis of microglia (MG) in the brain and other
tissue macrophages [19, 21, 27]. However, the role of
IRF8 for retinal MG (rMG) during homeostasis and neo-
vascular eye disease has not been elucidated so far. In
this study, we show that Irf8 is essential for a mature
rMG gene expression profile and influences MG morph-
ology,
migration
and
the
response
to
pathological
neovascularisation. Our results show that Irf8 is strongly expressed in
rMG in the steady state compared with other IRF family
members. The expression of Irf8 in rMG was even sub-
stantially higher than in brain MG (bMG), suggesting a
distinct and tissue-specific function of Irf8 in microglia
of the retina. Morphological analysis revealed that Irf8-
deficient mice exhibited an overall reduced branching of
rMG as well as a decreased MG cell number specifically
in the outer plexiform layer, which was already observed
during postnatal development. These findings recapitu-
late findings in the brain [21, 27, 39, 44] and point to a
migratory defect that is already present during postnatal
retinal layering and maintained into adulthood. To gain
further insight into the molecular changes in Irf8-defi-
cient rMG, we isolated rMG by flow cytometry and ana-
lysed their transcriptional profile by RNA sequencing. Our analysis revealed significant transcriptional differ-
ences between Irf8-potent and Irf8-deficient microglia,
which is consistent with several in vitro studies [25, 40]. We found that Sall1 was the most downregulated MG
signature gene in the retina of Irf8-deficient mice. This
is of particular functional importance since SALL1 is a
critical
transcription
factor
for
maintaining
the p
yp
Next, we analysed the DEG that were upregulated in
Irf8-deficient MG by GO cluster analysis and found an
activation of biological processes such as “defense re-
sponse” (GO:0006952, p.adj. < 8.4 × 10-6), “biological ad-
hesion” (GO:0022610, p.adj. < 2.9 × 10-4), “cell adhesion”
(GO:0007155, p.adj. < 6.1 × 10-4), “inflammatory re-
sponse” (GO:0006954, p.adj. < 9.5 × 10-6) and “positive
regulation
of cell
substrate
adhesion” (GO:0010811,
p.adj. < 1.9 × 10-3) in Irf8-deficient MG (Fig. 6C). Irf8 deficiency aggravates CNV formation H Fibronectin and Collagen IV are strongly associated in the CNV lesions in both Irf8 WT and
Irf8 KO mice Page 13 of 18 Page 13 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 compared with controls (Irf8 KO 96.6 ± 20.1; Irf8 WT
49.25 ± 5.3, p = 0.07) (Fig. 5D). retinal MG in Irf8-deficient mice compared with control
mice (data not shown). Consistent with the increased
number of Fn1 transcripts in Irf8-deficient MG, we
found significantly increased FN1 protein levels in the
RPE/choroid of Irf8-deficient mice (3314 ± 587.1 pg/μg
protein) compared with controls (1550 ± 268.5 pg/μg
protein, p < 0.05) using ELISA on tissue lysates (Fig. 6E,
F). Immunohistochemical studies showed that FN1 ex-
pression was restricted to the area of CNV lesions and
expressed by microglia at sites of CNV (Fig. 6G). In
addition to the enlarged collagen IV-positive CNV lesion
described above, Irf8-deficient mice showed a markedly
enlarged FN1-positive CNV lesion compared with WT
mice, indicating increased fibrosis (Fig. 6H). Transcriptional profile of Irf8 KO retinal microglia during
CNV formation In order to decipher the molecular mediators of en-
hanced CNV formation in Irf8 KO mice, we next iso-
lated CNV-associated rMG from Irf8 KO and control
mice by flow cytometry and analysed the cells using
RNA-seq. In total, we found 84 genes that were differen-
tially upregulated and 78 genes that were downregulated
in Irf8-deficient microglia 7 days following laser injury. In line with the RNA-seq analysis under homeostatic
conditions, we identified similar DEG that were down-
or upregulated in Irf8-deficient microglia after tissue in-
jury, such as Sall1, P2yr12 and Mrc1 (Fig. 6A). The
downregulation of P2ry12 in laser-treated Irf8 KO mice
prompted us to explore the expression of other puriner-
gic receptors which are critical for MG cell activation
and migration. Here, we found several other genes en-
coding purinergic receptors to be strongly downregu-
lated in Irf8-deficient MG, such as Adora1, P2ry12 and
P2ry13, underlining the proposed migration defect upon
laser injury (Fig. 6B). Furthermore, we analysed the ex-
pression of key M1 (Cd86, H2-Ab1, Tlr2) and M2 signa-
ture genes (CD163, Mrc1) in isolated CNV-associated
MG in Irf8-deficient mice and control animals (Suppl. Figure 5). We found that common M1 markers such as
Cd86, H2-Ab1 and Tlr2 were significantly downregu-
lated in Irf8-deficient MG compared with Irf8-potent
MG in the laser CNV model. On the other hand, some
of the common M2 markers, such as Cd163 and Mrc1,
were significantly upregulated in Irf8-deficient retinal
MG compared with Irf8-potent MG at sites of CNV sug-
gesting a M1 to M2 polarization in Irf8-deficient MG
compared with wild-type MG in the laser CNV model. Taken together, these studies show that Irf8-deficient
microglia exhibit a significantly altered expression profile
in the laser CNV model with downregulated migratory
genes, such as purinergic receptors, and upregulated
pro-fibrotic factors, such as Fn1. Discussion Furthermore, proliferation of
myeloid cells relies on IRF8 signalling [61] which could
explain the insufficient compensatory expansion of MG
in the OPL and contribute to the niche-dependent
phenotype with reduced microglial density especially in
the OPL in adult Irf8-deficient mice. Despite the aforementioned significant changes in MG
cell density and transcriptional profile, Irf8-deficient
mice exhibited a normal retinal structure, vascular sup-
ply, and physiological function. This is particularly sur-
prising as retinal MG are in close contact with retinal
vessels, especially during development, and are known to
shape the mature retinal vasculature [9, 13, 16, 18]. This
suggests that Irf8-deficient MG are functionally suffi-
cient to accompany physiological retinal development
and that minor developmental disturbances, which we
cannot completely rule out, can be compensated over
time. The fact that Irf8-deficient microglia exhibit tran-
scriptional changes that reduce their responsiveness
while maintaining a normal retinal phenotype, provides
an ideal setting to study the role of microglia in the de-
velopment of choroidal neovascularisation, which is
known to be associated with significant microglia activa-
tion and migration [35, 52, 59]. In the laser-induced CNV model, which mimics as-
pects of neovascular AMD, we found significantly in-
creased CNV lesion size under Irf8-deficient conditions. At the same time, the overall numbers of Irf8-deficient
Cx3cr1-GFP+ cells were significantly reduced around the
lesions compared with wild type. In line with this find-
ing, MG density in the IPL above CNV lesions was sig-
nificantly decreased in Irf8 KO mice compared with wild
type. These observations may be mediated either by a
migration defect caused by impaired purinergic signal-
ling [45] or by reduced microglial proliferation of Irf8-
deficient MG, or both, as discussed above. Interestingly,
the present study shows that impaired migration and a
lower number of MG in the vicinity of CNV are associ-
ated with a more severe CNV phenotype in Irf8-deficient
mice. This finding is in contrast to previous studies
showing that depletion of retinal or circulating myeloid
cells by clodronate is associated with reduced CNV size
suggesting an anti-angiogenic effect of myeloid cells on
CNV [14, 34, 51]. On the other hand, however, our re-
sults are consistent with reports showing that increased
accumulation of myeloid cells in neovascular lesions is
associated with decreased CNV and that myeloid cells
use FasL (CD95L) to inhibit CNV formation [2]. Discussion A
closer look at the DEG of three key GO clusters essential
for CNV development, namely "angiogenesis", "inflam-
matory response" and "cell adhesion", revealed Fibronec-
tin,
a profibrotic mediator
encoded by Fn1, as a
prominent upregulated gene in Irf8-deficient MG linking
all three biological processes (Fig. 6D). In addition, we
found a marked increase in the expression of other pro-
fibrotic factors such as Fgf2 and Spp1 and a subtle in-
crease in the expression of Tgfb1 in CNV-associated Page 14 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation (2021) 18:215 homeostatic gene expression pattern of MG and thus
regulates, for example, the expression of MG signature
genes such as Aif1, P2ry12 and Tmem119 [6, 33, 55]. Ac-
cordingly, we found Tmem119 and P2ry12 as signifi-
cantly reduced in Irf8-deficient rMG recapitulating the
findings from human brain microglia which suggest a
dependence of P2RY12 expression on IRF8 signalling
[5]. Notably, because SALL1 is not exclusively expressed
by microglia but also by astrocytes and oligodendrocytes
[8, 38, 48], non-cell autonomous effects of this gene can-
not be excluded. Further, single-cell RNA-seq (scRNA-
Seq) of conditional Irf8 KO mice (Fcgr1-Cre:Irf8fl/fl) re-
vealed a strong downregulation of Sall1, Aif1, Tmem119
and
P2ry12
in
bMG
while
other
genes,
typically
expressed in macrophages, like Mrc1, encoding the man-
nose receptor CD206, were upregulated [58]. In line with
this study, we found a strong upregulation of Mrc1 on
transcriptional level and the encoded mannose receptor
CD206 on protein level in Irf8-deficient rMG which is
consistent with data from Irf8-deficient bMG [44]. In
addition, other myeloid genes like Fcgr1, also known as
CD64, were downregulated on RNA and protein level. These findings strongly support common transcriptional
changes between retinal MG and brain MG under Irf8-
deficient conditions, as shown before in a direct com-
parison of wild-type rMG and bMG by scRNA-seq [59]. Furthermore, it underscores that retinal microglial dif-
ferentiation and maturation are highly dependent on a
defined transcriptional program instructed by PU.1 [16],
SALL1 [29] and IRF8. pathways of P2RY12 [40]. This hypothesis is supported
by in vitro studies showing a diminished entry of rMG
into retinal explant cultures following an interruption of
purinergic signalling [37]. Discussion These
seemingly contradictory contributions of myeloid cells
to CNV development could be reconciled by the poten-
tially different roles of resident MG and infiltrating To identify functionally related gene signatures, we
performed a gene ontology (GO) enrichment analysis
that revealed a downregulation of the clusters “cell mi-
gration“, “cell motility”, “cell adhesion” and “regulation of
cell proliferation” in adult Irf8-deficient rMG in the
steady state. The downregulation of these GO terms was
consistent with the reduced number of rMG in the OPL
in adult Irf8 KO mice, supporting previous in vitro data
describing IRF8 as an essential transcription factor for
microglial motility and migration [28, 39]. The signifi-
cant downregulation of genes relevant for cell migration
could also explain the observed developmental pheno-
type and point to an impaired Irf8-dependent perception
of guiding cues. During development, neurons con-
stantly release guiding cues, such as purines and other
extracellular nucleotides which guide microglia to colon-
ise the developing outer retina [1, 37]. Irf8-dependent
downregulation of sensors that detect these guiding
cues, such as Adora1 or P2yr12, may be responsible for
the disruption of purinergic signalling in Irf8-deficient
rMG, which attenuates their migratory capacity. Indeed,
in vitro cultivated brain MG lacking Irf8 had a strongly
reduced phosphorylation of AKT after ATP treatment,
thereby
diminishing
the
ATP-mediated
signalling Page 15 of 18 Page 15 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation deficient retinal microglia accumulating at CNV showed
less M1 and more M2 signature gene expression com-
pared with wild-type MG at CNV, suggesting increased
M1 to M2 polarization in Irf8-deficient MG. This is of
particular clinical interest as some studies have sug-
gested that more M2-like macrophages, which are as-
sumed to be pro-angiogenic, accumulate at the site of
wet compared with dry AMD and that the pathological
shift of macrophage polarization may contribute to the
pathogenesis of CNV in neovascular AMD [7]. The ex-
pression signatures or myeloid cells at the lesion site are
likely to be more complex than the aforementioned
polarization state; however, it confirms an altered signa-
ture of a set of well-known and characterized markers. Discussion Furthermore, GO cluster analysis showed that Irf8-defi-
cient rMG exhibit increased activity of biological pro-
cesses
that
are
critical
for
inflammation
and
cell
adhesion, which points to an interaction of microglia
with extracellular matrix components driving CNV for-
mation. We hereby identified Fibronectin, encoded by
the Fn1 gene, as being significantly higher expressed in
Irf8 KO rMG and linking the GO terms "cell adhesion",
"inflammatory response" and "angiogenesis". The immu-
nohistochemical and ELISA analyses confirmed that the
expression of Fibronectin protein was also significantly
increased at sites of CNV, suggesting that Irf8-deficient
MG or other cells affected by the loss of Irf8 contribute
to increased Fibronectin abundance at sites of CNV. Of
note, collagen IV fibers were reported to depend on an
established Fibronectin matrix directly co-localizing with
Fibronectin fibers in vitro that could be functionally re-
lated to our observation of increased CNV lesion size
promoted by higher Fibronectin expression in rMG [42,
43]. Furthermore, we found a marked increase in the
expression of the pro-fibrotic factors Fgf2 and Spp1
and a subtle increase in the expression of Tgfb1 in
CNV-associated
retinal
MG
in
Irf8-deficient
mice
compared with control mice. Since FGF2 and SPP1
are important mediators of scarring and have been
identified in human choroidal neovascular membranes
[17, 52], overexpression of these factors in retinal MG
may have further contributed to increased CNV lesion
size in Irf8-deficient mice. This is of particular inter-
est as pharmaceutical inhibition of SPP1 has been
shown to modulate CNV formation and inhibition of
FGF2 was associated with reduced CNV and subret-
inal fibrosis in a laser-induced mouse CNV model [3,
41, 52]. monocyte-derived macrophages. To investigate the dis-
tinct roles of these cellular populations, this study ex-
ploits Irf8-deficient mice which are characterized by a
near absence of circulating monocytes and, at the same
time, altered but present resident retinal microglia. The
enlarged CNV lesions in Irf8-deficient mice associated
with reduced numbers of Cx3cr1-GFP+ resident micro-
glia cells may thus be interpreted as a consequence of
dysfunctional pro-fibrotic Irf8-deficient MG or as a re-
sult of insufficient numbers of CNV-suppressing micro-
glia in Irf8-deficient animals. The latter may in turn
indicate a general protective role of wild-type microglia
which would be in line with previous studies [2, 46]. Discussion The
hypothesis that deficient microglia substantially influ-
ence CNV size in Irf8-deficient mice is further supported
by the head-shielded bone marrow transplantation ex-
periments performed in this study, which showed en-
larged CNV in Irf8-deficient animals compared with
controls despite successful reconstitution of Irf8-potent
peripheral monocytes. However, we cannot exclude the
possibility that other Irf8-expressing cell types in the ret-
ina or from the blood contribute to the increased CNV
lesion size in Irf8-deficient mice. Since Irf8 expression in
the retina was mainly restricted to retinal microglial
cells, which represent the most numerous myeloid cell
population in CNV, in contrast to a low number of infil-
trating peripheral monocytes [59], we consider this pos-
sibility
rather
unlikely. Furthermore,
heterozygous
Cx3cr1GFP/+ expression could have an impact on mye-
loid cells and be associated with an inflammatory pheno-
type. However, since both Irf8 wild-type and knockout
mice in our study were consistently heterozygous for
Cx3cr1, we consider this effect negligible and used the
Cx3cr1GFP/+ line to visualise Irf8 wild-type and knockout
mice in a comparable manner, since other standard
markers such as IBA1 are dysregulated and cannot be
used [44]. To explore potential mechanisms in Irf8-deficient ret-
inal MG contributing to CNV formation, we next per-
formed RNA-sequencing on sorted retinal MG at sites
of CNV. Among others, we found that purinergic receptors,
encoded by genes such as P2ry12, P2ry13 and Adora1,
were significantly downregulated in Irf8 KO rMG after
laser injury. Since these receptors are critical for the rec-
ognition of ATP released during tissue injury and thus
control cell migration, their downregulation may be at
least partly responsible for the reduced MG cell number
at sites of CNV in Irf8-deficient mice [12, 23, 30]. This
hypothesis is in line with in vitro work by Masuda et al. showing a downregulation of genes encoding purinergic
receptors, including P2ry12 and P2rx4, in Irf8-deficient
brain microglia which was associated with a migration
defect in vitro [39]. It is interesting to note that Irf8- Availability of data and materials The online version contains supplementary material available at https://doi. org/10.1186/s12974-021-02230-y. RNA sequencing data are available under the GSE accession number
GSE160845 and GSE182504. The online version contains supplementary material available at https://doi. org/10.1186/s12974-021-02230-y. The online version contains supple
org/10.1186/s12974-021-02230-y. Author details
1 1Medical Faculty, Eye Center, University Hospital, University of Freiburg,
Freiburg im Breisgau, Germany. 2Institute of Anatomy, Wuerzburg University,
Wuerzburg, Germany. 3Cardiology and Angiology, University Heart Center,
University of Freiburg, Freiburg im Breisgau, Germany. 4Medical Center and
Faculty of Medicine, Institute for Experimental Cardiovascular Medicine,
University Heart Center Freiburg Bad Krozingen, University of Freiburg,
Freiburg, Germany. 5Medical Faculty, Institute of Neuropathology, University
Hospital, University of Freiburg, Freiburg im Breisgau, Germany. 6CIBSS-Centre
for Integrative Biological Signalling Studies, University of Freiburg, Freiburg,
Germany. 7Medical Faculty, Center for Basics in NeuroModulation
(NeuroModulBasics), University of Freiburg, Freiburg im Breisgau, Germany. 8Signalling Research Centres BIOSS and CIBSS, University of Freiburg,
Freiburg im Breisgau, Germany. 9Institute of Anatomy, Leipzig University,
Leipzig, Germany. Additional file 3. Supplemental figure 3 Irf8 deficiency does not affect
the retinal structure, function and vasculature. A-C Representative color
fundus images (A), fluorescein angiography (B) and optical coherence
tomography (OCT) images of Irf8 WT and Irf8 KO mice. C) Both Irf8 WT
(blue, n=12) and Irf8 KO (red, n=12) mice displayed a regular retinal struc-
ture, a similar thickness of the inner nuclear layer and the outer retina at
100 and 200 μm from the optic nerve head in the optical coherence
tomographs. Data are shown as mean ± SEM. ONH = Optic nerve head. D Electroretinography (ERG). No significant difference was found between
the Irf8 WT (blue, n=9) and Irf8 KO (red, n=9) mice concerning the dark-
adapted scotopic and light-adapted photopic ERG measurements at dif-
ferent flash intensities. Data are shown as mean ± SEM. E Staining against
smooth muscle actin (SMA, red) reveals a comparable number of arteries
(Irf8 WT (n=5); Irf8 KO (n=7)) and major vessels (Irf8 WT (n=11); Irf8 KO (n=
13)) between both groups. Data are shown as mean ± SEM. F No signifi-
cant differences in vessel branch formation in the superficial (upper
panel) or deep plexus (lower panel) in the central or peripheral area of
the retina could be observed, compared between Irf8 WT (n=6) and Irf8
KO (n=7). Data are presented as mean ± SEM. Additional file 3. Supplemental figure 3 Irf8 deficiency does not affect
the retinal structure, function and vasculature. A-C Representative color
fundus images (A), fluorescein angiography (B) and optical coherence
tomography (OCT) images of Irf8 WT and Irf8 KO mice. Declarations Additional file 1. Supplemental figure 1 Irf8 is expressed predominantly
in retinal MG and in some bipolar or Müller cells. No Irf8-VENUS
expression could be detected co-localised with GFAP (A), βIII-Tubulin (B)
and Collagen IV (E), indicating that IRF8 is not expressed in retinal astro-
cytes, ganglion cells or vessels. All IBA1+ cells exhibited a strong Irf8-
VENUS signal (C) suggesting that all retinal MG express IRF8. Some
CHX10+ cells could be co-localised with Irf8-VENUS expression (D) dem-
onstrating that some bipolar cells or Müller cells express VENUS. Funding KK was supported by a project grant of the Fritz-Thyssen Foundation. CL, IH,
KK and MP were supported by the SFB/TRR167. Open Access funding en-
abled and organized by Projekt DEAL. Abbeviations
Ad
1 Ad Adora1: Adenosine A1 receptor; Aif1: Allograft inflammatory factor 1;
AMD: Age-related macular degeneration; ATP: Adenosine triphosphate;
CNS: Central nervous system; CNV: Choroidal neovascularization;
CSF1R: Colony stimulating factor 1 receptor; ELISA: Enzyme-linked
immunosorbent assay; ERG: Electroretinography; FFA: Fundus fluorescein
angiography; FN: Fibronectin; GO: Gene ontology; INL: Inner nuclear layer;
IRF: Interferon regulatory factor; MG: Microglia; OCT: Optical coherence
tomography; ONL: Outer nuclear layer; P2ry12: Purinergic receptor P2Y, G-
Protein couple 12; PR: Photoreceptors; Sall1: Spalt-like transcription factor 1;
Tmem119: Transmembrane 119; WT: Wild type Authors’ contributions PZ, AS, SB, JK, FL, YL, JK, FL, MB, AT, CH and PW performed experiments and
analysed data. JW analysed RNA-seq data. HA, GS, IH, KK and MP provided
scientific input. CL and PW supervised the project and wrote the manuscript. The authors read and approved the final manuscript. Conclusions In conclusion, this study identifies IRF8 as a critical me-
diator for the morphology, distribution and expression
profile of retinal microglia, and for transformation to a
reactive phenotype. The niche-dependent phenotype Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Page 16 of 18 Zhang et al. Journal of Neuroinflammation already present during postnatal development, the al-
tered morphology and the disturbed rMG distribution
did not lead to any impairment of retinal morphology
and function in the steady state, but to enlarged lesions
in the laser CNV model. This highlights the importance
of IRF8 and retinal MG for the development of patho-
logical neovascularisation in the eye and highlights the
potential of immunomodulatory therapeutic interven-
tions of rMG recruitment in retinal disease. Additional file 5. Supplemental figure 5: Polarisation markers expressed
by CNV-associated microglia. The M1 and M2 polarisation markers Cd86,
H2-Ab1, Tlr2, Cd163 and Mrc1 (CD206) are shown as transcripts per million
in comparison between Irf8 WT and KO under CNV conditions. Additional file 6. Supplemental table 1: List of primer sequences. Additional file 7. Supplemental table 2: List of antibodies used for
immunohistochemistry and flow cytometry. Additional file 5. Supplemental figure 5: Polarisation markers expressed
by CNV-associated microglia. The M1 and M2 polarisation markers Cd86,
H2-Ab1, Tlr2, Cd163 and Mrc1 (CD206) are shown as transcripts per million
in comparison between Irf8 WT and KO under CNV conditions. Additional file 7. Supplemental table 2: List of antibodies used for
immunohistochemistry and flow cytometry. Acknowledgements
ld l k
h We would like to thank Gabriele Prinz, Michael Bach and Daniel Böhringer
for excellent technical and bioinformatical assistance. We are grateful to
Frank Rosenbauer for providing Irf8-VENUS reporter mice. Competing interests
h
h
d
l The authors declare no conflict of interest in this study. The authors declare no conflict of interest in this study. Author details
1 C) Both Irf8 WT
(blue, n=12) and Irf8 KO (red, n=12) mice displayed a regular retinal struc-
ture, a similar thickness of the inner nuclear layer and the outer retina at
100 and 200 μm from the optic nerve head in the optical coherence
tomographs. Data are shown as mean ± SEM. ONH = Optic nerve head. D Electroretinography (ERG). No significant difference was found between
the Irf8 WT (blue, n=9) and Irf8 KO (red, n=9) mice concerning the dark-
adapted scotopic and light-adapted photopic ERG measurements at dif-
ferent flash intensities. Data are shown as mean ± SEM. E Staining against
smooth muscle actin (SMA, red) reveals a comparable number of arteries
(Irf8 WT (n=5); Irf8 KO (n=7)) and major vessels (Irf8 WT (n=11); Irf8 KO (n=
13)) between both groups. Data are shown as mean ± SEM. F No signifi-
cant differences in vessel branch formation in the superficial (upper
panel) or deep plexus (lower panel) in the central or peripheral area of
the retina could be observed, compared between Irf8 WT (n=6) and Irf8
KO (n=7). Data are presented as mean ± SEM. Received: 31 March 2021 Accepted: 4 August 2021 Received: 31 March 2021 Accepted: 4 August 2021 Ethics approval and consent to participate All animal experiments were authorized by the local animal care and use
committee under the respective EU, national, federal and institutional
regulations for animal experiments. Consent for publication
All authors consented to publish this article. Additional file 2. Supplemental figure 2 Temporal and spatial
distribution of retinal microglia during development. A Representative
pictures showing the numbers of microglia per field of view in Irf8 WT
and Irf8 KO mice in comparison between the ganglion cell and inner
plexiform layer (GCL/IPL) and the developing neuroblast layer (NBL) or
outer plexiform layer (OPL), respectively, at postnatal day (P) 1, P7 and in
adult mice. B Quantification thereof. Data are presented as mean ± SEM. 1.
Akhtar-Schäfer I, Wang L, Krohne TU, Xu H, Langmann T. Modulation of
three key innate immune pathways for the most common retinal
degenerative diseases. EMBO Mol Med. 2018;10(10). https://doi.org/10.152
52/emmm.201708259. References 1. Akhtar-Schäfer I, Wang L, Krohne TU, Xu H, Langmann T. Modulation of
three key innate immune pathways for the most common retinal
degenerative diseases. EMBO Mol Med. 2018;10(10). https://doi.org/10.152
52/emmm.201708259. 1. Akhtar-Schäfer I, Wang L, Krohne TU, Xu H, Langmann T. Modulation of
three key innate immune pathways for the most common retinal
degenerative diseases. EMBO Mol Med. 2018;10(10). https://doi.org/10.152
52/emmm.201708259. Additional file 4. Supplemental figure 4 Spatial distribution of retinal
microglia in CNV lesions. A Spatial distribution of microglia in the inner
plexiform layer (IPL) in lasered and unlasered Irf8 WT and Irf8 KO mice. B
Quantification of the numbers of microglia per field of view in the inner
plexiform layer in lasered and unlasered Irf8 WT and Irf8 KO mice. Data
are presented as mean ± SEM. Additional file 4. Supplemental figure 4 Spatial distribution of retinal
microglia in CNV lesions. A Spatial distribution of microglia in the inner
plexiform layer (IPL) in lasered and unlasered Irf8 WT and Irf8 KO mice. B
Quantification of the numbers of microglia per field of view in the inner
plexiform layer in lasered and unlasered Irf8 WT and Irf8 KO mice. Data
are presented as mean ± SEM. Additional file 4. Supplemental figure 4 Spatial distribution of retinal
microglia in CNV lesions. A Spatial distribution of microglia in the inner
plexiform layer (IPL) in lasered and unlasered Irf8 WT and Irf8 KO mice. B
Quantification of the numbers of microglia per field of view in the inner
plexiform layer in lasered and unlasered Irf8 WT and Irf8 KO mice. Data
are presented as mean ± SEM. Additional file 4. Chappell-Maor L, Kolesnikov M, Kim J-S, Shemer A, Haimon Z, Grozovski J,
et al. Comparative analysis of CreER transgenic mice for the study of brain
macrophages: a case study. Eur J Immunol. 2020;50(3):353–62. https://doi. org/10.1002/eji.201948342. 28. Kobayakawa K, Ohkawa Y, Yoshizaki S, Tamaru T, Saito T, Kijima K, et al. Macrophage centripetal migration drives spontaneous healing process after
spinal cord injury. Sci Adv. 2019;5:eaav5086. 29. Koso H, Nishinakamura R, Watanabe S. Sall1 regulates microglial
morphology cell autonomously in the developing retina. Adv Exp Med Biol. 2018;1074:209–15. https://doi.org/10.1007/978-3-319-75402-4_26. 9. Checchin D, Sennlaub F, Levavasseur E, Leduc M, Chemtob S. Potential role
of microglia in retinal blood vessel formation. Invest Ophthalmol Vis Sci. 2006;47(8):3595–602. https://doi.org/10.1167/iovs.05-1522. 30. Kronlage M, Song J, Sorokin L, Isfort K, Schwerdtle T, Leipziger J, et al. Autocrine purinergic receptor signaling is essential for macrophage
chemotaxis. Sci Signal. 2010;3:ra55. 10. Colijn JM, Buitendijk GHS, Prokofyeva E, Alves D, Cachulo ML, Khawaja AP,
et al. Prevalence of age-related macular degeneration in Europe: the past
and the future. Ophthalmology. 2017;124(12):1753–63. https://doi.org/10.101
6/j.ophtha.2017.05.035. 31. Lange C, Mowat F, Sayed H, Mehad M, Duluc L, Piper S, et al. Dimethylarginine dimethylaminohydrolase-2 deficiency promotes vascular
regeneration and attenuates pathological angiogenesis. Exp Eye Res. 2016;
147:148–55. https://doi.org/10.1016/j.exer.2016.05.007. 11. Combadiere C, Feumi C, Raoul W, Keller N, Rodero M, Pezard A, et al. CX3CR1-dependent subretinal microglia cell accumulation is associated with
cardinal features of age-related macular degeneration. J Clin Invest. 2007;
117:2920–8. 32. Lange CAK, Luhmann UFO, Mowat FM, Georgiadis A, West EL, Abrahams S,
et al. Von Hippel-Lindau protein in the RPE is essential for normal ocular
growth and vascular development. Dev Camb Engl. 2012;139:2340–50. 12. Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, Jung S, et al. ATP mediates
rapid microglial response to local brain injury in vivo. Nat Neurosci. 2005;
8(6):752–8. https://doi.org/10.1038/nn1472. 33. Lavin Y, Winter D, Blecher-Gonen R, David E, Keren-Shaul H, Merad M, et al. Tissue-resident macrophage enhancer landscapes are shaped by the local
microenvironment. Cell. 2014;159(6):1312–26. https://doi.org/10.1016/j.cell.2
014.11.018. 13. Dudiki T, Meller J, Mahajan G, Liu H, Zhevlakova I, Stefl S, et al. Microglia
control vascular architecture via a TGFβ1 dependent paracrine mechanism
linked to tissue mechanics. Nat Commun. 2020;11:1–16. 34. Liu J, Copland DA, Horie S, Wu W-K, Chen M, Xu Y, et al. Additional file 4. 2. Apte RS, Richter J, Herndon J, Ferguson TA. Macrophages inhibit
neovascularization in a murine model of age-related macular degeneration. PLoS Med. 2006;3(8):e310. https://doi.org/10.1371/journal.pmed.0030310. 2. Apte RS, Richter J, Herndon J, Ferguson TA. Macrophages inhibit
neovascularization in a murine model of age-related macular degeneration. PLoS Med. 2006;3(8):e310. https://doi.org/10.1371/journal.pmed.0030310. 3. Beguier F, Housset M, Roubeix C, Augustin S, Zagar Y, Nous C, et al. The
10q26 risk haplotype of age-related macular degeneration aggravates Page 17 of 18 Page 17 of 18 Page 17 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 subretinal inflammation by impairing monocyte elimination. Immunity. 2020;53:429–441.e8. subretinal inflammation by impairing monocyte elimination. Immunity. 2020;53:429–441.e8. 23. Haynes SE, Hollopeter G, Yang G, Kurpius D, Dailey ME, Gan W-B, et al. The
P2Y12 receptor regulates microglial activation by extracellular nucleotides. Nat Neurosci. 2006;9(12):1512–9. https://doi.org/10.1038/nn1805. 4. Boeck M, Thien A, Wolf J, Hagemeyer N, Laich Y, Yusuf D, et al. Temporospatial distribution and transcriptional profile of retinal microglia in
the oxygen-induced retinopathy mouse model. Glia. 2020;68(9):1859–73. https://doi.org/10.1002/glia.23810. 24. Holtschke T, Löhler J, Kanno Y, Fehr T, Giese N, Rosenbauer F, et al. Immunodeficiency and chronic myelogenous leukemia-like syndrome in
mice with a targeted mutation of the ICSBP gene. Cell. 1996;87(2):307–17. https://doi.org/10.1016/S0092-8674(00)81348-3. 5. Böttcher C, Schlickeiser S, Sneeboer MAM, Kunkel D, Knop A, Paza E, et al. Human microglia regional heterogeneity and phenotypes determined by
multiplexed single-cell mass cytometry. Nat Neurosci. 2019;22(1):78–90. https://doi.org/10.1038/s41593-018-0290-2. 25. Horiuchi M, Wakayama K, Itoh A, Kawai K, Pleasure D, Ozato K, et al. Interferon regulatory factor 8/interferon consensus sequence binding
protein is a critical transcription factor for the physiological phenotype of
microglia. J Neuroinflammation. 2012;9:227. 6. Buttgereit A, Lelios I, Yu X, Vrohlings M, Krakoski NR, Gautier EL, et al. Sall1 is
a transcriptional regulator defining microglia identity and function. Nat
Immunol. 2016;17(12):1397–406. https://doi.org/10.1038/ni.3585. 26. Jung S, Aliberti J, Graemmel P, Sunshine MJ, Kreutzberg GW, Sher A, et al. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and
green fluorescent protein reporter gene insertion. Mol Cell Biol. 2000;20(11):
4106–14. https://doi.org/10.1128/MCB.20.11.4106-4114.2000. 7. Cao X, Shen D, Patel MM, Tuo J, Johnson TM, Olsen TW, et al. Macrophage
polarization in the maculae of age-related macular degeneration: a pilot
study. Pathol Int. 2011;61(9):528–35. https://doi.org/10.1111/j.1440-1827.2011. 02695.x. 27. Kierdorf K, Erny D, Goldmann T, Sander V, Schulz C, Perdiguero EG, et al. Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-
dependent pathways. NatNeurosci. 2013;16:273–80. 8. Additional file 4. Myeloid cells
expressing VEGF and arginase-1 following uptake of damaged retinal
pigment epithelium suggests potential mechanism that drives the onset of
choroidal angiogenesis in mice. PloS One. 2013;8(8):e72935. https://doi. org/10.1371/journal.pone.0072935. 14. Espinosa-Heidmann DG, Suner IJ, Hernandez EP, Monroy D, Csaky KG,
Cousins SW. Macrophage depletion diminishes lesion size and severity in
experimental choroidal neovascularization. Invest Ophthalmol VisSci. 2003;
44(8):3586–92. https://doi.org/10.1167/iovs.03-0038. 15. Fantin A, Lampropoulou A, Senatore V, Brash JT, Prahst C, Lange CA, et al. VEGF165-induced vascular permeability requires NRP1 for ABL-mediated
SRC family kinase activation. J Exp Med. 2017;214(4):1049–64. https://doi. org/10.1084/jem.20160311. 35. Liyanage SE, Fantin A, Villacampa P, Lange CA, Denti L, Cristante E, et al. Myeloid-derived vascular endothelial growth factor and hypoxia-inducible
factor are dispensable for ocular neovascularization-brief report. Arterioscler
Thromb Vasc Biol. 2016;36(1):19–24. https://doi.org/10.1161/ATVBAHA.115.3
06681. 16. Fantin A, Vieira JM, Gestri G, Denti L, Schwarz Q, Prykhozhij S, et al. Tissue
macrophages act as cellular chaperones for vascular anastomosis
downstream of VEGF-mediated endothelial tip cell induction. Blood. 2010;
116(5):829–40. https://doi.org/10.1182/blood-2009-12-257832. 36. Lückoff A, Caramoy A, Scholz R, Prinz M, Kalinke U, Langmann T. Interferon-
beta signaling in retinal mononuclear phagocytes attenuates pathological
neovascularization. EMBO Mol Med. 2016;8(6):670–8. https://doi.org/10.152
52/emmm.201505994. 17. Frank RN, Amin RH, Eliott D, Puklin JE, Abrams GW. Basic fibroblast growth
factor and vascular endothelial growth factor are present in epiretinal and
choroidal neovascular membranes. Am J Ophthalmol. 1996;122(3):393–403. https://doi.org/10.1016/S0002-9394(14)72066-5. 37. Martín-Estebané M, Navascués J, Sierra-Martín A, Martín-Guerrero SM,
Cuadros MA, Carrasco M-C, et al. Onset of microglial entry into developing
quail retina coincides with increased expression of active caspase-3 and is
mediated by extracellular ATP and UDP. PloS One. 2017;12(8):e0182450. https://doi.org/10.1371/journal.pone.0182450. 18. Fruttiger M. Development of the retinal vasculature. Angiogenesis. 2007;
10(2):77–88. https://doi.org/10.1007/s10456-007-9065-1. 19. Goldmann T, Wieghofer P, Jordão MJC, Prutek F, Hagemeyer N, Frenzel K,
et al. Origin, fate and dynamics of macrophages at central nervous system
interfaces. Nat Immunol. 2016;17(7):797–805. https://doi.org/10.1038/ni.3423. 38. Masuda T, Amann L, Sankowski R, Staszewski O, Lenz M, Errico PD, et al. Novel Hexb-based tools for studying microglia in the CNS. Nat Immunol. 2020;21(7):802–15. https://doi.org/10.1038/s41590-020-0707-4. 20. Gupta N, Brown KE, Milam AH. Activated microglia in human retinitis pigmentosa,
late-onset retinal degeneration, and age-related macular degeneration. Exp Eye Res. 2003;76(4):463–71. https://doi.org/10.1016/S0014-4835(02)00332-9. 39. Masuda T, Iwamoto S, Mikuriya S, Tozaki-Saitoh H, Tamura T, Tsuda M, et al. Transcription factor IRF1 is responsible for IRF8-mediated IL-1β expression in reactive
microglia. J Pharmacol Sci. 2015;128(4):216–20. https://doi.org/10.1016/j.jphs.2015.08.002. 21. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. g
j
p
45. Nimmerjahn A, Kirchhoff F, Helmchen F. Resting microglial cells are highly
dynamic surveillants of brain parenchyma in vivo. Science. 2005;308(5726):
1314–8. https://doi.org/10.1126/science.1110647. 46. O’Koren EG, Mathew R, Saban DR. Fate mapping reveals that microglia and
recruited monocyte-derived macrophages are definitively distinguishable by
phenotype in the retina. Sci Rep. 2016;6(1):20636. https://doi.org/10.1038/
srep20636. 47. Prinz M, Jung S, Priller J. Microglia biology: one century of evolving
concepts. Cell. 2019;179(2):292–311. https://doi.org/10.1016/j.cell.2019.08.053. 48. Prinz M, Masuda T, Wheeler MA, Quintana FJ. Microglia and central nervous
system-associated macrophages-from origin to disease modulation. Annu
Rev Immunol. 2021;39(1):251–77. https://doi.org/10.1146/annurev-immunol-
093019-110159. 49. Rashid K, Akhtar-Schaefer I, Langmann T. Microglia in retinal degeneration. Front Immunol. 2019;10:1975. https://doi.org/10.3389/fimmu.2019.01975. 50. Robbie SJ, Lundh von Leithner P, Ju M, Lange CA, King AG, Adamson P,
et al. Assessing a novel depot delivery strategy for noninvasive
administration of VEGF/PDGF RTK inhibitors for ocular neovascular disease. Invest Ophthalmol Vis Sci. 2013;54(2):1490–500. https://doi.org/10.1167/
iovs.12-10169. 51. Sakurai E, Anand A, Ambati BK, Van RN, Ambati J. Macrophage depletion
inhibits experimental choroidal neovascularization. Invest Ophthalmol VisSci. 2003;44(8):3578–85. https://doi.org/10.1167/iovs.03-0097. 52. Schlecht A, Zhang P, Wolf J, Thien A, Rosmus D-D, Boneva S, et al. Secreted
phosphoprotein 1 expression in retinal mononuclear phagocytes links
murine to human choroidal neovascularization. Front Cell Dev Biol. 2021;8:
618598. 53. Schönheit J, Kuhl C, Gebhardt ML, Klett FF, Riemke P, Scheller M, et al. PU.1
level-directed chromatin structure remodeling at the Irf8 gene drives
dendritic cell commitment. Cell Rep. 2013;3(5):1617–28. https://doi.org/10.1
016/j.celrep.2013.04.007. 54. Schwarzer P, Kokona D, Ebneter A, Zinkernagel MS. Effect of inhibition of
colony-stimulating factor 1 receptor on choroidal neovascularization in
mice. Am J Pathol. 2020;190(2):412–25. https://doi.org/10.1016/j.ajpath.201
9.10.011. 55. Shemer A, Grozovski J, Tay TL, Tao J, Volaski A, Süß P, et al. Engrafted
parenchymal brain macrophages differ from microglia in transcriptome,
chromatin landscape and response to challenge. Nat Commun. 2018;9:5206. 56. Silverman SM, Wong WT. Microglia in the retina: roles in development,
maturity, and disease. Annu Rev Vis Sci. 2018;4(1):45–77. https://doi.org/1
0.1146/annurev-vision-091517-034425. 57. Tamura T, Kurotaki D, Koizumi S-I. Regulation of myelopoiesis by the
transcription factor IRF8. Int J Hematol. 2015;101(4):342–51. https://doi.org/1
0.1007/s12185-015-1761-9. 57. Tamura T, Kurotaki D, Koizumi S-I. Regulation of myelopoiesis by the
transcription factor IRF8. Int J Hematol. 2015;101(4):342–51. https://doi.org/1
0.1007/s12185-015-1761-9. 58. Additional file 4. Hagemeyer N, Kierdorf K, Frenzel K, Xue J, Ringelhan M, Abdullah Z, et al. Transcriptome-based profiling of yolk sac-derived macrophages reveals a
role for Irf8 in macrophage maturation. EMBO J. 2016;35(16):1730–44. https://doi.org/10.15252/embj.201693801. 40. Masuda T, Nishimoto N, Tomiyama D, Matsuda T, Tozaki-Saitoh H, Tamura T,
et al. IRF8 is a transcriptional determinant for microglial motility. Purinergic
Signal. 2014;10(3):515–21. https://doi.org/10.1007/s11302-014-9413-8. 41. Matsuda Y, Nonaka Y, Futakawa S, Imai H, Akita K, Nishihata T, et al. Anti-
angiogenic and anti-scarring dual action of an anti-fibroblast growth factor
2 aptamer in animal models of retinal disease. Mol Ther Nucleic Acids. 2019;
17:819–28. https://doi.org/10.1016/j.omtn.2019.07.018. 22. Härdtner C, Kornemann J, Krebs K, Ehlert CA, Jander A, Zou J, et al. Inhibition of macrophage proliferation dominates plaque regression in
response to cholesterol lowering. Basic Res Cardiol. 2020;115(6):78. https://
doi.org/10.1007/s00395-020-00838-4. Page 18 of 18 Page 18 of 18 Page 18 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 (2021) 18:215 Zhang et al. Journal of Neuroinflammation (2021) 18:215 61. Yoshida Y, Yoshimi R, Yoshii H, Kim D, Dey A, Xiong H, et al. The
transcription factor IRF8 activates integrin-mediated TGF-β signaling and
promotes neuroinflammation. Immunity. 2014;40(2):187–98. https://doi.org/1
0.1016/j.immuni.2013.11.022. 42. Miller CG, Budoff G, Prenner JL, Schwarzbauer JE. Minireview: fibronectin in
retinal disease. Exp Biol Med Maywood NJ. 2017;242(1):1–7. https://doi.org/1
0.1177/1535370216675245. 43. Miller CG, Pozzi A, Zent R, Schwarzbauer JE. Effects of high glucose on
integrin activity and fibronectin matrix assembly by mesangial cells. Mol Biol
Cell. 2014;25(16):2342–50. https://doi.org/10.1091/mbc.e14-03-0800. 62. Yu G, Wang L-G, Han Y, He Q-Y. clusterProfiler: an R package for comparing
biological themes among gene clusters. OMICS J Integr Biol. 2012;16(5):284–
7. https://doi.org/10.1089/omi.2011.0118. 62. Yu G, Wang L-G, Han Y, He Q-Y. clusterProfiler: an R package for comparing
biological themes among gene clusters. OMICS J Integr Biol. 2012;16(5):284–
7. https://doi.org/10.1089/omi.2011.0118. 44. Minten C, Terry R, Deffrasnes C, King NJC, Campbell IL. IFN regulatory factor
8 is a key constitutive determinant of the morphological and molecular
properties of microglia in the CNS. PloS One. 2012;7(11):e49851. https://doi. org/10.1371/journal.pone.0049851. Publisher’s Note Van Hove H, Martens L, Scheyltjens I, De Vlaminck K, Pombo Antunes AR,
De Prijck S, et al. A single-cell atlas of mouse brain macrophages reveals
unique transcriptional identities shaped by ontogeny and tissue
environment. Nat Neurosci. 2019;22(6):1021–35. https://doi.org/10.1038/s41
593-019-0393-4. 59. Wieghofer P, Hagemeyer N, Sankowski R, Schlecht A, Staszewski O, Amann
L, et al. Mapping the origin and fate of myeloid cells in distinct
compartments of the eye by single-cell profiling. EMBO J. 2021;40:e105123. 60. Wong WL, Su X, Li X, Cheung CMG, Klein R, Cheng C-Y, et al. Global prevalence
of age-related macular degeneration and disease burden projection for 2020
and 2040: a systematic review and meta-analysis. Lancet Glob Health. 2014;
2(2):e106–16. https://doi.org/10.1016/S2214-109X(13)70145-1.
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English
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Minimax and admissible adaptive two-stage designs in phase II clinical trials
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BMC Medical research methodology
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© 2016 The Author(s). 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. Shan et al. BMC Medical Research Methodology (2016) 16:90
DOI 10.1186/s12874-016-0194-3 Shan et al. BMC Medical Research Methodology (2016) 16:90
DOI 10.1186/s12874-016-0194-3 Open Access Abstract Background: Simon’s two-stage design is the most widely implemented among multi-stage designs in phase II
clinical trials to assess the activity of a new treatment in a single-arm study. In this two-stage design, the sample size
from the second stage is fixed regardless of the number of responses observed in the first stage. Methods: We develop a new minimax adaptive design for phase II clinical trials, by using the branch-and-bound
intelligent algorithm based on conditional error functions. Results: We compare the performance of the proposed design and competitors, including Simon’s minimax design,
and a modified Simon’s design that allows early stopping for futility or efficacy. The maximum sample size of the
proposed minimax adaptive design is guaranteed to be less than or equal to those from other existing designs. When
the proposed design has the same maximum sample size as others, it always has the smallest expected sample size. In
addition to the minimax adaptive design, we also introduce admissible adaptive designs determined from a Bayesian
perspective. Conclusions: The proposed adaptive minimax design can save sample sizes for a clinical trial. The minimum required
sample size is critical to reduce the cost of a project. Keywords: Adaptive design, Admissible design, Efficacy, Futility, Minimax design, Simon’s design : Adaptive design, Admissible design, Efficacy, Futility, Minimax design, Simon’s design when the treatment is indeed ineffective. For this reason, a
multi-stage design is often implemented, and among these
designs the most popular is Simon’s two-stage design [2]. Simon [2] proposed two optimal designs: the optimal
design with the expected sample size under the null (ESS0)
minimized, and the minimax design having the smallest
ESS0 among the designs with the maximum sample size
(MSS) minimized. Simon’s design allows early stopping
in the first stage for futility only. Recently, Mander and
Thompson [3] extended Simon’s design to allow stopping
for efficacy or futility by introducing an additional design
parameter that represents the stopping criteria for effi-
cacy in the first stage. It is guaranteed that the MSS of the
modified design is less than or equal to that of Simon’s. Minimax and admissible adaptive
two-stage designs in phase II clinical trials Guogen Shan1, Hua Zhang2 and Tao Jiang3* Methods Simon [2] proposed the widely used two-stage designs for
early phase II clinical trials with binary endpoints by test-
ing the response rate to make a conclusion of go or no-go
to the next trial phase of the study. In this study design, the
unacceptable response rate πu can be estimated from his-
torical data, and the acceptable response rate πa is often
the targeted response rate of a new treatment or ther-
apy, where πu < πa. For example, in the clinical trial for
urothelial cancer with neoadjuvant therapy [10], the unac-
ceptable and acceptable response rates are πu = 35 % and
πa = 50 %, respectively. The hypotheses to be tested are In phase II clinical trials, it is desirable to achieve the
primary goal of the study with the number of patients
minimized, as the cost of the study highly depends on
the number of patients. In addition, Institutional Review
Boards approve proposed studies based on the maxi-
mum possible number of patients that are needed to
address the scientific questions. Therefore, the minimax
design is preferable by researchers with the smallest MSS
as compared to the optimal design when the MSS dif-
ference between the two designs is not small. To our
best knowledge, no adaptive design based on the mini-
max criteria has been proposed for use in practice. Due
to the importance of such designs, we develop a new
minimax adaptive design with the monotonic property
respected in this article by using the branch-and-bound
algorithm [7] based on conditional type I and II error
rates. H0 : π ≤πu, against against against Ha : π ≥πa. Ha : π ≥πa. The null hypothesis is rejected for a large response rate. Let n1, n2, and n be the number of subjects enrolled in the
first stage, the second stage, and both stages combined,
respectively, and x1, x2, and x are the associated number
of responses observed from the study. In the clinical trial of the neoadjuvant therapy for
urothelial cancer [10], Simon’s minimax design was used
for sample size determination to achieve 80 % power
(β = 0.2) at the significance level of α = 0.1 when the
response rates were πu = 35 % and πa = 50 %. The
design was calculated as: (r1/n1, r/n) = (10/31, 21/49)
with the ESS0=40.8. Background In phase II clinical trials, a new treatment or a new therapy
is often assessed by measuring activity with dichotomized
endpoints, responding ’yes’ or ’no’ to the intervention. For Oncology clinical trials, the response criteria may be
determined by the Response Evaluation Criteria In Solid
Tumours (RECIST) [1]. The traditional experiment in
phase II Oncology trials is often conducted in a single arm
study, which is also popular in other studies, such as AIDS. All patients enrolled in the study are treated with the same
treatment, and their measurements are obtained at the
end of the study and compared to the priori estimate from
historical studies with the similar condition of experiment
and patients. From ethical and economical considerations,
a trial should be allowed to stop earlier after an interim
analysis to better protect patients, especially in situations In Simon’s design and the modified Simon’s design due
to Mander and Thompson [3], the second stage sample
size is always fixed and is not allowed to be modified as the
result observed from the first stage. To make a design flex-
ible and efficient, adaptive designs have been developed
to allow the second stage sample size to depend on first *Correspondence: jtao@263.net
3Department of Statistics, Zhejiang Gongshang University, Hangzhou, 310018
Zhejiang, China
Full list of author information is available at the end of the article Shan et al. BMC Medical Research Methodology (2016) 16:90 Shan et al. BMC Medical Research Methodology (2016) 16:90 Page 2 of 14 stage responders. It is easy to show that Simon’s opti-
mal design is a special case of adaptive designs, therefore,
the expected sample size of an optimal adaptive design is
always less than or equal to that of Simon’s design. Several
optimal adaptive designs have been developed for phase II
clinical trials, and the majority of them are based on the
optimal criteria with the smallest ESS0. Banerjee and Tsi-
atis [4] developed an optimal adaptive two-stage design
by using a Bayesian decision-theoretic construct to mini-
mize the expected loss through backward induction, with
type I and II error rates respected. The sample size savings
are small to modest when compared to Simon’s optimal
design. Later, Englert and Kieser [5] developed an opti-
mal adaptive two-stage design based on conditional error
functions [6] and an efficient search strategy [7]. Background Although
these adaptive designs guarantee the type I and II error
rates, these designs suffer from a counter-intuitive fea-
ture that the second stage sample size may increase as
the number of responses observed from the first stage
increases. Very recently, Shan et al. [8] developed an
optimal adaptive two-stage design with the monotonic-
ity property respected; the second stage sample size is
a non-increasing function of the first stage responders. This improvement makes it practical to use the optimal
adaptive design. The remainder of this article is organized as follows. In “Methods” Section, we introduce the detailed search
method for the optimal adaptive design when the first
stage and the MSS of the second stage sample size are
fixed, then present the approach to find the minimax
adaptive design. In “Results” Section we compare the MSS
and the ESS0 of the proposed minimax adaptive design
with competitors. A real clinical trial from a cancer study
is used to illustrate the proposed design at the end of
“Results” Section. Finally, we provide some remarks in
“Discussion and conclusions” Section. Methods A deci-
sion can not be made in the first stage when 11 < x1 ≤16,
and additionally n2 = n −n1 = 49 −32 = 17 patients will
be enrolled in the second stage. At the end of the study,
the null hypothesis will be rejected if x > 21. Otherwise,
it is concluded that the new treatment is not promising
enough to warrant further investigation. The ESS0 of the
minimax-EF design is 39.2 as compared to the ESS0 =
40.8 from Simon’s minimax design. In the two aforementioned designs, the second stage
sample size is not allowed to change with the number
of responses observed from the first stage. To improve
the efficiency and flexibility of a study, we propose a new
adaptive two-stage design based on the minimax crite-
ria from Simon’s, the design with the smallest ESS0 and
Table 1 The proposed adaptive minimax design for the
urothelial cancer trial with the neoadjuvant therapy with
(α, β, πu, πa) = (0.1, 0.2, 0.35, 0.5)
S
n2(S)
n(S)
r(S)
Minimax-EF design
≤11
0
32
0
12
17
49
21
13
17
49
21
14
17
49
21
15
17
49
21
16
17
49
21
≥17
0
32
0
Minimax adaptive design
≤9
0
28
0
10
21
49
21
11
21
49
21
12
21
49
21
13
21
49
21
14
19
47
20
15
18
46
20
≥16
0
28
0 MSS. In the proposed adaptive design, the second stage
sample size, n2(S), depends on the number of first stage
responses, S, and n2(S) is a non-increasing function of S,
specifically, n2(S1) ≥n2(S2) when S1 < S2. For a given
first stage sample size n1, the value of S ranges from 0
to n1: S = 0, 1, . . . , n1. Moreover, the associated critical
value for each S, r(S), also needs to be determined for the
adaptive design. Then, the proposed design is presented as design is less than that of the minimax design (35.2 agaisnt
40.8), but the MSS is much larger for the optimal design
as compared to the minimax design (58 against 49). p
g
(
g
)
A modified Simon’s design that allows early stopping
for futility or efficacy, was proposed by Mander and
Thompson [3] who introduced another design param-
eter, r2, as ((r1, r2)/n1, r/n). This design is referred to
as the Minimax-EF design. Methods For the aforementioned can-
cer study, the design can be calculated by using the
Stata package, simon2stage [3], as ((r1, r2)/n1, r/n)
=
((11, 16)/32, 21/49), see Table 1. With n1 = 32 patients
enrolled in the first stage of the study, the study will be
stopped for futility if x1 ≤11 or efficacy if x1 > 16. A deci-
sion can not be made in the first stage when 11 < x1 ≤16,
and additionally n2 = n −n1 = 49 −32 = 17 patients will
be enrolled in the second stage. At the end of the study,
the null hypothesis will be rejected if x > 21. Otherwise,
it is concluded that the new treatment is not promising
enough to warrant further investigation. The ESS0 of the
minimax-EF design is 39.2 as compared to the ESS0 =
40.8 from Simon’s minimax design. n1 and (n2(S), r(S)) , S = 0, 1, 2, · · · , n1, with a total of 2n1 + 3 unknown parameters. As pointed
out by many researchers [5, 8], it becomes quickly impos-
sible to estimate these parameters by enumerating all
attainable values of n2(S) and r(S), even after controlling
for the upper bound of the second stage sample size. Conditional error functions are frequently used in adap-
tive designs to fully use the information from the previous
stage, specifically, P(s|r(s), n2(s), π) = 1 −B (r(s) −s : n2(s), π) , where s is the observed first stage response under the
design parameters n2(s) and r(s), and B(x : y, z) is the
cumulative probability function of a binomial distribution
for observed value x with size y and probability z. Note
that P(s|π) = 0 or 1 when the study is terminated after
the first stage for futility or efficacy, respectively. As a spe-
cial case, it is always reasonable to stop the trial when no
response is observed from the first stage, P(0|π) = 0. It should be noted that the proposed design allows early
stopping in the first stage for either futility or efficacy just
as existing adaptive designs. In the two aforementioned designs, the second stage
sample size is not allowed to change with the number
of responses observed from the first stage. Methods The trial was allowed to stop for futil-
ity at the first stage if the number of first stage responses
x1 ≤10 was observed from a total of n1 = 31 patients. Otherwise, an additional n2 = n −n1 = 49 −31 = 18
patients were enrolled in the second stage, and at least
22 responses should be observed from total 49 patients,
x ≥22, in order to claim that the neoadjuvant therapy had
sufficient activity. The MSS of the minimax design was
49. An alterntive to the minimax design is Simon’s opti-
mal design whose ESS0 was the smallest among all designs
that met the design criteria. The design parameters for
the optimal design are: (r1/n1, r/n) = (7/20, 24/58) with
the ESS0 = 35.2. As expected, the ESS0 of the optimal Both minimax and optimal designs have been widely
used in clinical trials. It is often the case that the expected
sample size of the minimax design is much larger than that
of the optimal design, although the minimax design has
a smaller maximum sample size. To compromise between
the maximum sample size and the expected sample size
under the null, an admissible adaptive design was pro-
posed by Jung et al. [9], which was implemented in Java
language by them. By using the Bayes risk function as in
Jung et al. [9], we propose a new admissible adaptive two-
stage design that is between the minimax adaptive design
and the optimal adaptive design. Shan et al. BMC Medical Research Methodology (2016) 16:90 Page 3 of 14 Page 3 of 14 Shan et al. BMC Medical Research Methodology (2016) 16:90
design is less than that of the minimax design (35.2 agaisnt
40.8), but the MSS is much larger for the optimal design
as compared to the minimax design (58 against 49). A modified Simon’s design that allows early stopping
for futility or efficacy, was proposed by Mander and
Thompson [3] who introduced another design param-
eter, r2, as ((r1, r2)/n1, r/n). This design is referred to
as the Minimax-EF design. For the aforementioned can-
cer study, the design can be calculated by using the
Stata package, simon2stage [3], as ((r1, r2)/n1, r/n)
=
((11, 16)/32, 21/49), see Table 1. With n1 = 32 patients
enrolled in the first stage of the study, the study will be
stopped for futility if x1 ≤11 or efficacy if x1 > 16. Methods (1) is used in
two folders. The function is first used to find all satisfied
designs with the smallest MSS, minmax(n1+n2(s),s=0,1,...,n1). The second is to identify the minimax adaptive design as
the one from these in the previous step with the smallest
ESS0. function. Two procedures are recursively utilized in the
algorithm to identify the optimal design. The first pro-
cedure is the branching process that splits the problem
into several complement problems. The conditional type
I error functions are used in this step to split problems. Although it is not a requirement to sort the elements in
in the design search, it helps to reduce the computational
intensity to sort them by n2(S) in an ascending order, and
P(S|πu) in an increasing order. The ordering of n2(S) is
used to meet the monotonicity feature of the proposed
design. We start the design search with a fixed first stage sam-
ple size n1 and the MSS n. Then, the MSS of the second
stage is n2,max = n −n1. It is easy to show that n2(S)
≤n2,max and r(S) ≤n2(S). The optimal design needs
to be searched over a triangle space for each S, φ(S) =
{(n2(S), r(S)) : r(S) ≤n2(S) ≤n2,max}, where S =
0, 1, 2, · · · , n1. The complete search space is a product of
these triangle spaces, The second procedure, the bounding procedure, com-
putes boundary values of constraint functions. Let
O(S, WS) be the WS-th conditional error function in the
when the number of responses is S in the first stage. Sup-
pose the current branching outcome from the branching
procedure is at S = k (φ(0) × φ(1) × · · · × φ(n1)) . As the first stage sample size n1 increases, the size of this
complete search space increases exponentially. Therefore,
it is not feasible to conduct this naive search to identify
the optimal design. O(0, 1),O(1, W1),O(2,W2), . . . ,O(k,Wk), O(k+1,1), . . . ,O(n1,1). (2) (2) When no response is observed from the first stage, the
trial is assumed to be stopped for futility, that is repre-
sented by O(0, 1). Note that we use n2(S, WS) and r(S, WS)
to replace n2(S) and r(S) in the design search. Methods The objec-
tive function at the current branching step is calculated
as It is much more complicated to search for an opti-
mal solution over a two-dimensional space than a one-
dimensional space. For this reason, Englert and Kieser [5]
suggested using the union of all type I conditional error
functions and (0, 1), referred to as , as the parameter
space. For each element in , it contains the informa-
tion of n2(S) and r(S) as in the two-dimensional space. That said, it is equivalent to determine the conditional
type I error value for S and (r(S), n2(S)). It is still not fea-
sible to conduct a grid search over the parameter space
(a(n1 + 1) −dimensional space) due to the fact that the
size of the parameter space increases very quickly as n1
and n2,max go up. f =
k
s=0
(n1 + n2(s, ws))×b(s : n1, πu)+
n1
s=k+1
n1b(s : n1, πu). The overall goal is to find the values of W for each S in
O(S, W) that minimizes the objective function as min
O(S,WS),S=0,1,...,n1
f . min
O(S,WS),S=0,1,...,n1
f . In order to overcome the computational burden, the
branch-and-bound algorithm [7], an intelligent algorithm,
is considered when searching for the optimal design over
a one-dimensional space on each S. This algorithm can
be used to search for the optimal design with or with-
out constraints [5, 8]. The monotonicity restriction in the
optimal adaptive design search by Shan et al. [8] is an
important feature that makes a design usable in practice. The second stage sample size is a non-increasing func-
tion of the number of responses observed from the first
stage: n2(S1) ≥n2(S2) when S1 < S2. This monotonicity
restriction is respected in the proposed minimax adaptive
design. Two constraints need to be satisfied in the design search Two constraints need to be satisfied in the design search αmin =
k
s=0
P (s|r(s, ws), n2(s, ws), πu) × b(s : n1, πu), and βmin = 1 −
k
s=0
P (s|r(s, ws), n2(s, ws), πa) × b(s : n1, πa)
−
n1
s=k+1
b(s : n1, πa). These two constraints help to determine the set of feasi-
ble solutions, and discard the candidates that do not lead
to the optimal design. Methods To improve
the efficiency and flexibility of a study, we propose a new
adaptive two-stage design based on the minimax crite-
ria from Simon’s, the design with the smallest ESS0 and Table 1 The proposed adaptive minimax design for the
urothelial cancer trial with the neoadjuvant therapy with
(α, β, πu, πa) = (0.1, 0.2, 0.35, 0.5)
S
n2(S)
n(S)
r(S)
Minimax-EF design
≤11
0
32
0
12
17
49
21
13
17
49
21
14
17
49
21
15
17
49
21
16
17
49
21
≥17
0
32
0
Minimax adaptive design
≤9
0
28
0
10
21
49
21
11
21
49
21
12
21
49
21
13
21
49
21
14
19
47
20
15
18
46
20
≥16
0
28
0 Table 1 The proposed adaptive minimax design for the
urothelial cancer trial with the neoadjuvant therapy with For each design, the type I and II error rates are then
calculated from conditional error functions as α =
n1
s=0
P(s|r(s), n2(s), πu) × b(s : n1, πu),
and
β = 1 −
n1
s=0
P(s|r(s), n2(s), πa) × b(s : n1, πa), β = 1 −
n1
s=0
P(s|r(s), n2(s), πa) × b(s : n1, πa), where b(.) is the density function of a binomial distribu-
tion. All designs with guaranteed type I and II error rates,
are candidates for the optimal design. Often, multiple
designs meet the design criteria, and an additional criteria
should be applied in order to find the optimal design. The
criteria used in the proposed adaptive minimax design is
the smallest ESS0 and MSS, where b(.) is the density function of a binomial distribu-
tion. All designs with guaranteed type I and II error rates,
are candidates for the optimal design. Often, multiple
designs meet the design criteria, and an additional criteria
should be applied in order to find the optimal design. The
criteria used in the proposed adaptive minimax design is
the smallest ESS0 and MSS, min
max(n1+n2(s),s=0,1,...,n1) ESS0,
(1) (1) where ESS0 = n1
s=0[ n1 + n2(s)] ×b(s : n1, πu) is the
expected sample size under the null for the design with
n1 and max(n1 + n2(s), s = 0, 1, . . . , n1) as the first stage Shan et al. BMC Medical Research Methodology (2016) 16:90 Page 4 of 14 sample size and the MSS. The min in Eq. Methods As pointed out, it is time consuming to compute the
actual type I and II error rates for each element in the
parameter space, and the branch-and-bound algorithm is
able to finish the design search in a timely manner by dis-
carding elements that do not lead to the optimal design,
which is the key idea of this intelligent algorithm. When
the sample sizes (n1, n) are given, the ESS0 is the objective The minimax-EF design is a special case of the mini-
max adaptive design, therefore, the MSS of the minimax-
EF design is the upper bound of the proposed adaptive
design. For this reason, we start the search with the MSS, Page 5 of 14 Shan et al. BMC Medical Research Methodology (2016) 16:90 in parameter space , is useful to meet the monotonic
relationship between the n2(S) and S in searching for the
design. Among these obtained optimal adaptive designs,
the one with the smallest ESS0 is the adaptive mini-
max design when the MSS is nt. From the relationship
between the proposed design and the minimax-EF design,
it is guaranteed that an optimal adaptive design will be
obtained when MSS is nt. The MSS is then decreased by
1, and the optimal adaptive design is searched again with
the MSS=nt −1. This procedure will be continued until no
optimal design is obtained from three consecutive MMS
values, say n∗−1, n∗−2, and n∗−3. Then, n∗is the nt, which is the MSS of the minimax-EF design. For this
given MSS, say nt, the possible number of subjects from
the first stage, n1, ranges between 1 and nt −1. The search
for n1 = 1 and nt −1 as the first stage sample sizes are
excluded for practical reasons: it is not realistic to enroll
only one patient to make a decision. For each sample size configuration (n1, nt), the algo-
rithm is applied for the design search. If the study is
stopped for futility when S ≤s −1, then we assign
n2(s, Ws) = n −n1 to guarantee that the MSS is exactly
nt. It should be noted that the MSS could occur at mul-
tiple S values. Methods The ascending order of n2(S) for elements Table 2 Comparison between three optimal designs for expected sample size ESS0 at α = 0.05 given πa −πu = 0.2 and 0.15
Minimax
Optimal
Simon
Minimax-EF
Adaptive
Adaptive
πu
πa
Power
n
ESS0
n
ESS0
n
ESS0
n
ESS0
πa −πu = 20 %
0.1
0.3
0.8
25
19.51
24
20.30
23
20.94
29
14.85
0.9
33
26.18
33
23.96
33
23.93
35
22.38
0.2
0.4
0.8
33
22.25
32
24.93
32
23.22
37
20.48
0.9
45
31.23
44
35.68
44
33.39
53
29.74
0.3
0.5
0.8
39
25.69
36
30.68
36
29.31
46
23.45
0.9
53
36.62
50
42.47
50
41.03
60
34.08
0.4
0.6
0.8
39
34.44
39
34.33
39
26.86
46
24.39
0.9
54
38.06
54
38.03
53
42.65
66
35.64
0.5
0.7
0.8
37
27.74
37
26.90
37
26.87
43
23.33
0.9
53
36.11
51
41.14
51
37.74
59
33.45
0.6
0.8
0.8
35
20.77
33
23.97
33
22.13
38
20.28
0.9
45
35.90
45
33.30
45
31.36
52
28.74
0.7
0.9
0.8
26
23.16
26
23.11
25
18.00
27
14.82
0.9
32
22.66
32
22.66
32
22.64
36
20.80
πa −πu = 15 %
0.1
0.25
0.8
40
28.84
38
33.94
38
28.87
43
24.49
0.9
55
40.03
53
47.87
53
41.29
62
36.45
0.2
0.35
0.8
53
40.44
53
40.41
53
40.33
63
34.87
0.9
77
58.42
76
66.51
74
59.58
87
50.80
0.3
0.45
0.8
65
49.63
64
51.32
64
48.08
77
41.33
0.9
88
78.51
88
78.45
88
68.29
104
59.96
0.4
0.55
0.8
70
60.07
69
54.17
69
49.84
82
44.05
0.9
94
78.88
94
76.30
94
74.20
106
63.84
0.5
0.65
0.8
68
66.11
68
66.05
67
58.41
81
43.01
0.9
93
75.00
93
72.20
93
69.84
109
61.87
0.6
0.75
0.8
62
43.79
62
42.89
61
45.26
69
38.53
0.9
84
73.20
84
73.13
84
64.00
97
54.99
0.7
0.85
0.8
49
34.44
49
34.36
49
33.00
59
29.78
0.9
68
48.52
65
50.46
65
48.78
78
42.60 Shan et al. BMC Medical Research Methodology (2016) 16:90 Page 6 of 14 Table 3 Proposed optimal adaptive designs for πa = πu + 0.2 at α = 0.05. Methods Simon’s minimax design (r1/n1, r/n), and the minimax that
stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference
Power = 80 %
Power = 90 %
S
n2(S)
n(S)
r(S)
S
n2(S)
n(S)
r(S)
πu = 0.2
Simon:(4/18, 10/33)
Simon:(5/24, 13/45)
Minimax-EF:((2, 6)/15, 10/32)
Minimax-EF:((4, 9)/25, 13/44)
New:n1 = 19
New:n1 = 23
≤4
0
19
0
≤4
0
23
0
5
13
32
10
5
21
44
12
6
13
32
10
6
21
44
13
7
13
32
9
7
21
44
13
8
13
32
10
8
21
44
13
9
11
30
10
9
21
44
13
≥10
0
19
0
10
15
38
11
≥11
0
23
0
πu = 0.3
Simon:(6/19, 16/39)
Simon:(7/24, 21/53)
Minimax-EF:((8, 13)/27, 15/36)
Minimax-EF:((11, 17)/37, 20/50)
New:n1 = 20
New:n1 = 32
≤5
0
20
0
≤9
0
32
0
6
16
36
14
10
18
50
19
7
16
36
15
11
18
50
20
8
16
36
15
12
18
50
20
9
16
36
15
13
18
50
20
10
16
36
15
14
18
50
20
11
16
36
15
15
18
50
20
12
14
34
15
16
18
50
20
≥13
0
20
0
17
11
43
18
≥18
0
32
0
πu = 0.4
Simon:(17/34, 20/39)
Simon:(12/29, 27/54)
Minimax-EF:((17, 19)/34, 20/39)
Minimax-EF:((12, 19)/29, 27/54)
New:n1 = 16
New:n1 = 35
≤6
0
16
0
≤14
0
35
0
7
23
39
20
15
18
53
26
8
23
39
20
16
18
53
27
9
23
39
20
17
18
53
27
10
23
39
20
18
18
53
27
11
23
39
21
19
18
53
26
12
22
38
20
20
17
52
26
13
16
32
18
21
17
52
26
14
9
25
16
22
17
52
26
15
5
21
15
23
17
52
27
16
3
19
16
≥24
0
35
0 Table 3 Proposed optimal adaptive designs for πa = πu + 0.2 at α = 0.05. Simon’s minimax design (r1/n1, r/n), and the minimax that
stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference Page 7 of 14 Shan et al. BMC Medical Research Methodology (2016) 16:90 Table 3 Proposed optimal adaptive designs for πa = πu + 0.2 at α = 0.05. Methods Simon’s minimax design (r1/n1, r/n), and the minimax that
stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) Table 3 Proposed optimal adaptive designs for πa = πu + 0.2 at α = 0.05. Simon’s minimax design (r1/n1, r/n), and the minimax that
stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) Table 3 Proposed optimal adaptive designs for πa = πu + 0.2 at α = 0.05. Simon’s minimax design (r1/n1, r/n), and the minimax that
stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) p
p
p
g
a
u +
g ( 1/ 1, / ),
stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued)
πu = 0.5
Simon:(12/23, 23/37)
Simon:(14/27, 32/53)
Minimax-EF:((10, 15)/20, 23/37)
Minimax-EF:((17, 23)/34, 31/51)
New:n1 = 20
New:n1 = 28
≤10
0
20
0
≤14
0
28
0
11
17
37
23
15
23
51
30
12
17
37
23
16
23
51
31
13
17
37
23
17
23
51
31
14
17
37
23
18
23
51
31
15
15
35
22
19
23
51
31
≥16
0
20
0
20
23
51
31
21
21
49
29
22
6
34
22
≥23
0
28
0
πu = 0.6
Simon:(8/13, 25/35)
Simon:(15/26, 32/45)
Minimax-EF:((10, 14)/17, 24/33)
Minimax-EF:((15, 20)/25, 32/45)
New:n1 = 15
New:n1 = 23
≤9
0
15
0
≤14
0
23
0
10
18
33
24
15
22
45
32
11
18
33
24
16
22
45
32
12
17
32
23
17
21
44
31
13
16
31
22
18
21
44
31
14
14
29
21
19
21
44
31
15
14
29
21
20
10
33
24
21
10
33
25
22
8
31
24
≥23
0
23
0
πu = 0.7
Simon:(19/23, 21/26)
Simon:(13/18, 26/32)
Minimax-EF:((19, 20)/23, 21/26)
Minimax-EF:((13, 18)/18, 26/32)
New:n1 = 13
New:n1 = 18
≤9
0
13
0
≤13
0
18
0
10
12
25
21
14
14
32
26
11
12
25
21
15
14
32
26
12
12
25
20
16
14
32
26
13
7
20
16
17
14
32
26
≥17
3
21
18
minimum MSS, and the optimal design associated with
n∗is the final minimax adaptive two-stage design. It is
For the candidates of an admissible design, the f
step is to identify the MSS values of the minimax ad minimum MSS, and the optimal design associated with
n∗is the final minimax adaptive two-stage design. It is
obvious that n∗≤nt. For the candidates of an admissible design, the first
step is to identify the MSS values of the minimax adap-
tive design and the optimal adaptive design by Shan et al. Shan et al. BMC Medical Research Methodology (2016) 16:90 Page 8 of 14 Table 4 Proposed optimal adaptive designs for πa = πu + 0.15 at α = 0.05. Table 3 Proposed optimal adaptive designs for πa = πu + 0.2 at α = 0.05. Simon’s minimax design (r1/n1, r/n), and the minimax that
stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) Simon’s minimax design (r1/n1, r/n), and the minimax
that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference
Power=80 %
Power=90 %
S
n2(S)
n(S)
r(S)
S
n2(S)
n(S)
r(S)
πu = 0.1
Simon:(2/22, 7/40)
Simon:(3/31, 9/55)
Minimax-EF:((4, 6)/33, 7/38)
Minimax-EF:((6, 8)/47, 9/53)
New:n1 = 18
New:n1 = 33
≤1
0
18
0
≤3
0
33
0
2
20
38
7
4
20
53
8
3
20
38
7
5
20
53
9
4
19
37
6
6
20
53
9
5
19
37
6
7
18
51
8
6
18
36
6
8
17
50
8
≥7
0
18
0
≥9
0
33
0
πu = 0.2
Simon:(6/31, 15/53)
Simon:(8/42, 21/77)
Minimax-EF:((6, 13)/31, 15/53)
Minimax-EF:((13, 18)/62, 21/76)
New:n1 = 31
New:n1 = 47
≤6
0
31
0
≤9
0
47
0
7
22
53
15
10
27
74
20
8
22
53
15
11
27
74
20
9
22
53
15
12
27
74
20
10
22
53
15
13
27
74
20
11
22
53
15
14
26
73
20
12
21
52
15
15
26
73
20
≥13
0
31
0
16
26
73
20
17
25
72
20
18
14
61
18
≥19
0
47
0
πu = 0.3
Simon:(16/46, 25/65)
Simon:(27/77, 33/88)
Minimax-EF:((13, 19)/43, 25/64)
Minimax-EF:((27, 33)/77, 33/88)
New:n1 = 32
New:n1 = 51
≤9
0
32
0
≤15
0
51
0
10
32
64
24
16
37
88
33
11
32
64
25
17
37
88
33
12
32
64
25
18
37
88
33
13
32
64
25
19
37
88
33
14
32
64
25
20
37
88
33
15
31
63
24
21
37
88
33
16
30
62
24
22
37
88
33
17
29
61
24
23
37
88
33
18
24
56
22
24
37
88
34
≥19
0
32
0
25
36
87
33
26
34
85
33
27
34
85
33
≥28
0
51
0 Table 4 Proposed optimal adaptive designs for πa = πu + 0.15 at α = 0.05. Simon’s minimax design (r1/n1, r/n), and the minimax
that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference Page 9 of 14 Shan et al. BMC Medical Research Methodology (2016) 16:90 Table 4 Proposed optimal adaptive designs for πa = πu + 0.15 at α = 0.05. Table 3 Proposed optimal adaptive designs for πa = πu + 0.2 at α = 0.05. Simon’s minimax design (r1/n1, r/n), and the minimax that
stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) Simon’s minimax design (r1/n1, r/n), and the minimax
that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) Table 4 Proposed optimal adaptive designs for πa = πu + 0.15 at α = 0.05. Simon’s minimax design (r1/n1, r/n), and the minimax
that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued)
πu = 0.4
Simon:(28/59, 34/70)
Simon:(24/62, 45/94)
Minimax-EF:((16, 23)/41, 34/69)
Minimax-EF:((21, 31)/55, 45/94)
New:n1 = 37
New:n1 = 52
≤15
0
37
0
≤20
0
52
0
16
32
69
33
21
42
94
44
17
32
69
34
22
42
94
45
18
32
69
34
23
42
94
45
19
32
69
34
24
42
94
45
20
32
69
34
25
42
94
45
21
31
68
33
26
42
94
45
22
31
68
33
27
42
94
45
23
31
68
33
28
42
94
45
24
21
58
29
29
42
94
45
≥25
0
37
0
30
42
94
45
31
39
91
43
≥32
0
52
0
πu = 0.5
Simon:(39/66, 40/68)
Simon:(28/57, 54/93)
Minimax-EF:((39, 40)/66, 40/68)
Minimax-EF:((30, 38)/59, 54/93)
New:n1 = 54
New:n1 = 55
≤28
0
54
0
≤28
0
55
0
29
13
67
39
29
38
93
54
30
13
67
40
30
38
93
54
31
13
67
40
31
38
93
54
32
13
67
40
32
38
93
54
33
13
67
40
33
38
93
54
34
13
67
39
34
38
93
54
35
13
67
39
35
38
93
53
36
13
67
39
36
38
93
54
37
9
63
38
37
38
93
53
≥38
0
54
0
≥38
0
55
0
πu = 0.6
Simon:(18/30, 43/62)
Simon:(48/72, 57/84)
Minimax-EF:((16, 22)/27, 43/62)
Minimax-EF:((48, 53)/72, 57/84)
New:n1 = 32
New:n1 = 58
≤19
0
32
0
≤37
0
58
0
20
29
61
42
38
26
84
57
21
29
61
42
39
26
84
57
22
29
61
42
40
26
84
57
23
28
60
42
41
26
84
57
24
28
60
42
42
25
83
57
25
28
60
42
43
25
83
57
26
27
59
41
44
23
81
56
27
27
59
41
≥45
0
58
0
28
15
47
34
≥29
0
32
0 Page 10 of 14 Shan et al. BMC Medical Research Methodology (2016) 16:90 Table 4 Proposed optimal adaptive designs for πa = πu + 0.15 at α = 0.05. Table 4 Proposed optimal adaptive designs for πa = πu + 0.15 at α = 0.05. Simon’s minimax design (r1/n1, r/n), and the minimax
that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) Simon’s minimax design (r1/n1, r/n), and the minimax
that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) πu = 0.7
Simon:(16/23, 39/49)
Simon:(33/44, 53/68)
Minimax-EF:((16, 21)/23, 39/49)
Minimax-EF:((29, 35)/41, 51/65)
New:n1 = 25
New:n1 = 37
≤18
0
25
0
≤26
0
37
0
19
24
49
39
27
28
65
51
20
24
49
39
28
28
65
51
21
24
49
39
29
28
65
51
22
23
48
38
30
28
65
51
23
8
33
26
31
27
64
50
≥24
0
25
0
32
27
64
50
33
24
61
48
≥34
0
37
0 q with n −ESS0 as the slope and ESS0 as the intercept. As
ESS0 is always less than n, T is an increasing function of q. [8], nmin and nopt that are in the range of the MSS of
an admissible design. Secondly, for each given MSS, n,
between nmin and nopt, the optimal design with the small-
est ESS0 is calculated by using the algorithm aforemen-
tioned. The sample size information, n and ESS0, are used
in calculating the Bayes risk function [8], nmin and nopt that are in the range of the MSS of
an admissible design. Secondly, for each given MSS, n,
between nmin and nopt, the optimal design with the small-
est ESS0 is calculated by using the algorithm aforemen-
tioned. The sample size information, n and ESS0, are used
in calculating the Bayes risk function Results BMC Medical Research Methodology (2016) 16:90 Page 11 of 14 Table 5 Probability of early termination at the first stage for the
designs with πa = πu + 0.2 and 80 % power
πu
Simon
Minimax-EF
New adaptive design
0.2
0.716
0.402
0.674
0.3
0.666
0.582
0.417
0.4
0.913
0.921
0.527
0.5
0.661
0.589
0.589
0.6
0.647
0.554
0.597
0.7
0.946
0.949
0.579 Table 5 Probability of early termination at the first stage for the
designs with πa = πu + 0.2 and 80 % power the minimax-EF design. When the proposed design has
the same MSS as either of the two minimax designs, the
ESS0 of the proposed design is always smaller. For exam-
ple, the saving of the ESS0 from the proposed design as
compared to the minimax-EF design when they have the
same MSS in Table 2, ranges from 0.03 to 10.16, with
an average of 3.07 patients. The optimal adaptive design
is included as a reference, and the MSS of this design
is generally larger than that of the proposed minimax
adaptive design, with a range from 2 to 16, and an aver-
age of 9.3 patients from all the configurations studied in
Table 2. We present the proposed minimax adaptive design with
specific design parameters in Tables 3 and 4 for πa =
πu + 0.2 and πa = πu + 0.15, respectively. The pre-
specified type I error rate is set as α = 0.05, and two type
II error rates are studied, β = 0.1 and 0.2. We present the
minimum adaptive design for various values of πu, from
0.2 to 0.7. For example, for the design to achieve 90 %
power with πu = 0.6, πa = 0.8 as in Table 3, Simon’s min-
imax design, the minimax-EF design and the proposed
adaptive minimax design are displayed in Fig. 1: the MSS
of the study (n(S)) VS the number of responses from the
first stage (S). Simon’s minimax design is calculated as
(n1, n, r1, r) = (26, 45, 15, 32) with the MSS=45 and the
ESS0 = 35.90. The minimax-EF design is found to be
((r1, r2)/n1, r/n) = ((15, 20)/25, 32/45) with 25 patients
enrolled in the first stage, and a possible total sample size
of 25. Results We compare performance of the proposed minimax adap-
tive design, Simon’s minimax design, the minimax-EF
design, and the optimal adaptive design due to Shan
et al. [8]. The first three designs are minimax designs,
while the last one is under the optimal criteria. The first T = q × n + (1 −q) × ESS0 = (n −ESS0)q + ESS0, where q is a pre-specified weight value, q ∈[ 0, 1]. It can be
seen that the Bayes risk function T is a linear function of The number of responses in the first stage: S
Maximum sample size: n(S)
0
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
20
23
26
29
32
35
38
41
44
47
50
53
Proposed adaptive minimax design
Simon’s minimax design
Minimax−EF design
Fig. 1 The comparison among Simon’s minimax design, the minimax-EF design and the proposed adaptive minimax design for the design with
parameters (α, β, πu, πa) = (0.05, 0.1, 0.6, 0.8). The maximum sample size, n(S), is plotted as a function of the number of responses from the first
stage, S 0
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 The number of responses in the first stage: S n among Simon’s minimax design, the minimax-EF design and the proposed adaptive minimax design for the design with
πa) = (0.05, 0.1, 0.6, 0.8). The maximum sample size, n(S), is plotted as a function of the number of responses from the first Fig. 1 The comparison among Simon’s minimax design, the minimax-EF design and the proposed adaptive minimax design for the design with
parameters (α, β, πu, πa) = (0.05, 0.1, 0.6, 0.8). The maximum sample size, n(S), is plotted as a function of the number of responses from the first
stage, S Shan et al. Results The trial will be stopped for futitlity when S ≤
15 or efficacy when S > 20 out of n1 = 25 patients
in the first stage. The ESS0 is 33.3 for this design. For
the proposed minimax adaptive design, the second stage design and the last design are adaptive designs. To the
best of our knowledge, we do not find a direct com-
petitor in the category of adaptive two-stage designs
under the minimax criteria. Simon’s minimax design is the
most commonly used design under the minimax criteria,
thus it is included in the comparison. The minimax-
EF design stops for either futility or efficacy in the first
stage. This stopping rule is also applied in the proposed
design, thus, this design is also included in the compar-
ison. Simon’s design only allows stopping of the trial at
the first stage for futility, and the other three designs
allows the stoppage for either futility or efficacy in the first
stage. The MSS and the ESS0 are compared in Table 2 for
the proposed minimax adaptive design, and the other
three competitors when πa −πu = 0.2, and 0.15 at the
significance level of α = 0.05. As expected, the pro-
posed minimax adaptive design has a smaller or the same
MSS as compared to the Simon’s minimax design and 0.0
0.2
0.4
0.6
0.8
1.0
30
40
50
60
q
The Bayes risk function: T
Design with n=50
Design with n=51
Design with n=52
Design with n=53
Design with n=54
Design with n=55
Design with n=56
Design with n=57
Design with n=58
Design with n=59
Design with n=60
Fig. 2 The Bayes risk function as a function of the weight value q in searching for an admissible adaptive design for (α, β, πu, πa) = (0.05, 0.1, 0.3, 0.5) Fig. 2 The Bayes risk function as a function of the weight value q in searching for an admissible adaptive design for (α, β, πu, πa) = (0.05, 0.1, 0.3, 0.5) Shan et al. Application pp
We revisit the urothelial cancer trial with the neoad-
juvant therapy [10]. Simon’s minimax design was used
for study design to attain 80 % power at the signifi-
cance level of α = 0.1. The research team expected a
15 % increase in response rate as compared to the priori
estimated response rate πu = 35 %. The design param-
eters using Simon’s minimax design are: (n1, n, r1, r) =
(31, 49, 10, 21) with the ESS0=40.8. The minimax-EF
design is: ((r1, r2)/n1, r/n) = ((11, 16)/32, 21/49). The
design parameters, (n1, n(S), r(S)), for the proposed adap-
tive minimax design are presented in Table 1, and also
plotted in Fig. 3. They all have the same maximum sample
size 49, but the expected sample size under the null for the
proposed design is smaller, 38.9 VS 40.8(Simon’s design),
and 39.2 (the minimax-EF design). The adaptive design is
also flexible to allow the second stage sample size and its
associted critical value to depend on the result from the
first stage. As suggested by one of the reviewers, we compare the
probability of early termination (PET) at the first stage for
these designs. The PET is defined as the probability of a
study that is stopped at the first stage due to either futil-
ity or efficacy. We present the PET of the three designs
with πa = πu + 0.2 and 80 % power in Table 5. The
PET of the new adaptive design is always less than that by
Simon’s minimax design in these cases. There is no clear
relationship between the minimax-EF design and the new
design with regard to the PET. It can be seen that the PET
of the new design is more consistent as compared to the
competitors. Results BMC Medical Research Methodology (2016) 16:90 Page 12 of 14 Table 6 Admissible adaptive designs for (α, β, πu, πa) = (0.05,
0.1, 0.3, 0.5) Table 6 Admissible adaptive designs for (α, β, πu, πa) = (0.05,
0.1, 0.3, 0.5)
Interval of q
n
ESS0
Comment
[ 0.000, 0.040]
60
34.08
Optimal design
[ 0.040, 0.105]
59
34.12
[ 0.105, 0.132]
57
34.36
[ 0.132, 0.468]
54
34.81
[ 0.468, 0.580]
53
35.69
[ 0.580, 0.721]
51
38.45
[ 0.721, 1.000]
50
41.03
Minimax design sample size is allowed to change as a function of the first
stage responders, and the relationship is monotonic. The
first stage sample size is n1 = 23 and the maximum sam-
ple size is 45 and this maxmum sample size only occurs
for the cases with S = 15 and 16 responders observed
from the first stage. The trial is terminated at the end of
the first stage for futility or efficacy for S ≤14 or S ≥23,
respectively. In such cases, the MSS is the first stage sam-
ple size, which is 23. When the first stage response is
between 15 and 22, the corresponding second stage sam-
ple size n2(S) is presented in Table 3. As compared to
the other designs, the adaptive minimax design has the
smallest expected sample size and the smallest first stage
sample size in this particular example. In the proposed
design, the second stage sample size is a non-increasing
function of the number of responses from the first stage,
not a constant as in Simon’s design and the Minimax-EF
design. It can be seen that although the adaptive optimal
design has the smallest expected sample size as compared
to others, the MSS of the adaptive optimal design is
often much larger than that of the proposed adaptive
minimax. Abbreviations
ESS
h ESS0, the expected sample size under the null; MMS, maximum sample size Discussion and conclusions To reduce
the computational time, one may use a backward search
method as in this article, starting with the maximum sam-
ple size from the minimax-EF design. In addition, when
the proposed design and other designs have the same
MSS, the expected sample size under the null of the
proposed design is always smaller than others. g
Randomized clinical trials are used in clinical tri-
als by comparing the new treatment or therapy to the
best available treatment for disease. Randomized clin-
ical trials are preferable in the majority of studies to
reduce the selection bias and confounding effects, thus
capturing the true effectiveness of the new treatment. The widely used two-stage design for a two-arm study
with binary outcomes is the one due to Thall et al. [16], that does not allow the second stage sample size
to change from the results of the first stage. We will
extend the adaptive approach from the one-arm study to
this two-arm study to develop a new adaptive two-stage
design for a randomized clinical trial with dichotomous
endpoints. The proposed adaptive design assumes a monotonic
relationship between the second stage sample size and
the first stage result. In practice, an investigator may
want to accrue more patients in the second stage when
the number of response from the first stage is large, to
obtain as much information as possible from the clini-
cal study. In this case, an additional constraint can be
added during the design search to meet the investiga-
tor’s requirement: the second stage sample sizes are the
same when S is above Sc, where Sc can be determined by
the new constraint from the investigator. The new con-
straint added in the design search should be clinically
meaningful. Discussion and conclusions Figure 2 displays the lines of the Bayes risk function T as
a function of q for each design to attain 90 % power at the
significance level of 0.05, with πu = 0.3 and πa = 0.5. The
minimax adaptive design and the optimal adaptive design
are presented in Table 3, with nmin = 50 and nopt = 60. Therefore, a total of 11 lines are displayed in the figure
to represent the optimal designs when n is between 50
and 60. In order to identify an admissible design for a
given range of q, one has to first compute the intersections
among these 11 lines, and the maximum number of inter-
sections between 0 and 1 is
11
2
= 55. Within these 55
intersections, 2 of them are out of the range of [ 0, 1],
this leads to a total of 53 intersections between 0 and
1. After sorting the x-values of these intersections, the
design among these 11 designs is the admissible design
for a given range of q when this design has the smallest T
over this range, see Table 6. It can be seen that the optimal
adaptive design is the admissible design when q is close to
0, and the minimax design is the admissible design when
q is close to 1. We develop a new minimax adaptive two-stage design
for use in phase II clinical trials to assess the new treat-
ment’s activity. The software program to implement the
adaptive designs in this article is written in the statisti-
cal language, R [11–14], and it is available per request
from the first author (guogen.shan@unlv.edu) or the cor-
responding author (jtao@263.net). We are also working
together to develop a new R package to implement the
adaptive minimax and admissible designs from this arti-
cle and the adaptive optimal design by Shan et al. [8]. The
proposed design allows the second stage sample size and
its associated critical value to depend on the result from
the first stage. The proposed design satisfies the mono-
tonicity property of the relationship between the second
stage sample size and the first stage responders, which is
an important feature for a practical application. The MSS of the proposed adaptive minimax design
is always less than or equal to that of the minimax-EF
design. We consider this as an important advantage of Shan et al. Acknowledgment g
The authors are very grateful to the Editor, and two referees for their insightful
comments that help improve the manuscript. The naive point estimate for the probability of response
rate is calculated as the number of responses divided by
the total number of patients, and it is well known that this
estimate is biased. In the traditional Simon’s design, Jung
and Kim [15] derived the uniformly minimum variance
unbiased estimate for the probability of response based on
the Rao-Blackwell theorem. To the best of our knowledge,
no unbiased estimate for the probability of response Discussion and conclusions BMC Medical Research Methodology (2016) 16:90 Page 13 of 14 The number of responses in the first stage: S
Maximum sample size: n(S)
0
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
22
25
28
31
34
37
40
43
46
49
52
55
58
Proposed adaptive minimax design
Simon’s minimax design
Minimax−EF design
Fig. 3 For the urothelial cancer trial with parameters (α, β, πu, πa) = (0.1, 0.2, 0.35, 0.5), the design parameters for Simon’s minimax design, the
minimax-EF design and the proposed adaptive minimax design. The maximum sample size, n(S), is plotted as a function of the number of
responses from the first stage, S Fig. 3 For the urothelial cancer trial with parameters (α, β, πu, πa) = (0.1, 0.2, 0.35, 0.5), the design parameters for Simon’s minimax design, the
minimax-EF design and the proposed adaptive minimax design. The maximum sample size, n(S), is plotted as a function of the number of
responses from the first stage, S Fig. 3 For the urothelial cancer trial with parameters (α, β, πu, πa) = (0.1, 0.2, 0.35, 0.5), the design parameters for Simon’s minimax design, the
minimax-EF design and the proposed adaptive minimax design. The maximum sample size, n(S), is plotted as a function of the number of
responses from the first stage, S has been proposed in an adaptive two-stage design set-
ting. This may be due to the complexity of an adaptive
design as compared to the traditional sample size fixed
design. the minimax adaptive design to reduce the computational
intensity as compared to adaptive designs based on the
optimal criteria [8], where the upper bound of the sample
size has to be set in the design search process. To reduce
the computational time, one may use a backward search
method as in this article, starting with the maximum sam-
ple size from the minimax-EF design. In addition, when
the proposed design and other designs have the same
MSS, the expected sample size under the null of the
proposed design is always smaller than others. the minimax adaptive design to reduce the computational
intensity as compared to adaptive designs based on the
optimal criteria [8], where the upper bound of the sample
size has to be set in the design search process. Funding g
Shan’s research is partially supported by grants from the National Institute of
General Medical Sciences from the National Institutes of Health:
P20GM109025, P20GM103440, and 5U54GM104944. Zhang’s work was
supported by the Zhejiang Provincial Natural Science Foundation of China
(grant no. LY15F020001) and the National Natural Science Foundation of
China (grant no. 61170099). Page 14 of 14 Page 14 of 14 Shan et al. BMC Medical Research Methodology (2016) 16:90 Shan et al. BMC Medical Research Methodology (2016) 16:90 13. Shan G, Ma C. Unconditional tests for comparing two ordered
multinomials. Stat Methods Med Res. 2016;25(1):241–54. doi:10.1177/0962280212450957. http://dx.doi.org/10.1177/
0962280212450957. 14. Shan G, Moonie S, Shen J. Sample size calculation based on efficient
unconditional tests for clinical trials with historical controls. J Biopharm
Stat. 2016;26(2):240–9. http://view.ncbi.nlm.nih.gov/pubmed/25551261. 15. Jung SHH, Kim KMM. On the estimation of the binomial probability in
multistage clinical trials. Stat Med. 2004;23(6):881–96. doi:10.1002/sim.1653. http://dx.doi.org/10.1002/sim.1653. 16. Thall PF, Simon R, Ellenberg SS. Two-Stage Selection and Testing
Designs for Comparative Clinical Trials. Biometrika. 1988;75(2):303–10. http://www.jstor.org/stable/2336178. 13. Shan G, Ma C. Unconditional tests for comparing two ordered
multinomials. Stat Methods Med Res. 2016;25(1):241–54. doi:10.1177/0962280212450957. http://dx.doi.org/10.1177/
0962280212450957. Availability of data and materials This is a manuscript to develop novel study designs, therefore, no real data is
involved. Authors’ contributions The idea for the paper was originally developed by GS and TJ. GS and HZ
computed and analysed the adaptive minimax and admissible two-stage
designs in this paper. GS, HZ and TJ drafted the manuscript, revised the paper
critically and approved the final version. 15. Jung SHH, Kim KMM. On the estimation of the binomial probability in
multistage clinical trials. Stat Med. 2004;23(6):881–96. doi:10.1002/sim.1653. http://dx.doi.org/10.1002/sim.1653. Received: 18 March 2016 Accepted: 22 July 2016 Received: 18 March 2016 Accepted: 22 July 2016 Competing interests 16. Thall PF, Simon R, Ellenberg SS. Two-Stage Selection and Testing
Designs for Comparative Clinical Trials. Biometrika. 1988;75(2):303–10. http://www.jstor.org/stable/2336178. 16. Thall PF, Simon R, Ellenberg SS. Two-Stage Selection and Testing
Designs for Comparative Clinical Trials. Biometrika. 1988;75(2):303–10. http://www.jstor.org/stable/2336178. The authors declare that they have no competing interests. References 1. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R,
Dancey J, Arbuck S, Gwyther S, Mooney M, et al. New response
evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur J Cancer. 2009;45(2):228–47. doi:10.1016/j.ejca.2008.10.026. http://dx.doi.org/10.1016/j.ejca.2008.10.026. 1. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R,
Dancey J, Arbuck S, Gwyther S, Mooney M, et al. New response
evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur J Cancer. 2009;45(2):228–47. doi:10.1016/j.ejca.2008.10.026. http://dx.doi.org/10.1016/j.ejca.2008.10.026. 1. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R,
Dancey J, Arbuck S, Gwyther S, Mooney M, et al. New response
evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur J Cancer. 2009;45(2):228–47. doi:10.1016/j.ejca.2008.10.026. http://dx.doi.org/10.1016/j.ejca.2008.10.026. 2. Simon R. Optimal two-stage designs for phase II clinical trials. Control Clin
Trials. 1989;10(1):1–10. http://view.ncbi.nlm.nih.gov/pubmed/2702835. 2. Simon R. Optimal two-stage designs for phase II clinical tria 2. Simon R. Optimal two-stage designs for phase II clinical trials. Control Clin
Trials. 1989;10(1):1–10. http://view.ncbi.nlm.nih.gov/pubmed/2702835. 3. Mander AP, Thompson SG. Two-stage designs optimal under the
alternative hypothesis for phase II cancer clinical trials. Contemp Clin
Trials. 2010;31(6):572–8. http://view.ncbi.nlm.nih.gov/pubmed/20678585. p
g
p
4. Banerjee A, Tsiatis AA. Adaptive two-stage designs in phase II clinical
trials. Stat Med. 2006;25(19):3382–95. http://view.ncbi.nlm.nih.gov/
pubmed/16479547. 5. Englert S, Kieser M. Optimal adaptive two-stage designs for phase II
cancer clinical trials. Biom J. 2013;55(6):955–8. http://view.ncbi.nlm.nih. gov/pubmed/23868324. 5. Englert S, Kieser M. Optimal adaptive two-stage designs for phase II
cancer clinical trials. Biom J. 2013;55(6):955–8. http://view.ncbi.nlm.nih. gov/pubmed/23868324. 6. Englert S, Kieser M. Improving the flexibility and efficiency of phase II
designs for oncology trials. Biometrics. 2012;68(3):886–92. http://view.ncbi.nlm.nih.gov/pubmed/22150825. http://view.ncbi.nlm.nih.gov/pubmed/22150825. 7. Wolsey LA. Integer Programming, 1edn: Wiley-Interscience; 1998. http://www.worldcat.org/isbn/0471283665. 7. Wolsey LA. Integer Programming, 1edn: Wiley-Interscience; 1998. http://www.worldcat.org/isbn/0471283665. 8. Shan G, Wilding GE, Hutson AD, Gerstenberger S. Optimal adaptive
two-stage designs for early phase II clinical trials. Statist Med. 2016;35(8):
1257–66. doi:10.1002/sim.6794. http://dx.doi.org/10.1002/sim.6794. 8. Shan G, Wilding GE, Hutson AD, Gerstenberger S. Optimal adaptive
two-stage designs for early phase II clinical trials. Statist Med. 2016;35(8):
1257–66. doi:10.1002/sim.6794. http://dx.doi.org/10.1002/sim.6794. Author details 1Epidemiology and Biostatistics Program, Department of Environmental and
Occupational Health, School of Community Health Sciences, University of
Nevada Las Vegas, 89154 Las Vegas, NV, USA. 2School of Computer and
Information Engineering, Zhejiang Gongshang University, Hangzhou, 310018
Zhejiang, China. 3Department of Statistics, Zhejiang Gongshang University,
Hangzhou, 310018 Zhejiang, China. Hangzhou, 310018 Zhejiang, China. 13. Shan G, Ma C. Unconditional tests for comparing two ordered
multinomials. Stat Methods Med Res. 2016;25(1):241–54.
doi:10.1177/0962280212450957. http://dx.doi.org/10.1177/
0962280212450957. 13. Shan G, Ma C. Unconditional tests for comparing two ordered
multinomials. Stat Methods Med Res. 2016;25(1):241–54.
doi:10.1177/0962280212450957. http://dx.doi.org/10.1177/
0962280212450957.
14. Shan G, Moonie S, Shen J. Sample size calculation based on efficient
unconditional tests for clinical trials with historical controls. J Biopharm
Stat. 2016;26(2):240–9. http://view.ncbi.nlm.nih.gov/pubmed/25551261.
15. Jung SHH, Kim KMM. On the estimation of the binomial probability in
multistage clinical trials. Stat Med. 2004;23(6):881–96.
doi:10.1002/sim.1653. http://dx.doi.org/10.1002/sim.1653.
16. Thall PF, Simon R, Ellenberg SS. Two-Stage Selection and Testing
Designs for Comparative Clinical Trials. Biometrika. 1988;75(2):303–10.
http://www.jstor.org/stable/2336178. 14. Shan G, Moonie S, Shen J. Sample size calculation based on efficient
unconditional tests for clinical trials with historical controls. J Biopharm
Stat. 2016;26(2):240–9. http://view.ncbi.nlm.nih.gov/pubmed/25551261. Submit your next manuscript to BioMed Central
and we will help you at every step: 9. Jung SHH, Lee T, Kim K, George SL. Admissible two-stage designs for
phase II cancer clinical trials. Stat Med. 2004;23(4):561–9. http://view.ncbi. nlm.nih.gov/pubmed/14755389. 9. Jung SHH, Lee T, Kim K, George SL. Admissible two-stage designs for
phase II cancer clinical trials. Stat Med. 2004;23(4):561–9. http://view.ncbi. nlm.nih.gov/pubmed/14755389. • We accept pre-submission inquiries
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p 10. Siefker-Radtke AO, Dinney CP, Shen Y, Williams DL, Kamat AM,
Grossman HB, Millikan RE. A phase 2 clinical trial of sequential
neoadjuvant chemotherapy with ifosfamide, doxorubicin, and
gemcitabine followed by cisplatin, gemcitabine, and ifosfamide in locally
advanced urothelial cancer. Cancer. 2013;119(3):540–7. doi:10.1002/cncr.27751. http://dx.doi.org/10.1002/cncr.27751. 10. Siefker-Radtke AO, Dinney CP, Shen Y, Williams DL, Kamat AM,
Grossman HB, Millikan RE. A phase 2 clinical trial of sequential
neoadjuvant chemotherapy with ifosfamide, doxorubicin, and
gemcitabine followed by cisplatin, gemcitabine, and ifosfamide in locally
advanced urothelial cancer. Cancer. 2013;119(3):540–7. doi:10.1002/cncr.27751. http://dx.doi.org/10.1002/cncr.27751. 11. Shan G, Wang W. ExactCIdiff: An R Package for Computing Exact
Confidence Intervals for the Difference of Two Proportions. The R Journal. 2013;5(2):62–71. 12. Shan G. Exact Statistical Inference for Categorical Data, 1edn: Academic
Press; 2015. http://www.worldcat.org/isbn/0081006810. 12. Shan G. Exact Statistical Inference for Categorical Data, 1edn: Academic
Press; 2015. http://www.worldcat.org/isbn/0081006810.
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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0018397&type=printable
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English
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Non-AIDS Associated Kaposi's Sarcoma: Clinical Features and Treatment Outcome
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PloS one
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Abstract Competing Interests: The authors have declared that no competing interests exist. * E-mail: claus.garbe@med.uni-tuebingen.de To date it remains unclear whether KS itself is a true
malignancy or rather just a reactive proliferation [5]. Introduction Kaposi’s sarcoma (KS) was first described in 1872 by the
Hungarian dermatologist Moritz Kaposi [1]. It is a rare neoplasm
of lymphatic endothelial cells frequently evident as multiple
vascular cutaneous and mucosal nodules. Lymph node and
visceral manifestation is seen in cases of strong immunosuppres-
sion or aggressive disease. This study focuses on non-AIDS related KS of the skin i.e. cutaneous manifestations of KS in HIV-negative patients present-
ed to the Department of Dermatology, University of Tuebingen
Medical Center between 1987 and 2009. Lena Jakob, Gisela Metzler, Ko-Ming Chen, Claus Garbe* Department of Dermatology, Eberhard-Karls-University, Tuebingen, Germany Department of Dermatology, Eberhard-Karls-University, Tuebingen, Germany PLoS ONE | www.plosone.org Abstract Background: Kaposi’s sarcoma (KS) in HIV negative patients is rare and has to be distinguished from AIDS associated KS. Two groups are at risk to develop non-AIDS related KS: elderly men mainly of Mediterranean origin and persons with
iatrogenic immunosuppression. Patients and Methods: In order to define risk-groups and major clinical features we retrospectively evaluated clinical data of
all patients with non-AIDS associated KS presenting to the Department of Dermatology, University Hospital Tuebingen
between 1987 and 2009. Data were extracted from the tumor registry of the Comprehensive Cancer Center Tuebingen and
from patient records. Results: 20 patients with non-AIDS KS have been identified. The average age at KS onset was 66.6 years; the male-to-female-
ratio was 3:1. Most of the patients were immigrants from Mediterranean or Eastern European countries (60%). 15 cases of
classic KS versus 5 cases of iatrogenic KS were observed. In 95% of the cases, KS was limited to the skin, without mucosal,
lymph node or visceral manifestation. KS lesions were in all cases multiple and mostly bilateral, the most common
localization was the skin of the lower extremities. Tumor control was achieved in nearly all cases by the use of local or
systemic therapy. No patient died from KS. Conclusions: Unlike KS in AIDS patients, non-AIDS associated KS is a rather localized process which rarely involves lymph
nodes or organs. It is mostly seen in elderly males from Mediterranean or Eastern European countries and in most cases
responsive on local or systemic therapeutic strategies. Citation: Jakob L, Metzler G, Chen K-M, Garbe C (2011) Non-AIDS Associated Kaposi’s Sarcoma: Clinical Features and Treatment Outcome. PLoS ONE 6(4): e18397. doi:10.1371/journal.pone.0018397 r G, Chen K-M, Garbe C (2011) Non-AIDS Associated Kaposi’s Sarcoma: Clinical Features and Treatment Outcome. PLoS ONE 6(4): e18397
0018397 Editor: H. Peter Soyer, The University of Queensland, Australia Editor: H. Peter Soyer, The University of Queensland, Australia Received January 1, 2011; Accepted February 28, 2011; Published April 12, 2011 Copyright: 2011 Jakob et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Materials and Methods Four groups are at risk to develop KS: elderly males of
Mediterranean and Eastern European lineage; children and adults
from central Africa; persons who are iatrogenically immunocom-
romised; and homosexual men infected with human immunode-
ficiency virus (HIV) [2]. Major differences in clinical presentation
and in prognosis among those groups have lead to the following
classifications: classical KS, endemic or so called African KS,
iatrogenic KS and AIDS-KS. This is a retrospective descriptive study of a series of 20 patients
admitted consecutively for KS, from 1987 to 2009 in the
Department of Dermatology, University of Tuebingen Medical
Center. Informed consent was not obtained for all patients as the
entire data was analyzed anonymously. This applies according to
the German Medical Association’s professional code of conduct
and has been approved by the Ethics Committee, University of
Tuebingen (Figure S1). Chang et al. discovered human herpes virus 8, (HHV-8), also
known as Kaposi’s sarcoma associated herpes virus, which is
strongly implicated in the pathogenesis of all types of KS [3]. The
herpes virus is considered necessary but not sufficient for the
development of KS, which is a multistep process including not
only HHV-8 infection, but also genetic and angiogenic factors, as
well as the production of several inflammatory cytokines [4]. In order to define risk-groups and major clinical features we
retrospectively identified cases registered by the tumor registry of
the Comprehensive Cancer Center Tuebingen and evaluated
clinical data documented in the hospital records. All cases presented histopathologically approved KS lesions of
the skin and negative HIV-1/2 screening by macro enzyme 1 April 2011 | Volume 6 | Issue 4 | e18397 Non-AIDS Associated Kaposi’s Sarcoma Non-AIDS Associated Kaposi’s Sarcoma Figure 1. Brownish macules and plaques on the foot of a
patient with classic KS. doi:10.1371/journal.pone.0018397.g001 immunoassay. Histological diagnosis was in most cases completed
by immunohistochemical tests such as HHV-8 staining, or
immunostaining with antibodies against endothelial markers D2-
40, CD31 and CD34. Most patients underwent tumor staging by
lymph node and abdominal ultrasound as well as chest x-ray. Demographic features such as origin, age at onset, gender of the
patient, as well as clinical features such as clinical subtype of KS and
localization of lesions were evaluated. Furthermore treatment
modalities, results and tumor recurrence in the time of observation
were recorded. Treatment outcome was classified according to the
Response Evaluation Criteria in Solid Tumors (RECIST Guide-
lines) [6]. Results vascular channels in the dermis below an integral epidermis. The
so-called promontory sign is sometimes found in patch stage
lesions and denotes vascular spaces surrounding pre-existing blood
vessels (Figure 3). Perivascular lymphoplasmocytic cells as well as
extravasated erythrocytes and hemosiderin deposits are charac-
teristic for patch stage. 20 cases of non-AIDS KS were identified in this study. Mean
age at diagnosis of the group was 66.6 year-old (SD = 15.36). The
youngest patient was 36-year-old and iatrogenically immunosup-
pressed; the oldest developed his classic KS by the age of 90. Mean age at onset of patients with classic KS was 69.6 years
(SD = 12.09). Mean age at onset of patients with classic KS was 69.6 years
(SD = 12.09). As KS progresses to the plaque stage bizarre vessels with an
atypical endothelial lining dissect the collagen tissue (Figure 4). In
the nodular stage spindled endothelial cells predominate forming
slit-like vascular spaces containing erythrocytes and hyaline
globules. Figure
5
and
6
demonstrate
a
well-circumscript
spindle-cell tumor with poorly defined slit-like vascular spaces. A male predominance in KS – 75% male patients (n = 15) versus
25% female patients (n = 5) was shown with a male/female ratio of
3:1. 60% (n = 12) of the patients observed in Tuebingen were
immigrants from Mediterranean and Eastern European countries
(all first generation) versus eight German patients. Figure 7 shows D2-40 immune staining of lymphatic endothelial
cells revealing arborizing vascular structures and lymphocytic cell
infiltration. Figure 8 displays HHV-8 stained atypical endothelial
cells. Regarding the clinical Subtype 15 cases of classic KS and five
cases of iatrogenic KS were identified. Iatrogenic immunosuppression was in one case used for a liver
transplant-patient and four patients were under immunosuppres-
sive therapy due to autoimmune diseases i.e. Behc¸et’s disease,
myasthenia gravis, chronic membranous glomerulonephritis and
systemic sarcoidosis. Immunosuppressive medication included
systemic corticosteroids in all five cases in addition to azathioprine
in one case and ciclosporin A in two cases. Treatment modalities included local therapy such as surgery
and radiotherapy as well as systemic immune therapy with
interferon-a-2a. Interferon-a-2a was applied by subcutaneous
injection using 3 million international units three times weekly. One patient developed neutralizing antibodies and therefore his
therapy was switched to Interferon alphacon (consensus interferon
9 mg daily, then every second day). The duration of administration One patient was categorized as classic KS even though
presenting a primary nodal peripheral T-cell lymphoma causing
immunosuppression. Materials and Methods One patient had a stable disease course and therefore did
not need treatment and one patient refused therapy. Median time of
follow-up for all patients were four years (SEM = 7.86 months). In 7
cases time of observation was determined by intercurrent death. Causes of death were other than KS in all cases. Statistical package of social sciences 16.0 software (SPSS Inc.,
Chicago, IL, U.S.A.) was used to calculate means and standard
deviations. Statistical package of social sciences 16.0 software (SPSS Inc.,
Chicago, IL, U.S.A.) was used to calculate means and standard
deviations. Figure 1. Brownish macules and plaques on the foot of a
patient with classic KS. doi:10.1371/journal.pone.0018397.g001 PLoS ONE | www.plosone.org Discussion of interferon ranged from five weeks to six years. In one case with
post-transplant KS immunosuppressive medication was switched
from ciclosporin A to sirolimus resulting in a partial response. One
patient had a stable disease course and therefore did not need
treatment and one patient refused therapy. Non-AIDS KS is considered a rare disease, but incidence varies
according to individual factors such as origin, sex, age and
immune status of the patient. Between 1987 and 2009, only 20 cases of non-AIDS related KS
were observed at the Department of Dermatology in Tuebingen,
Germany. This is in line with the low incidence of cases appearing
in North-Western Europe. Treatment outcome was evaluated according to the Response
Evaluation Criteria in Solid Tumors (RECIST-Guidelines). The term
‘‘complete response’’ means clearance of KS lesions on later visits
when compared with the first lesions on admission. ‘‘Partial response’’
equals at least 30 percent decrease in the sum of the longest diameter
of target lesions whereas ‘‘stable disease’’ involves less than 30 percent
decrease. The terms complete and partial response as well as stable
disease implicate the absence of new lesions or of progressive lesions. This was achieved in all cases. No patient presented a progressive
disease course initially nor died of KS. Four patients developed
multiple local recurrences. Two of them had primarily received a
local therapy and one interferon-a-2a. No data regarding the incidence of KS in Germany currently
exists. Iscovich et al. analyzed reports of classic KS from different
cancer registries throughout the world between 1988 and 1992. Low incidence rates were found in Denmark (0.18 per million men
aged over 65 per year), intermediate rates in France, and especially
high rates in Sardinia and Sicily (13.2 per million men aged over
65 per year) [7]. It is worth mentioning that the majority of the non-AIDS KS in
Tuebingen (12 out of 20) have been found in first generation
immigrants from Mediterranean countries with presumably higher
incidence rates e.g. in Turkey and Italy (1 per 100,000 men per Clinical features and treatment outcome of non-AIDS KS are
presented in Table 1. Figure 4. Plaque stage KS with bizarre vessels dissecting the
upper dermis. There is erythrocyte extravasation and hemosiderin
pigmentation. doi:10.1371/journal.pone.0018397.g004 Figure 6. Tumor stage KS: Close up view. doi:10.1371/journal.pone.0018397.g006 Figure 4. Plaque stage KS with bizarre vessels dissecting the
upper dermis. There is erythrocyte extravasation and hemosiderin
pigmentation. doi:10.1371/journal.pone.0018397.g004 Figure 6. Results However, immunosuppression was in this
case not iatrogenically induced. Figure 2. Patch stage classic KS: Red to brownish irregularly-
shaped macules and plaques. doi:10.1371/journal.pone.0018397.g002 In 95% (n = 19) of the cases, KS was limited to the skin, without
mucosal, lymph node or visceral manifestation. One patient presented mucosal lesions of the oral and genital
region as well as inguinal lymph node invasion. KS lesions were multiple in all patients (n = 20), no patient
presented just one singular lesion. 70% of the patients (n = 14) were
affected on both hemispheres of the body and 30% (n = 6)
presented unilateral lesions. The most frequent manifestation was the skin of the lower leg
(n = 8), six patients were affected on their feet and five patients on
all four extremities. All in all 95% of the patients were affected on
the skin of the lower extremities. Figure 1 and 2 present a patch stage classic KS characterized by
several brownish irregularly-shaped maculae as well as the
existence of a few partly indurated plaques. Figure 2. Patch stage classic KS: Red to brownish irregularly-
shaped macules and plaques. doi:10.1371/journal.pone.0018397.g002 All stages of KS lesions were observed within the group: The
initial patch stage exhibits an irregular proliferation of jagged April 2011 | Volume 6 | Issue 4 | e18397 2 Figure 3. Patch stage KS with promontory sign. Dilated irregular
vascular channels surround a pre-existing vessel. doi:10.1371/journal.pone.0018397.g003 Non-AIDS Associated Kaposi’s Sarcoma Figure 5. Tumor stage KS: Well-circumscript spindle-cell tumor. Erythrocytes lie within poorly defined slit-like vascular spaces. doi:10.1371/journal.pone.0018397.g005 Figure 5. Tumor stage KS: Well-circumscript spindle-cell tumor. Erythrocytes lie within poorly defined slit-like vascular spaces. doi:10.1371/journal.pone.0018397.g005 Figure 3. Patch stage KS with promontory sign. Dilated irregular
vascular channels surround a pre-existing vessel. doi:10.1371/journal.pone.0018397.g003 Discussion Patients with widespread
disease may need systemic chemotherapeutic or immunologic
medication. Positive results have been found for pegylated
liposomal doxorubicin, danaurubicin, paclitaxel and interferon a
[24,25]. In patients with iatrogenic KS, immunosuppressive
medication may be reduced or modified with the considerate
possibility of grafts being rejected with insufficient immunosup-
pression [26]. Gender presents another factor which strongly influences KS
manifestation. Former male-to-female ratios from 10:1 to 15:1 had
been reported in classic KS [9]. More recent studies showed
gender ratios ranging from 1:1 in England up to 4:1 in Italy
[10,11]. Iscovich and colleagues ascertained a lower gender
difference in populations with lower incidence rates [7]. The
Tuebingen case group showed a significant male predominance
with a male-to-female ratio of 3:1. Just as other investigations classic KS was the most frequent
subtype we found. Iatrogenic KS was with 25% a considerately
highly represented subtype. This may be an effect of mismatch in
classification. Other studies call this subtype post transplant-KS
and patients with KS under immunosuppressive therapy for Between 1987 and 2009 single KS lesions have been surgically
removed and irradiated. The immune stimulating, antiviral and
anti-proliferative properties of interferon-a-2a have been used for
both systemic and intralesional therapy. In one patient with post-
transplant KS immunosuppressive medication was switched from
ciclosporin to the m-TOR inhibitor sirolimus. Sirolimus has
immunosuppressive, anti-angiogenic and anti-neoplastic potential
[27]. Current studies confirm it’s positive effects on iatrogenic KS
[28]. Figure 8. HHV-8 staining of atypical endothelial cells. doi:10.1371/journal.pone.0018397.g008 We achieved complete responses in half of the patients. The rest
presented partial remissions or stable disease courses. No patient
died of KS. Due to the small number of cases we were not able to
demonstrate differences between treatments, neither were we able
to provide outcome diagnosis between different subtypes. Four
patients developed multiple local recurrences. Three of them had
primarily received a surgical therapy and one had primarily been
treated with interferon-a-2a. In summary this study analyzed the clinical manifestation,
treatment and outcome of all patients with non-AIDS associated
KS patients presenting to the Department of Dermatology,
University Hospital Tuebingen, between 1987 and 2009. In
addition to HHV-8 infection, individual factors like origin, age, sex
and immune status of the patient seem to have an impact on the
development of KS. Discussion Tumor stage KS: Close up view. doi:10.1371/journal.pone.0018397.g006 April 2011 | Volume 6 | Issue 4 | e18397 April 2011 | Volume 6 | Issue 4 | e18397 PLoS ONE | www.plosone.org 3 Non-AIDS Associated Kaposi’s Sarcoma Figure 7. D2-40 staining of arborizing vascular structures;
irregular lymphatic vessels. doi:10.1371/journal.pone.0018397.g007 autoimmune disorders may not be listed in this group. However
the increased risk of KS in iatrogenically immunosuppressed
patients is well documented [12–14]. One of our patients presented with a nodal peripheral T-cell
lymphoma, a hematological malignancy that possibly implicates
immunosuppression. Over fourfold significant increase of Kaposi’s
sarcoma in patients with lymphohematopoietic malignancies have
been reported in the literature [15]. Presumably immunodeficien-
cy of any kind; iatrogenic, malignant or HIV-induced is a
considerable factor in the development of KS. Clinically, non-AIDS KS mostly presents itself as multiple
bilateral cutaneous lesions of the lower limb [7]. We found the
lesions to be multiple in 100% of the cases, they were mostly
bilateral (70%) and the lower extremity was clearly the most
affected localization (95%). Only one patient with extracutaneous
lesions was identified. This is in line with the results of Hong and
Lee who compared characteristics of KS in HIV positive and
negative subjects [16]. Figure 7. D2-40 staining of arborizing vascular structures;
irregular lymphatic vessels. doi:10.1371/journal.pone.0018397.g007 KS can be seen as a systemic disease with mutilocular
occurrence of vascular tumors. Thus the therapeutic administra-
tion of KS differs essentially from the management of most other
neoplastic diseases. In comparison with other tumors KS therapy
comprises
growth
control
rather
than
elimination
without
presenting a palliative situation. A standard therapeutic guideline
does not exist as the therapeutic options have to be chosen
depending on subtype and stage of the disease as well as on the
immune status of the patient [17]. year [8]). Immigrants from those two countries also represent the
two largest ethnic minorities in Germany. Mean age at onset of our 20 patients was 66.6 years. Focusing
exclusively on classic KS an average age of 69.6 years was found,
which is comparable to more comprehensive investigations like the
Italian 870-cases study conducted by Dal Maso et al. showing a
mean age of 72 years [8]. Treatment modalities comprise local therapy for example
surgery, radiotherapy and local chemotherapy such as injections
of vinca alkaloids or local immune therapy by interferon,9 cis
retinoid acid or imiquimod [18–23]. April 2011 | Volume 6 | Issue 4 | e18397 Discussion In contrast to AIDS-associated KS, KS in
HIV negative patients appears less aggressive, mostly limited to the
skin and well-responsive on local or systemic therapeutic strategies. Figure 8. HHV-8 staining of atypical endothelial cells. doi:10.1371/journal.pone.0018397.g008 April 2011 | Volume 6 | Issue 4 | e18397 PLoS ONE | www.plosone.org April 2011 | Volume 6 | Issue 4 | e18397 4 Non-AIDS Associated Kaposi’s Sarcoma Table 1. Clinical features and treatment outcome. Patient
Age
Origin
Subtypea
Localizationb
Treatmentc
Resultd
Recurrence
P1
44
Italy
immune
lower legs
sirolimus
PR
-
P2
87
Germany
classic
lower legs
IFNa
SD
-
P3
76
Romania
classic
lower legs
Rx, IFNa
SD
-
P4
70
Germany
immune
lower legs
Rx, IFNa
CR
-
P5
59
Turkey
classic
extremities
excs. CR
multiple
P6
69
Italy
classic
lower leg
No need
-
-
P7
48
Egypt
immune
lower legs
excs. CR
multiple
P8
78
Germany
classic
extremities, mucosa, LN
Rx, excs. SD
multiple
P9
51
Turkey
classic
foot
excs. CR
-
P10
45
Italy
classic
foot
IFNa
PR
-
P11
89
Germany
classic
feet
Rx, excs. SD
-
P12
36
Turkey
immune
extremities
IFNa
CR
-
P13
69
Italy
classic
extremities
drop-out
-
-
P14
71
Germany
classic
extremities
IFNa
SD
-
P15
70
Italy
immune
feet
Rx
PR
-
P16
68
Germany
classic
feet
IFNa
CR
multiple
P17
62
Turkey
classic
feet
IFNa
CR
-
P18
70
Italy
classic
lower leg
IFNa, excs. CR
-
P19
79
Germany
classic
nose
excs. CR
-
P20
90
Germany
classic
lower legs
Rx
CR
- p
p
p
doi:10.1371/journal.pone.0018397.t001 Author Contributions Conceived and designed the experiments: CG LJ. Performed the
experiments: LJ. Analyzed the data: LJ KMC. Contributed reagents/
materials/analysis tools: GM CG. Wrote the paper: LJ CG KMC GM. Figure S1
Ethical vote for the study. References 1. Kaposi M (1872) Idiopathisches multiples Pigmentsarkom der Haut. Arch
Dermatol Syph 4: 265–272. 1. Kaposi M (1872) Idiopathisches multiples Pigmentsarkom der Haut. Arch
Dermatol Syph 4: 265–272. 14. Penn I (1979) Kaposi’s sarcoma in organ transplant recipients: report of 20 cases. Transplantation 27: 8–11. 2. Antman K, Chang Y (2000) Kaposi’s sarcoma. N Engl J Med 342: 1027–1038. 15. Royle JS, Baade P, Joske D, Fritschi L (2010) Risk of second cancer after
lymphohematopoietic neoplasm. Int J Cancer. 3. Chang Y, Moore PS (1996) Kaposi’s Sarcoma (KS)-associated herpesvirus and
its role in KS. Infect Agents Dis 5: 215–222. 16. Hong A, Lee CS (2002) Kaposi’s sarcoma: clinico-pathological analysis of
human immunodeficiency virus (HIV) and non-HIV associated cases. Pathol
Oncol Res 8: 31–35. 4. Ensoli B, Sgadari C, Barillari G, Sirianni MC, Sturzl M, et al. (2001) Biology of
Kaposi’s sarcoma. Eur J Cancer 37: 1251–1269. 5. Goh SG, Calonje E (2008) Cutaneous vascular tumours: an update. Histopathology 52: 661–673. 17. Vogt T, Brockmeyer N, Kutzner H, Schofer H (2008) Short German guidelines:
angiosarcoma and Kaposi sarcoma. J Dtsch Dermatol Ges 6 Suppl 1: S19–S24. 18. Trakatelli M, Katsanos G, Ulrich C, Kalabalikis D, Sotiriadis D, et al. (2010)
Efforts to counteract locally the effects of systemic immunosupression: a review
on the use of imiquimod, a topical immunostimulator in organ transplant
recipients. Int J Immunopathol Pharmacol 23: 387–396. 6. Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, et al. (2000)
New guidelines to evaluate the response to treatment in solid tumors. European
Organization for Research and Treatment of Cancer, National Cancer Institute
of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 92:
205–216. 19. Celestin Schartz NE, Chevret S, Paz C, Kerob D, Verola O, et al. (2008)
Imiquimod 5% cream for treatment of HIV-negative Kaposi’s sarcoma skin
lesions: A phase I to II, open-label trial in 17 patients. J Am Acad Dermatol 58:
585–591. 7. Iscovich J, Boffetta P, Franceschi S, Azizi E, Sarid R (2000) Classic kaposi
sarcoma: epidemiology and risk factors. Cancer 88: 500–517. 8. Dal ML, Polesel J, Ascoli V, Zambon P, Budroni M, et al. (2005) Classic
Kaposi’s sarcoma in Italy, 1985–1998. Br J Cancer 92: 188–193. . Dal ML, Polesel J, Ascoli V, Zambon P, Budroni M, et al. (20 20. FDA (1998) KS drug goes to FDA. Food and Drug Administration. GMHC
Treat Issues 12: 7. 28. Stallone G, Schena A, Infante B, Di PS, Loverre A, et al. (2005) Sirolimus for
Kaposi’s sarcoma in renal-transplant recipients. N Engl J Med 352: 1317–1323. Non-AIDS Associated Kaposi’s Sarcoma 27. Sehgal SN (2003) Sirolimus: its discovery, biological properties, and mechanism
of action. Transplant Proc 35: 7S–14S. 25. Brambilla L, Romanelli A, Bellinvia M, Ferrucci S, Vinci M, et al. (2008)
Weekly paclitaxel for advanced aggressive classic Kaposi sarcoma: experience in
17 cases. Br J Dermatol 158: 1339–1344. 26. Montagnino G, Bencini PL, Tarantino A, Caputo R, Ponticelli C (1994) Clinical
features and course of Kaposi’s sarcoma in kidney transplant patients: report of
13 cases. Am J Nephrol 14: 121–126. 25. Brambilla L, Romanelli A, Bellinvia M, Ferrucci S, Vinci M, et al. (2008)
Weekly paclitaxel for advanced aggressive classic Kaposi sarcoma: experience in
17 cases. Br J Dermatol 158: 1339–1344.
26. Montagnino G, Bencini PL, Tarantino A, Caputo R, Ponticelli C (1994) Clinical
features and course of Kaposi’s sarcoma in kidney transplant patients: report of
13 cases. Am J Nephrol 14: 121–126. 27. Sehgal SN (2003) Sirolimus: its discovery, biological properties, and mechanism
of action. Transplant Proc 35: 7S–14S.
28. Stallone G, Schena A, Infante B, Di PS, Loverre A, et al. (2005) Sirolimus for
Kaposi’s sarcoma in renal-transplant recipients. N Engl J Med 352: 1317–1323. PLoS ONE | www.plosone.org References Kaposi’s sarcoma in Italy, 1985–1998. Br J Cancer 92: 188–193. 9. Ronchese F, Kern AB (1953) Kaposi’s sarcoma (angioreticulomatosis). Postgrad
Med 14: 101–111. 21. Schwartz RA, Micali G, Nasca MR, Scuderi L (2008) Kaposi sarcoma: a
continuing conundrum. J Am Acad Dermatol 59: 179–206. 10. Grulich AE, Beral V, Swerdlow AJ (1992) Kaposi’s sarcoma in England and
Wales before the AIDS epidemic. Br J Cancer 66: 1135–1137. g
J
22. Szajerka T, Jablecki J (2007) Kaposi’s sarcoma revisited. AIDS Rev 9: 230–236. 11. Geddes M, Franceschi S, Barchielli A, Falcini F, Carli S, et al. (1994) Kaposi’s
sarcoma in Italy before and after the AIDS epidemic. Br J Cancer 69: 333–336. 23. Brambilla L, Bellinvia M, Tourlaki A, Scoppio B, Gaiani F, et al. (2010)
Intralesional vincristine as first-line therapy for nodular lesions in classic Kaposi
sarcoma: a prospective study in 151 patients. Br J Dermatol 162: 854–859. 12. Hoshaw RA, Schwartz RA (1980) Kaposi’s sarcoma after immunosuppressive
therapy with prednisone. Arch Dermatol 116: 1280–1282. sarcoma: a prospective study in 151 patients. Br J Dermatol 162: 8 24. Di LG, Kreuter A, Di TR, Guarini A, Romano C, et al. (2008) Activity and
safety of pegylated liposomal doxorubicin as first-line therapy in the treatment of
non-visceral classic Kaposi’s sarcoma: a multicenter study. J Invest Dermatol
128: 1578–1580. 13. Klepp O, Dahl O, Stenwig JT (1978) Association of Kaposi’s sarcoma and prior
immunosuppressive therapy: a 5-year material of Kaposi’s sarcoma in Norway. Cancer 42: 2626–2630. PLoS ONE | www.plosone.org April 2011 | Volume 6 | Issue 4 | e18397 April 2011 | Volume 6 | Issue 4 | e18397 5 PLoS ONE | www.plosone.org April 2011 | Volume 6 | Issue 4 | e18397 6
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Within-Canopy Experimental Leaf Warming Induces Photosynthetic Decline Instead of Acclimation in Two Northern Hardwood Species
|
Frontiers in forests and global change
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Within-canopy experimental leaf warming induces photosynthetic
Within-canopy experimental leaf warming induces photosynthetic
decline instead of acclimation in two northern hardwood species
decline instead of acclimation in two northern hardwood species
Kelsey R. Carter Kelsey R. Carter
Michigan Technological University Molly A. Cavaleri
Michigan Technological University Follow this and additional works at: https://digitalcommons.mtu.edu/forestry-fp
Part of the Forest Sciences Commons
Recommended Citation
Recommended Citation
Carter, K. R., & Cavaleri, M. A. (2018). Within-canopy experimental leaf warming induces photosynthetic
decline instead of acclimation in two northern hardwood species. Frontiers in Forests and Global Chang
1(11). http://dx.doi.org/10.3389/ffgc.2018.00011
Retrieved from: https://digitalcommons.mtu.edu/forestry-fp/64
Follow this and additional works at: https://digitalcommons.mtu.edu/forestry-fp
Part of the Forest Sciences Commons Follow this and additional works at: https://digitalcommons.mtu.edu/forestry-fp Part of the Forest Sciences Commons Part of the Forest Sciences Commons Edited by:
Kouki Hikosaka,
Tohoku University, Japan Reviewed by:
Mary A. Heskel,
Macalester College, United States
Joseph Ronald Stinziano,
University of New Mexico,
United States *Correspondence:
Kelsey R. Carter
kcarter@mtu.edu Specialty section:
This article was submitted to
Forest Ecophysiology,
a section of the journal
Frontiers in Forests and Global
Change
Received: 03 September 2018
Accepted: 03 December 2018
Published: 19 December 2018 Specialty section:
This article was submitted to
Forest Ecophysiology,
a section of the journal
Frontiers in Forests and Global
Change Received: 03 September 2018
Accepted: 03 December 2018
Published: 19 December 2018 Within-Canopy Experimental Leaf
Warming Induces Photosynthetic
Decline Instead of Acclimation in Two
Northern Hardwood Species Northern hardwood forests are experiencing higher temperatures and more extreme
heat waves, potentially altering plant physiological processes. We implemented in-situ
leaf-level warming along a vertical gradient within a mature forest canopy to investigate
photosynthetic acclimation potential of two northern hardwood species, Acer saccharum
and Tilia americana. After 7 days of +3◦C warming, photosynthetic acclimation was
assessed by measuring differences between heated and control photosynthetic rates
(Aopt) at leaf optimum temperatures (Topt). We also measured the effects of warming and
height on maximum rates of Rubisco carboxylation, stomatal conductance, transpiration,
and leaf traits: leaf area, leaf mass per area, leaf nitrogen, and leaf water content. We found no evidence of photosynthetic acclimation for either species, but rather
Aopt declined with warming overall. We found slight shifts in LMA and Narea, leaf
traits associated with photosynthetic capacity, after 1 week of experimental warming. T. americana LMA and Narea was lower in the upper canopy heated leaves than
in the control leaves, contributing a shift in Narea height distribution in the heated
leaves. T. americana showed evidence of greater resiliency to warming, with greater
thermoregulation, physiological plasticity, and evapotranspiration. As expected, Aopt of
A. saccharum increased with height, but Aopt of T. americana was highest in the sub
canopy, possibly due to constraints on leaf water balance and photosynthetic capacity
in the upper canopy. Thus, models relying on canopy height or light environment may
incorrectly estimate vertical variation of photosynthetic capacity. If these species are
not able to acclimate to warmer temperatures, we could see alteration of plant carbon
balance of these two key northern hardwood species. Keywords: Acer saccharum, canopy, experimental warming, leaf traits, photosynthesis, Tilia americana, thermal
acclimation Recommended Citation
Recommended Citation Carter, K. R., & Cavaleri, M. A. (2018). Within-canopy experimental leaf warming induces photosynthetic
decline instead of acclimation in two northern hardwood species. Frontiers in Forests and Global Change,
1(11). http://dx.doi.org/10.3389/ffgc.2018.00011
Retrieved from: https://digitalcommons.mtu.edu/forestry-fp/64 Follow this and additional works at: https://digitalcommons.mtu.edu/forestry-fp Part of the Forest Sciences Commons Part of the Forest Sciences Commons ORIGINAL RESEARCH
published: 19 December 2018
doi: 10.3389/ffgc.2018.00011 Citation: Carter KR and Cavaleri MA (2018)
Within-Canopy Experimental Leaf
Warming Induces Photosynthetic
Decline Instead of Acclimation in Two
Northern Hardwood Species. Front. For. Glob. Change 1:11. doi: 10.3389/ffgc.2018.00011 Heat waves are expected to become more frequent in the northern hemisphere compared to the
southern hemisphere with continued climate change (Meehl and Tebaldi, 2004; Gershunov et al.,
2009). Northern hardwood forests are already experiencing more extreme temperature events,
potentially altering both plant photosynthetic and respiratory capacities and reducing ecosystem
level primary productivity (Ciais et al., 2005; Bastos et al., 2014; Filewod and Thomas, 2014). December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org Northern Hardwood Experimental Warming Carter and Cavaleri In addition, there is evidence that upper canopy leaves in
mid-latitude ecosystems are currently operating near their
thermal thresholds (O’Sullivan et al., 2017; Mau et al., 2018),
and photosynthetic decline could be exacerbated in temperate
ecosystems due to their characteristically hot, dry summers. Both the overall elevated temperatures and increased number
of heat waves could also contribute to shifts or reductions
of species ranges (Thomas et al., 2004; Jump et al., 2006). Specifically, climate change is expected to impact the abundance
and distribution of northern hardwood key species, such as Acer
saccharum, Fagus grandifolia, and Tilia americana, with some
evidence predicting a decline or shift to higher latitudes (Iverson
et al., 2008; Tang and Beckage, 2010; Treyger and Nowak, 2011),
while other models predict the expansion of species, such as A. saccharum (Walker et al., 2002). Understanding how warmer
temperatures will affect northern hardwood species’ physiology
will give us a better understanding of how these forests will
respond to climate change. plants with higher rates of stomatal conductance tend to have
a higher capacity for temperature regulation due to higher
rates of evapotranspiration (Lu et al., 1998), which could, in
turn, allow for maintained rate of photosynthesis with higher
air temperatures (Michaletz et al., 2016) or prevent longer
term damage that can occur when temperatures exceed high
temperature thresholds (Urban et al., 2017; Drake et al., 2018). A plant’s ability to cope with stressors, either through
photosynthetic
acclimation
or
thermal
regulation,
can
vary between species and across canopy vertical gradients. Species with low leaf area, complex leaves, and high stomatal
conductance, and therefore high thermoregulation capacity,
can maximize carbon gain by preventing excessively high leaf
temperatures (Michaletz et al., 2016; Fauset et al., 2018). Citation: Species
that have high trait plasticity may hold a thermal regulation
advantage if they are able to adjust traits in their upper canopy
leaves. In addition to differences in thermal regulation across a
vertical gradient, leaves in the upper canopy may have a greater
ability to acclimate than leaves lower in the canopy. Leaves that
are exposed to one stressor (e.g., light, drought) have been shown
to have a higher plasticity to respond to another stressor (e.g.,
temperature) (Havaux, 1992; Niinemets et al., 1999). This has
important implications for canopy gradients, as upper canopy
leaves are often exposed to multiple stressors at a given time. p
g
Photosynthetic thermal acclimation could help alleviate
some of the negative impacts of supraoptimal temperatures. Photosynthesis
rates
generally
increase
with
increasing
measurement temperatures up to an optimum rate (Aopt),
after which rates will decline (Berry and Bjorkman, 1980). Declines in net photosynthesis above this optimum temperature
(Topt) occur as a result of several processes, including increased
thylakoid
membrane
permeability
(Bukhov
et
al.,
1999;
Zhang et al., 2009), Rubisco activase dysfunction (Wang and
Portis, 1992; Salvucci et al., 2001; Zhang et al., 2002), higher
rates of photorespiration (Ku and Edwards, 1978), stomatal
closure (Farquhar and Sharkey, 1982), and higher rates of
daytime respiration [reviewed in (Sage and Kubien, 2007)]. Photosynthetic acclimation occurs either through a shift in
Topt to a higher temperature, or through a greater capacity
to photosynthesize at optimum temperatures (i.e., higher
rates of Aopt) (Way and Yamori, 2014). Mechanisms involved in
photosynthetic thermal acclimation include increased membrane
stability through physical changes in the thylakoid membrane
structure (Huner, 1988; Havaux et al., 1996) and production of
more stable isoforms of Rubisco activase (Salvucci et al., 2001;
Portis, 2003). In addition to physiological acclimation through
positive shifts in Topt and/or Aopt, some plants can regulate
their leaf temperatures through convective cooling. Leaf traits,
such as leaf area and shape can influence leaf thermal regulation
(Vogel, 1970). Leaves with smaller areas and high complexity,
for example, can have lower boundary layer thickness, which can
lead to increased evaporative cooling (Gurevitch and Shuepp,
1990; Nicotra et al., 2008; Leigh et al., 2017). Abbreviations: Amax, light saturated net photosynthesis (µmol m−2 s−1); Aopt,
photosynthetic rate at the temperature optima (µmol m−2 s−1); Eleaf , leaf
evapotranspiration; gs, stomatal conductance (µmol m−2 s−1); LMA, leaf mass
per area (g cm−2); Narea, nitrogen per leaf area (g m−2); Nmass, nitrogen per
leaf mass (mg g−1); Tair, air temperature (◦C); Tleaf , leaf temperature (◦C);
TLeafMax, maximum leaf temperature (◦C); Topt, optimum temperature (◦C);
WUE, instantaneous water use efficiency (Amax/Eleaf ); WUEint, intrinsic water use
efficiency (Amax/gs); ˆV cmax, maximum rate of Rubisco carboxylation; VPD, vapor
pressure deficit (kPa); 1T, temperature difference between heated and control leaf
(◦C). Frontiers in Forests and Global Change | www.frontiersin.org Citation: In addition, Although northern hemisphere ecosystems are expected to
experience drastic changes in their temperature regimes, there
is convincing evidence that temperate forests will be able
to photosynthetically acclimate to moderate climate warming
(Gunderson et al., 2000, 2010; Cunningham and Read, 2002;
Turnbull et al., 2004; Way and Oren, 2010; Sendall et al., 2015b). In experiments lasting from 2 weeks to three full growing seasons,
seedlings and saplings in temperate hardwoods have been shown
to acclimate to moderate temperature increases (Gunderson
et al., 2000, 2010; Cunningham and Read, 2002; Sendall
et al., 2015b); however, photosynthetic acclimation in a mature
temperate hardwood forest canopy is yet to be experimentally
examined. Globally, acclimation most commonly occurs through
positive shifts in Topt and through upregulated photosynthesis
at the new growth temperature (Way and Yamori, 2014). The
modes of photosynthetic acclimation vary in temperate species,
with most studies finding a positive shift in Topt (Cunningham
and Read, 2002; Gunderson et al., 2010; Sendall et al., 2015b). More specifically, studies on A. saccharum seedlings have found
acclimation either through positive shifts in Topt (Sendall et al.,
2015b) or Aopt (Gunderson et al., 2000). Understanding within-
canopy physiological acclimation is particularly important within
the global carbon cycle, as upper canopy leaves have high
photosynthetic capacity (Carswell et al., 2000; Meir et al., 2002),
and can cycle a disproportional amount of carbon in closed-
canopy ecosystems (Ellsworth and Reich, 1993). Barriers to canopy warming studies include cost and energy
required for mature tree-scale warming; therefore, most in-situ
temperate ecosystem warming studies have focused on warming
the forest understory (de Frenne et al., 2010; Melillo et al., 2011;
Fu et al., 2013; Jarvi and Burton, 2013; also reviewed in Chung
et al., 2013; Marchin et al., 2016; Noh et al., 2016) or early
successional growth (Rollinson and Kaye, 2012; Rich et al., 2015). December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 2 Northern Hardwood Experimental Warming Carter and Cavaleri Environmental Information, 2018). Mean annual rainfall was
86.6 cm and mean annual snowfall is 564 cm. The soil is classified
as Michigamme coarse loam (NRCS Soil Survey Staff, 2018). Site
description can be found in Potvin and Lilleskov (2017). Fewer studies have looked at the effects of warming on temperate
forest canopies beyond the seedling developmental stage. Leaf Warming g
Individual leaves were heated using 100 watt, 120VAC silicon
heating pads (24100, Kat’s, Five Star Manufacturing Group Inc.,
Springfield, TN). Leaf temperature was monitored using 30 AWG
copper-constantan thermocouple wire [TT-T-30 SLE(ROHS)],
OMEGA Engineering, Inc., Norwalk, CT, USA) wired to a solid-
state thermocouple multiplexer (AM25T, Campbell Scientific
Inc., Logan, UT, USA) connected to a data logger (CR1000,
Campbell Scientific Inc.). Leaf thermocouples were adhered
on the abaxial side of the heated and control leaves using
breathable medical tape (Slot et al., 2016). Thermocouples were
extended using 24 AWG copper- constantan thermocouple
wire or 20 AWG for thermocouples that extended more
than 15 meters, to ensure that current resistance did not
exceed 100 (TT-T-20 and TT-T-24 OMEGA Engineering Inc.)
using thermocouple connectors (SMPW-CC-T-MF, OMEGA
Engineering Inc.). Heating pad temperature was controlled using
a 24-380VAC SSR-25 DA solid state relay module (SSR-25 DA,
Fotek Controls Co., Taiwan) wired into a digital output module
(SDM-CD 16D, Campbell Scientific Inc.). The heating pad turned
offwhen the heated leaf temperature reached more than 3◦C
above the control leaf temperature. A datalogger heating program
monitored leaf temperature every 15 s, and instantaneous leaf
temperatures were recorded every 2 min. The heating pads were
attached to a metal frame 7–12 cm below the leaf and secured to
the metal frame using metal mesh, which also prevented direct
exposure of thermocouple to the heating pad (Figure 1). Control
and heated leaves were selected to ensure that the heated leaf was Design g
Experimental warming was conducted on two species, T. americana and A. saccharum, at three canopy positions:
understory, sub canopy, and upper canopy (0–2, 6–8, and 12–
14 m). Canopy scaffolding (Contur Modular Scaffold, BilJax,
Archbold, OH, USA) enabled access to one A. saccharum tree
[12.5 m height, 15.6 cm diameter at breast height (DBH)] and
two T. americana trees (7.5 and 14 m height; 7.0 and 16.9 cm
DBH, respectively). T. americana sub and upper canopy warming
was conducted on separate individuals, while A. saccharum sub
and upper canopy warming was on the same individual tree. Understory measurements were conducted on three individual
saplings per species, ranging from 0.3 to 2.1 m height, located
adjacent to the canopy scaffolding. The upper canopy leaves
sampled were partially shaded late in the day by an adjacent 16 m
tall emergent tree. g
y
g
We had two primary study objectives: (1) to develop and
test a novel leaf warming device, and (2) to assess whether two
northern hardwood species, T. americana and A. saccharum,
could acclimate to 7 days of leaf-level +3◦C warming. While
short-term warming treatments may result in more conservative
acclimation responses than warming for longer time periods,
studies have found that temperate tree Topt can adjust to
seasonal temperature variations within 1–5 days (Gunderson
et al., 2010; Sendall et al., 2015b). In addition, Smith and Dukes
(2017) recently found photosynthetic biochemical acclimation
to experimental warming after 7 days. We hypothesized that (i)
both species would be able to photosynthetically acclimate to
warmer temperatures through shifts in both Topt and Aopt, (ii)
T. americana would have a higher resiliency, through higher
thermoregulation and higher trait plasticity, to warming in
the upper canopy leaves due to characteristic higher stomatal
conductance and lower leaf area (Thomas, 2010), and (iii) leaves
in the upper canopy for both species would have a higher capacity
to acclimate than leaves at lower heights. Three fully-developed leaves per species per height (18 total)
were heated day and night +3.0◦C above a nearby control leaf for
7 days. Understory and sub canopy warming was conducted from
July 14–21, 2016, and upper canopy warming was conducted
August 23–30, 2016. As a result of heater malfunction, one T. americana upper canopy leaf experienced a total of 19 fewer
hours of warming than the other heated leaves. Citation: Studies
have implemented warming on immature trees using warming
chambers (Gunderson et al., 2010), by pumping heated air
through tubes and into an immature tree canopy (Bauerle et al.,
2009), or through passive heating (Yamaguchi et al., 2016). Smaller scale within-canopy warming, either through branch
or leaf warming, is a practical method to investigate the plant
physiological effects of warming in forest canopies (Cavaleri et al.,
2015). Heated cables have been used to warm mature temperate
tree branches (Nakamura et al., 2010), and large, infrared heaters
have been implemented within canopies to warm branches and
leaves (Nakamura et al., 2016). As far as we are aware, there have
only been two examples of leaf-level warming in mature forests
canopies, both in tropical ecosystems (Doughty, 2011; Slot et al.,
2014). These studies used resistance wires covered in aluminum
foil (Doughty, 2011) or heat rope and infrared reflective frames
(Slot et al., 2014) to heat upper canopy leaves. Leaf-level warming
studies can give us important information on the physiological
responses of forest ecosystem upper canopies that are currently
unattainable through in-situ ecosystem-level warming studies. Study Site y
This study was conducted in a mature secondary growth
northern hardwood stand dominated by white ash (Fraxinus
americana L), sugar maple (Acer saccharum Marshall), basswood
(Tilia americana L), and northern red oak (Quercus rubra L),
located at the USDA Forest Service Northern Research Station,
Forestry Sciences Laboratory in Houghton, MI, USA (N47◦6′
52.884′′, W 88◦32′ 52.332′′). In 2013, the basal area was 27
m2 ha−1 and the stand density was 2,960 trees ha−1. Mean
stand height was 14 m, with a stand age from 50 to 60 years. A. saccharum comprised 10% of the stand basal area, while T. americana comprised 20% stand basal area. Site elevation is
243 m. The previous 30 years average monthly air temperature
ranged from −8.65 to 15.57◦C (NOAA National Centers for Frontiers in Forests and Global Change | www.frontiersin.org December 2018 | Volume 1 | Article 11 3 Northern Hardwood Experimental Warming Carter and Cavaleri temperatures above 33◦C, VPD often reached above 2 kPa. At
temperatures below 20◦C, VPD was often slightly below 1 kPa. FIGURE 1 | Example of a leaf heater positioned underneath an understory Tilia
americana leaf. To gain insight into mechanistic drivers of temperature
response and acclimation, we measured a variety of leaf traits
on all experimental leaves, including leaf mass per area (LMA),
leaf water content, leaf nitrogen on both area and mass bases
(Narea and Nmass, respectively), % leaf carbon (%C), maximum
rate of Rubisco carboxylation (Vcmax), stomatal conductance
(gs), and leaf evapotranspiration (Eleaf ). Stomatal conductance
and
evapotranspiration were
measured
concurrently
with
photosynthesis using the LI6400 (Li-COR Inc.). After completing
gas exchange measurements, sampled leaves were placed in a
sealed plastic bag in an ice cooler for no longer than 2 h, weighed
for fresh mass, and immediately placed in a −20◦C freezer. At
the conclusion of the experiment, frozen leaves were thawed
and measured for leaf area using a desktop scanner (HP Deskjet
4480) and ImageJ v1.50 image analysis software (Schneider et al.,
2012). Leaves were placed in a 60◦C drying oven for at least 72 h
for dry mass (g). LMA was obtained by dividing the dry mass
(g) by total leaf area (cm2). Leaf water content was calculated
by subtracting dry mass (g) from fresh mass (g), dividing by
fresh mass (g), and multiplying by 100 to calculate percent water
content (%). Study Site Dried leaves were ground to a fine powder in
a ball bearing grinder (8000 M Mixer/Mill, Spex Sample Prep,
Metuchen, NJ, USA) and analyzed for % carbon (C) and %
nitrogen (N) using a combustion analyzer (ESC 4010, Costech
Analytical Technologies Inc., Valencia, CA, USA). FIGURE 1 | Example of a leaf heater positioned underneath an understory Tilia
americana leaf. exposed to a similar ambient environment to the control leaf,
including height and shade. Leaf Scorching Due to an artifact of this leaf warming method, the temperature
difference between paired heated and control leaves (1T)
was often >3◦C before the digital output module turned the
heater off. Leaf scorching, defined as visible leaf necrosis, was
assessed on all heated leaves to account for possible damage to
photosynthetic apparatus caused by spikes in leaf temperature. Percent leaf area scorched was calculated on scanned leaf images
using ImageJ software. Environmental Monitoring Air temperature and relative humidity were monitored using
HOBO sensors (U23 Pro V2, Onset Corp, Bourne, MA, USA)
placed on the canopy access scaffolding at heights of 0.5, 6.25,
and 12.5 m. In July, the air temperature sensors were placed
on the southeast side of the scaffolding and were moved to the
northwest side of the tower in August. Air temperature and
relative humidity were also measured in an adjacent open field
(Vaisala temperature and relative humidity probe HMP50-L,
Campbell Scientific Inc.). Gas Exchange and Leaf Traits g
After
1
week
of
experimental
warming,
gas
exchange
(net
photosynthesis,
stomatal
conductance,
and
leaf
evapotranspiration) response to temperature was measured
on each individual heated and control leaf. Gas exchange
measurements were conducted using an open-system LI6400
infrared gas analyzer fitted with a 6400-88 expanded temperature
kit (Li-COR Inc., Lincoln, NE, USA). Photosynthetic response
to temperature was measured at nine temperatures (17, 20,
23, 25, 30, 33, 35, 37◦C); although we were unable to reach
37◦C for some measurements. Due to difficulties reaching a
low enough temperature to extract the parameter Topt, we
included an additional 15◦C temperature measurement to A. saccharum understory and sub canopy temperature curves. Based on photosynthetic light response curves measured prior
to leaf warming (data not shown), photosynthetic photon flux
density was controlled at 800 µmol m−2 s−1 for the understory
and sub canopy, and 1,200 µmol m−2 s−1 for the upper canopy
leaves. CO2 concentration was controlled at 400 ppm. Flow
was controlled between 200 and 500 µmol m−2 s−1 to keep the
vapor pressure deficit (VPD) between 1 and 2 kPa; although, at December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org Data Analysis Differences between Aopt, Topt = −b
2a
(2) (2) Aopt is extracted by setting Tleaf = Topt in Equation (1) and
solving for Amax. The polynomial regression model was not able
to capture Topt of 9 of the 18 A. saccharum curves. For these
curves, we took the temperature at the maximum Amax value
from each individual curve and treated this value as Topt. The
inability to fit positive polynomial curves to these data is likely
due to the very low response to temperature in A. saccharum
understory and sub canopy leaves (Figure 2A); which, can likely,
in part, be due to the very low stomatal conductance recorded in
these leaves (Figure S1A). To examine if the response of water use efficiency (WUE)
to temperature varied between treatments, species, and canopy
position, we calculated instantaneous water use efficiency
(WUE), calculated as Amax/Eleaf , and intrinsic water use
efficiency (WUEint), calculated as Amax/gs. WUE and WUEint
response to temperature was modeled using a mixed effects
model where Tleaf , species, treatment, and canopy position were
the fixed effects and individual tree was the random effect. WUE
slopes and intercepts were extracted and compared with post-
hoc contrasts using the FSA package (Ogle, 2018) in R Statistical
software (R Core Team, 2015). In order to explain any possible differences in photosynthetic
rates between heated and control leaves, stomatal conductance
at the photosynthetic optimum temperature (gs at Topt),
maximum
rate of
Rubisco
carboxylation
(Vcmax)
at the
photosynthetic optimum temperature, and equation parameters
of evapotranspiration (Eleaf ) response to temperature was
compared between treatment, species, and canopy position. gs
was modeled using Equation (1) and substituting gs for Amax
(Figures S1A,B). In the cases where Topt was extracted at the
maximum Amax value, gs at Topt was also taken as this leaf
temperature. To identify environmental differences between species and
across canopy vertical gradients, we investigated maximum and
mean leaf temperatures between species and maximum and mean
leaf and air temperatures along the canopy vertical gradient. Average and maximum leaf temperatures of control leaves were
compared for each canopy position and species using two-way
ANOVAs and post-hoc contrasts. Average and maximum air
temperatures were compared at each canopy position using a
one-way ANOVA and Tukey’s post-hoc mean separation. The
canopy air temperature and relative humidity sensors were
moved between the July and August sampling. Data Analysis Warming device efficacy was determined by examining the
average 1T across species, canopy position, sample month, and
time of day (daytime or nighttime). To assess the effect of
species and height on temperature spiking, heated leaf maximum
temperature (TLeafMax) and the frequency of time points where
heated leaves were 10◦C higher than control leaves (1T > 10◦C)
for each leaf pair were compared between species and canopy
positions using two-way ANOVAs and post-hoc contrasts. p
g
y
p
Photosynthetic acclimation is denoted by a positive shift in
Topt or an increase in the photosynthetic rate at Topt (Aopt). In order for a Topt shift to result in enhanced photosynthetic
performance, an upregulation of photosynthesis at the new
growth temperature must also occur. For example, a positive shift
in Topt could occur at the same time as decreased Aopt, which is
considered a detractive adjustment to photosynthesis, as opposed
to constructive adjustments that would occur with positive shifts
in Topt and Aopt (Way and Yamori, 2014). Topt and Aopt were
determined by fitting individual temperature response curves to December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 4 Northern Hardwood Experimental Warming Carter and Cavaleri regression equation where the leaf temperature was exponentially
transformed to each response curve (Figures 2C,D): regression equation where the leaf temperature was exponentially
transformed to each response curve (Figures 2C,D): the second order polynomial regression model (Cavieres et al.,
2000) (Figures 2A,B): Amax = aTleaf 2 + bTleaf + c
(1) (1) Eleaf = β0 + β1eTleaf
(4) Eleaf = β0 + β1eTleaf
(4) (4) where Amax is light saturated CO2 assimilation (µmol m−2 s−1)
at leaf measurement temperature (Tleaf ) (◦C). Topt is calculated
from the first derivative of the polynomial equation: where β0 is the intercept and β1 describes the exponential rise
in Eleaf with increasing temperature. Differences between Aopt,
Topt, ˆV cmax at Topt, gs at Topt, Eleaf (intercept, β0), and Eleaf
(exponential rise, β1) values between treatment, species, and
canopy position were compared using mixed effects models
that accounted for individual tree as the random effect and
species, treatment, and canopy position as the fixed effects. Mean
separation was compared using post-hoc contrasts. where β0 is the intercept and β1 describes the exponential rise
in Eleaf with increasing temperature. Data Analysis To account for
differences in canopy temperatures between sample months, we
used a Welch’s t-test to measure the difference between in July
and August using air temperature measurements collected in an
adjacent open field. Vcmax was calculated for each Amax value using the one point
method (De Kauwe et al., 2016a,b). The one-point method works
under the assumption that light saturated photosynthesis (Amax)
is limited by Rubisco carboxylation instead of RuBP regeneration,
or photosynthetic electron transport. The constants for the
apparent Vcmax ( ˆV cmax) were estimated based on Bernacchi
et al. (2001) estimation of Michalis constants for CO2 and
O2 temperature dependencies. The CO2 compensation point
was estimated from Crous et al. (2013). Because the one-point
method uses internal CO2 concentration to calculate ˆVcmax, we
removed all data points that had CO2 concentration <50 and
>500 ppm, which resulted in the removal of 12 out of 192 data
points (4.1% of the data). ˆV cmax was fitted to Tleaf using an
Arrhenius equation (Medlyn et al., 2002) (Figures S1 C,D): Leaf trait differences between treatments, species, and canopy
positions were analyzed to investigate possible drivers of
photosynthetic rates across canopy positions and photosynthetic
acclimation. Leaf traits (Nmass, Narea, leaf area, LMA, %C, and leaf
water content) were compared for differences across treatment,
species, and canopy position using a mixed effects model where
treatment, species, and canopy position were the fixed effects and
individual tree was the random effect and post-hoc contrasts. In
order to elucidate drivers of photosynthetic rates and leaf water
content, Aopt responses to leaf traits (LMA, Narea, and Nmass) and
leaf water content correlation with Eleaf (Intercept) were assessed
for differences in species and treatment using mixed effects
models with tree as the random effect. All statistical analyses
were performed using R Statistical Software (R Core Team, 2015). Mixed effects models were analyzed using the “nlme” package in
R (Pinheiro et al., 2018). f (Tk) = k25exp
Ea(Tk −298)
(298RTk)
(3) (3) Where Tk is the temperature in Kelvin, k25 is the rate of ˆV cmax at
25 ◦C, and Ea is the activation energy, or exponential rise, of the
ˆV cmaxto increasing temperature. ˆV cmax at Topt was calculated by
substituting k25 and Ea, estimated from individual temperature
response curves, and substituting Topt (K) into Equation (3). Frontiers in Forests and Global Change | www.frontiersin.org RESULTS Mean differences between heated and control leaves (1T) across
species, canopy position, sample month (July and August), and
daytime vs. nighttime ranged from 2.91 to 3.14◦C (Table S2). Temperatures of the warmed leaves were more variable than
controls (Figure 3A); however, average 1T values were close to
3◦C at all canopy positions (Table S2; Figure 3B). Data Analysis p
Evapotranspiration
(Eleaf )
parameters
were
estimated
for each individual Eleaf -Tleaf
response curve by fitting a December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 5 Northern Hardwood Experimental Warming Carter and Cavaleri FIGURE 2 | Photosynthetic (Amax) and leaf evapotranspiration (Eleaf) response to leaf temperature (Tleaf). Individual temperature response curves were fit to
polynomial equations for (A) A. saccharum and (B) T. americana photosynthetic response. Exponential transformations of leaf temperature were fit to the
evapotranspiration response for (C) A. saccharum and (D) T. americana. Dotted lines depict individual curves in the upper canopy, dashed lines depict curves in the
sub canopy, and solid lines represent understory temperature response curves. Control leaves are represented by blue lines while red lines represent heated leaves. FIGURE 2 | Photosynthetic (Amax) and leaf evapotranspiration (Eleaf) response to leaf temperature (Tleaf). Individual temperature response curves were fit to
polynomial equations for (A) A. saccharum and (B) T. americana photosynthetic response. Exponential transformations of leaf temperature were fit to the
evapotranspiration response for (C) A. saccharum and (D) T. americana. Dotted lines depict individual curves in the upper canopy, dashed lines depict curves in the
sub canopy, and solid lines represent understory temperature response curves. Control leaves are represented by blue lines while red lines represent heated leaves. Frontiers in Forests and Global Change | www.frontiersin.org Air Temperature, Leaf Temperature, and
Warming Device Performance Maximum and mean daily leaf and air temperatures were
consistent across all canopy heights for both of our study species. Neither daily maximum nor daily mean air temperatures differed
across canopy positions (Figures S2A,C; Table S1), and we found
no difference in mean daily Tair between canopy months when
measured in an adjacent open field [20.15 ± 1.31◦C for July,
19.56 ± 0.80◦C for August (mean ± SEM)] (Table S1). Mean
daily Tleaf of unheated foliage did not differ across canopy
position or between species (Figure S2B; Table S1). Control
leaf maximum daily temperatures (TLeafMax) showed an almost
significant species × canopy position interaction; however,
post-hoc mean separation found no significant differences
(Figure S2D; Table S1). Moderate temperature spiking did occur in all heated leaves,
and we found some evidence of leaf scorch, primarily in A. saccharum. The occurrences of temperature spiking differed
across canopy positions and between species. While A. saccharum
heated leaf TLeafMax was higher in the upper canopy than the
sub canopy and understory, T. americana TLeafMax did not vary
with canopy height (Figure 4A). There was no difference in A. saccharum and T. americana heated TLeafMax in the understory or
sub canopy, but A. saccharum heated TleafMax was higher than T. americana in the upper canopy by ∼9◦C. The mean % frequency
1T > 10◦C was <1.2% for all canopy positions for both species
(Figure 4B), and there were no effects of species, canopy position,
or their interaction (Table 1). Leaf scorching was found on 5
of the 18 total heated leaves: four A. saccharum and one T. The
leaf-level
warming
device
effectively
increased
temperatures of treated leaves +3◦C compared to paired
control leaves over 24 h for both species at all canopy positions. December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 6 Northern Hardwood Experimental Warming Carter and Cavaleri FIGURE 3 | Leaf heater performance. (A) Average of heated and control leaf temperatures (Tleaf) at three canopy positions (Acer saccharum and Tilia americana
combined) and (B) example of the variation in Tleaf for one heated and one control T. americana leaf over 24 h on August 25, 2016. Control Tleaf is depicted by the
black lines and heated Tleaf is depicted by gray lines. Upper canopy is represented by dotted lines, sub canopy is depicted by solid lines, and understory is depicted
by dashed lines. Air Temperature, Leaf Temperature, and
Warming Device Performance FIGURE 3 | Leaf heater performance. (A) Average of heated and control leaf temperatures (Tleaf) at three canopy positions (Acer saccharum and Tilia americana
combined) and (B) example of the variation in Tleaf for one heated and one control T. americana leaf over 24 h on August 25, 2016. Control Tleaf is depicted by the
black lines and heated Tleaf is depicted by gray lines. Upper canopy is represented by dotted lines, sub canopy is depicted by solid lines, and understory is depicted
by dashed lines. americana. A. saccharum showed some degree of scorching on
one leaf in the upper canopy (9% of leaf area scorched), two leaves
in the sub canopy (10% and 2%), and one leaf in the understory
(17%). Only one T. americana leaf, located in the understory,
exhibited scorching on 1% of its leaf area. canopy positions and species (Figures 6, 7). Neither the mixed
effects model nor post-hoc contrast detected any differences
between species or between canopy positions in the values of Topt
(Figure 6A; Table 2). A. saccharum upper canopy Aopt was twice
that of understory and sub canopy levels, while T. americana sub
and upper canopy Aopt values were more than double understory
Aopt (Figure 6B). Rates of Aopt of T. americana were greater than
rates of A. saccharum in the upper and sub canopy (Figure 6B). ˆV cmax at Topt was higher in T. americana than A. saccharum at
all canopy positions. T. americana sub canopy ˆV cmax was double
the rate of the understory and upper canopy and A. saccharum
ˆV cmax at Topt was consistent throughout all canopy positions
(Figure 6C). Stomatal conductance at optimum temperatures (gs
at Topt) showed similar patterns with species and canopy position
as did Aopt; however, there were no differences between species in
the understory and upper canopy (Figure 7A). A. saccharum Eleaf
intercept followed a similar pattern to A. saccharum gs at Topt,
where the upper canopy had higher rates of Eleaf compared to
the understory and sub canopy. T. americana Eleaf intercept was
highest in the sub canopy and the upper canopy Eleaf intercept
was higher than the understory (Figure 7B). Frontiers in Forests and Global Change | www.frontiersin.org Leaf Level Acclimation and Within-Canopy
Differences in Gas Exchange Parameters g
There was no evidence of photosynthetic acclimation for either
A. saccharum or T. americana after 1 week of experimental
warming, but there was evidence of overall reduced rates
of photosynthetic capacity in the warmed leaves, indicating
detractive adjustment with experimental warming. We found
no warming treatment effects on optimum temperature for
photosynthesis (Topt), stomatal conductance (gs) at Topt,
maximum rate of Rubisco carboxylation ( ˆV cmax) at Topt,
or leaf evapotranspiration (Eleaf ) parameters for either study
species (Table 2; Figure 5). We did, however, find a significant
overall treatment effect for rates of photosynthesis at optimum
temperatures, where warmed leaves showed slightly lower Aopt
than control leaves (p = 0.020; Table 2; Figure 5B). The mixed effects model comparing the effects of Tleaf ,
treatment, species, and canopy position on both instantaneous
water use efficiency (WUE, Amax/Eleaf ) and intrinsic water use
efficiency (WUEint, Amax/gs) showed interaction effects across
most variables (Table S3); however, post-hoc analyses primarily
distinguished differences between A. saccharum sub canopy
heated and control leaves (Figure S3). Overall, WUE and WUEint Optimum temperature was consistent throughout all canopy
positions, while patterns with height of both optimum rates of
photosynthesis, stomatal conductance, evapotranspiration, and
the rate of Rubisco carboxylation differed by species. There were
no significant treatment interactions for Topt, Aopt, ˆV cmax at Topt,
gs at Topt, or the intercept term of Eleaf (Table 2); therefore,
treatments were pooled and analyzed for differences between December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 7 Northern Hardwood Experimental Warming Carter and Cavaleri TABLE 1 | Two-way ANOVA p-value results of heated leaf temperature spiking. Max daily
heated Tleaf
% Frequency
1T > 10◦C
Species
0.002**
0.907
Canopy position
0.271
0.296
Species × Canopy position
<0.001***
0.447
Heated leaf daily maximum leaf temperature and percentage of occurrences where the
difference heated and control Tleaf was >10◦C (1T > 10◦C). p-value results of treatment,
canopy position, and the interaction effect of species and canopy position. **p < 0.01,
***p < 0.001. Understory and sub canopy daily maximum Tleaf n = 9, where n is the
number of days used to calculate the mean. A. saccharum upper canopy n = 8, T. americana upper canopy n = 10. FIGURE 4 | Summary of heated leaf temperature spiking. Leaf Traits ***Denotes results of contrast between
species at each canopy position, where p < 0.001. Overall, both LMA and Narea increased with canopy height
and had opposing treatment responses between A. saccharum
and T. americana, whereas, Nmass and %C had no treatment
effect and the height response differed between species. Canopy
position had a strong positive effect on both A. saccharum and
T. americana LMA and Narea (Figures 8A–D). LMA showed
evidence of a ∼22% decrease with warming, but only in the
upper canopy and only for T. americana (near significant 3-way
interaction; Table 3; Figure S4B), while A. saccharum showed
no treatment effect on LMA at any canopy position (Table 3;
Figure S4A). Both species showed Narea treatment effects in the
upper canopy only, but in opposite directions (Figures S4C,D),
a pattern which mirrored LMA (Figures S4A,B). A. saccharum
upper canopy Narea was greater than sub canopy or understory
for both heated and control leaves (Figures 8C,D). T. americana
control leaf Narea steadily increased from the understory to the
upper canopy (Figure 8C), while heated leaf Narea increased
from the understory to sub canopy but did not increase from
the sub to upper canopy (Figure 7D). This resulted in a
39% higher Narea for T. americana upper canopy control leaf
than the sub canopy leaf (p = <0.001, Figure 8B); however,
heated sub and upper canopy leaves did not differ in Narea
(Figure 8C). Heated upper canopy Narea was slightly greater
than control leaf Narea (14%, p = 0.019) in A. saccharum leaves
(Figure S4C). A. saccharum Nmass did not change with height,
while Nmass of T. americana was greatest in the sub canopy
(Figures 8E,F). T. americana Nmass was significantly greater
than A. saccharum at all canopy positions (Figures 8E,F). There were weakly correlated with Tleaf , and significant correlations
only occurred for some species, canopy position, and treatment
combinations (Table S4). The only significant A. saccharum
WUE vs. Tleaf correlation occurred in heated leaves located
in sub (WUE p = 0.016 and WUEint p <
0.001) and upper
(WUE p = 0.011) canopy, which all had decreasing slopes
(Table S4; Figures S3A,C). T. americana water use efficiency
tended to have a negative response to temperature, where only
the sub canopy heated leaf (both WUE and WUEint) and the
upper canopy heated leaf (WUEint only) did not decrease with
increasing temperature (Table S4; Figures S3B–D). Leaf Level Acclimation and Within-Canopy
Differences in Gas Exchange Parameters (A) Maximum daily
heated leaf temperature (Tleaf) and (B) percent frequency of time points where
the difference in control and associated heated leaf was >10◦C (1T > 10◦C)
for Acer saccharum (open circles) and Tilia americana (filled circles) at each
canopy position. Error bars denote SEM. Letters above and below error bars
denote results of post-hoc contrasts. Contrasts between canopy position of A. saccharum is represented by capital letters, T. americana contrasts are
represented by lower case letters. ***Denotes results of contrast between
species at each canopy position, where p < 0.001. TABLE 1 | Two-way ANOVA p-value results of heated leaf temperature spiking. leaf intercept was higher than the control leaf (Figure S3A). There were very few differences detected between species
(Table S3). Leaf Traits Experimental warming had no effect on leaf area or leaf
water content; however, both leaf traits were higher for T. americana than A. saccharum. A. saccharum leaf area did not
vary with height (Table 3; Figure 7C); however, leaf area in the T. americana sub canopy was ∼60% higher than in the understory
or upper canopy and was greater than that of A. saccharum in
both sub and upper canopy (Figure 7C). For both species, leaf
water content declined linearly with increasing canopy height,
and was greater for T. americana than A. saccharum at all canopy
positions (Table 3; Figure 7D). FIGURE 4 | Summary of heated leaf temperature spiking. (A) Maximum daily
heated leaf temperature (Tleaf) and (B) percent frequency of time points where
the difference in control and associated heated leaf was >10◦C (1T > 10◦C)
for Acer saccharum (open circles) and Tilia americana (filled circles) at each
canopy position. Error bars denote SEM. Letters above and below error bars
denote results of post-hoc contrasts. Contrasts between canopy position of A. saccharum is represented by capital letters, T. americana contrasts are
represented by lower case letters. ***Denotes results of contrast between
species at each canopy position, where p < 0.001. FIGURE 4 | Summary of heated leaf temperature spiking. (A) Maximum daily
heated leaf temperature (Tleaf) and (B) percent frequency of time points where
the difference in control and associated heated leaf was >10◦C (1T > 10◦C)
for Acer saccharum (open circles) and Tilia americana (filled circles) at each
canopy position. Error bars denote SEM. Letters above and below error bars
denote results of post-hoc contrasts. Contrasts between canopy position of A. saccharum is represented by capital letters, T. americana contrasts are
represented by lower case letters. ***Denotes results of contrast between
species at each canopy position, where p < 0.001. FIGURE 4 | Summary of heated leaf temperature spiking. (A) Maximum daily
heated leaf temperature (Tleaf) and (B) percent frequency of time points where
the difference in control and associated heated leaf was >10◦C (1T > 10◦C)
for Acer saccharum (open circles) and Tilia americana (filled circles) at each
canopy position. Error bars denote SEM. Letters above and below error bars
denote results of post-hoc contrasts. Contrasts between canopy position of A. saccharum is represented by capital letters, T. americana contrasts are
represented by lower case letters. Leaf Traits Treatment
0.489
0.020*
0.319
0.546
0.173
0.364
Species
0.244
<0.001***
0.012*
<0.001***
0.001**
0.364
Canopy position
0.573
<0.001***
<0.001***
0.001**
<0.001***
0.386
Treatment × Species
0.112
0.741
0.272
0.419
0.862
0.330
Treatment × Canopy Position
0.971
0.817
0.886
0.836
0.187
0.385
Species × Canopy position
0.858
0.001**
0.032*
0.006**
0.015
0.430
Treatment × Species × Canopy position
0.923
0.925
0.879
0.851
0.572
0.387
*Denotes significance at p < 0.05. ** Denotes significance at p < 0.01. *** Denotes significance at p < 0.001. The full names for the symbols are provided in the abbreviations list. n = 3
(n = 2 for A. saccharum heated understory and sub canopy ˆV cmax at Topt). FIGURE 5 | Optimum temperature for photosynthesis (Topt), the photosynthetic rate at Topt (Aopt), the maximum rate of Rubisco carboxylase ( ˆV cmax) at Topt, and
stomatal conductance (gs) at Topt for the heated and control leaves. (A) A. saccharum and T. americana Topt for control (filled) and heated (no fill) leaves, (B) Aopt for
both species, (C) ˆV cmax) at Topt for both species, and (D) gs at Topt for both species. Heated and control leaves are pooled across all canopy positions for each
species individually. Error bars denote SEM. *denotes results mixed effects model comparing treatment, species, and canopy position. *p < 0.05, ***p < 0.001. n = 18. FIGURE 5 | Optimum temperature for photosynthesis (Topt), the photosynthetic rate at Topt (Aopt), the maximum rate of Rubisco carboxylase ( ˆV cmax) at Topt, and
stomatal conductance (gs) at Topt for the heated and control leaves. (A) A. saccharum and T. americana Topt for control (filled) and heated (no fill) leaves, (B) Aopt for
both species, (C) ˆV cmax) at Topt for both species, and (D) gs at Topt for both species. Heated and control leaves are pooled across all canopy positions for each
species individually. Error bars denote SEM. *denotes results mixed effects model comparing treatment, species, and canopy position. *p < 0.05, ***p < 0.001. n = 18. species differences in the slope response of any gas exchange
parameters to any leaf traits; however, there were differences
in intercepts (Table S6). T. americana had a higher Aopt per
LMA intercept (4.03 µmol m−2 s−1) than A. saccharum (−0.938
µmol m−2 s−1, p =
0.003). A. saccharum had higher Aopt
per Nmass intercept (−1.94 µmol m−2 s−1) than T. Leaf Traits The mixed
effects model showed some treatment effects; however, post-
hoc analysis only distinguished treatment differences between
A. saccharum sub canopy (WUE slope p = 0.030, WUE
intercept p < 0.001, WUEint slope p =
0.004, and WUEint
intercept p < 0.001) and T. americana upper canopy WUE
intercept (p = 0.050) (upper case letters, Table S4). A. saccharum
sub canopy heated leaf had a more negative slope than
the control leaf, while T. americana upper canopy heated December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 8 Northern Hardwood Experimental Warming Carter and Cavaleri TABLE 2 | P-value results for mixed effect model comparing treatment, species, canopy position, and the interactions between all three variables, with individual tree as
the random effect, for Topt, Aopt, gs at Topt, and ˆV cmax at Topt. TABLE 2 | P-value results for mixed effect model comparing treatment, species, canopy position, and the interactions between all three variables, with individual tree as
the random effect, for Topt, Aopt, gs at Topt, and ˆV cmax at Topt. TABLE 2 | P value results for mixed effect model comparing treatment, species, canopy position, and the interactions between all three variables, with individual tree as
the random effect, for Topt, Aopt, gs at Topt, and ˆV cmax at Topt. Topt
Aopt
gs at Topt
Vcmax at Topt
Eleaf intercept (β0)
Eleaf exponential rise (β1)
Treatment
0.489
0.020*
0.319
0.546
0.173
0.364
Species
0.244
<0.001***
0.012*
<0.001***
0.001**
0.364
Canopy position
0.573
<0.001***
<0.001***
0.001**
<0.001***
0.386
Treatment × Species
0.112
0.741
0.272
0.419
0.862
0.330
Treatment × Canopy Position
0.971
0.817
0.886
0.836
0.187
0.385
Species × Canopy position
0.858
0.001**
0.032*
0.006**
0.015
0.430
Treatment × Species × Canopy position
0.923
0.925
0.879
0.851
0.572
0.387
*Denotes significance at p < 0.05. ** Denotes significance at p < 0.01. *** Denotes significance at p < 0.001. The full names for the symbols are provided in the abbreviations list. n = 3
(n = 2 for A. saccharum heated understory and sub canopy ˆV cmax at Topt). Frontiers in Forests and Global Change | www.frontiersin.org Leaf Traits FIGURE 6 | Optimum temperature for photosynthesis (Topt), the FIGURE 6 | Optimum temperature for photosynthesis (Topt), the
photosynthetic rate at Topt (Aopt), maximum rate of Rubisco carboxylation
( ˆV cmax) at Topt at each canopy position for each species. (A) Topt, (B) Aopt,
(C) ˆV cmax at Topt for Acer saccharum (open circles) and Tilia americana (filled
circles) leaves in thee understory, sub canopy, and upper canopy. Variables at
each canopy position includes both heated and control leaves because there
were no significant interactions present in the mixed effects model. Error bars
denote SEM. Letters above and below error bars denote results of post-hoc
contrasts. Contrasts between canopy position of A. saccharum is represented
by capital letters, T. americana contrasts are represented by lower case letters. ** and *** denote results of contrasts between species at each canopy
position. **p < 0.01, ***p < 0.001. n = 6 for Topt, Aopt, ˆV cmax at Topt (T. americana and A. saccharum upper canopy), gs at Topt, and Eleaf intercept. n = 5 for Vcmax at Topt understory and sub canopy. FIGURE 6 | Optimum temperature for photosynthesis (Topt), the
photosynthetic rate at Topt (Aopt), maximum rate of Rubisco carboxylation
( ˆV cmax) at Topt at each canopy position for each species. (A) Topt, (B) Aopt,
(C) ˆV cmax at Topt for Acer saccharum (open circles) and Tilia americana (filled
circles) leaves in thee understory, sub canopy, and upper canopy. Variables at
each canopy position includes both heated and control leaves because there
were no significant interactions present in the mixed effects model. Error bars
denote SEM. Letters above and below error bars denote results of post-hoc
contrasts. Contrasts between canopy position of A. saccharum is represented
by capital letters, T. americana contrasts are represented by lower case letters. ** and *** denote results of contrasts between species at each canopy
position. **p < 0.01, ***p < 0.001. n = 6 for Topt, Aopt, ˆV cmax at Topt (T. americana and A. saccharum upper canopy), gs at Topt, and Eleaf intercept. n = 5 for Vcmax at Topt understory and sub canopy. FIGURE 7 | Leaf water traits and fluxes for each species at different canopy
positions. (A) gs at Topt, (B) Eleaf intercept, (C) Leaf area, and (D) leaf water
content for Acer saccharum (open circles) and Tilia americana (filled circles) by
canopy position. Leaf Traits Leaf area and leaf water content for each canopy position
includes both heated and control leaves because there were no significant
interactions present in the mixed effects model. Error bars denote SEM. Letters above and below error bars denote results of post-hoc contrasts. Contrasts between canopy position of A. saccharum is represented by capital
letters, T. americana contrasts are represented by lower case letters. *denotes
results of contrast between species at each canopy position. *p < 0.05,
**p < 0.01, ***p < 0.001. n = 6. heated leaf had double the slope of the control leaf (p = 0.043,
Table S6). Leaf Traits americana
(−4.49 µmol m−2 s−1, p = 0.032); however, there were no
differences in Aopt per Narea between the two species (p = 0.485). T. americana also had a slightly higher leaf water content per
Eleaf (intercept) than A. saccharum (p = 0.026; Figure S5). The
only significant treatment interaction occurred for Aopt response
to Narea where post-hoc comparison showed that T. americana were no significant treatment effects or treatment interaction
effects found for Nmass (Table 3; Figures S4E,F). A. saccharum
had a higher %C than T. americana in the understory and
mid canopy control leaves, but not in the heated leaves. %C
tended to increase with canopy height for A. saccharum and
T. americana control leaves but was consistent throughout the
canopy in the heated leaves (Figures 8G,H). There were also
no significant treatment or treatment interaction effects for %C
(Table 3; Figures S4G,H). All photosynthetic leaf gas exchange parameters and leaf traits
(Aopt vs. LMA, Narea, and Nmass) were correlated, while leaf water
content and Eleaf intercept was not (Table S5). There were no December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 9 Northern Hardwood Experimental Warming Carter and Cavaleri FIGURE 6 | Optimum temperature for photosynthesis (Topt), the
photosynthetic rate at Topt (Aopt), maximum rate of Rubisco carboxylation
( ˆV cmax) at Topt at each canopy position for each species. (A) Topt, (B) Aopt,
(C) ˆV cmax at Topt for Acer saccharum (open circles) and Tilia americana (filled
circles) leaves in thee understory, sub canopy, and upper canopy. Variables at
each canopy position includes both heated and control leaves because there
were no significant interactions present in the mixed effects model. Error bars
denote SEM. Letters above and below error bars denote results of post-hoc
contrasts. Contrasts between canopy position of A. saccharum is represented
by capital letters, T. americana contrasts are represented by lower case letters. ** and *** denote results of contrasts between species at each canopy
position. **p < 0.01, ***p < 0.001. n = 6 for Topt, Aopt, ˆV cmax at Topt (T. americana and A. saccharum upper canopy), gs at Topt, and Eleaf intercept. n = 5 for Vcmax at Topt understory and sub canopy. Potential Resilience to Future Warming:
Comparing Species While there are disadvantages
to heating individual leaves instead of whole plants, leaf-level
warming studies can give us important information on the
physiological responses of forest ecosystem upper canopies
that are currently unattainable through in-situ ecosystem-level
warming studies. Physiological acclimation has also been found
to occur more readily in plant tissues that are not fully developed
(Turnbull et al., 1993); however, warming individual plant
tissues, instead of whole plants, introduces carbon sink-source
interactions that are difficult to parse from one another. Fully-
developed leaves lose their capacity to import carbohydrates
upon maturation (Turgeon, 2006). As such, mature leaves can
only use the carbohydrates they produce themselves for both
maintenance and growth. In contrast, developing leaves are able
to import carbohydrates from other parts of the plant. Therefore,
our leaves were treated as independent units with respect to
whole-tree source-sink dynamics, and by warming mature leaves
our results were not affected by changes in sink activity of our
experimental leaves. In addition to canopy gradient plasticity, our results could
have been confounded with ontogeny. All of our understory
measurements were conducted on saplings, while the canopy
warming was implemented on reproductively mature trees. Ontogeny could affect our results because trees that are fully
shaded might allocate their resources differently than canopy
trees with both sun and shade leaves. Both of our study species
have been found to have high photosynthetic capacities and leaf
area at intermediate size classes (Thomas, 2010). A. saccharum
leaf area and photosynthesis have been found to increase with
increasing plant size (Sendall et al., 2015a), a result which is
similar to what we found in our A. saccharum leaves. LMA
also tends to increase with increasing plant age, focusing leaf
construction toward longer-live leaves with high photosynthetic
capacity (Valladares and Niinemets, 2008). Particularly with
shade tolerant species, resource allocation differs as trees mature
and become less light limited (Sendall et al., 2015a). This suggests
that plant age, in combination with canopy position, could have
played a role in our results between the understory and canopy
trees. While leaf area could have been affected by ontogeny Instead of Aopt thermally acclimating to warmer temperatures,
we found evidence of photosynthetic decline in the heated
leaves (Figure 5B). Other warming studies conducted on A. Potential Resilience to Future Warming:
Comparing Species T. americana
showed
a
greater
resiliency
to
warming
compared to A. saccharum, possibly through more efficient
thermoregulation. Maximum temperatures of A. saccharum
heated leaves were higher in the upper canopy, while T. americana maximum temperatures were consistent between
canopy positions (Figure 4A). A. saccharum also had more leaf
scorching than T. americana. Lower leaf temperatures and less
evidence of leaf scorch suggests that T. americana may have
a greater thermoregulation ability than A. saccharum. This is
further supported by species differences in leaf area, stomatal
conductance at Topt, and evapotranspiration. Smaller leaves can
promote convective cooling by allowing higher transpiration
rates induced through a thinner boundary layer (Michaletz et al.,
2016; Fauset et al., 2018). T. americana leaf area is lower in the
upper canopy compared with the sub canopy, while A. saccharum
leaf area is consistent across all canopy positions (Figure 7C). Lower T. americana leaf area combined with high Eleaf could
allow higher thermoregulation (Figure 7B). These results are
similar to other studies, which have found that Tilia species have
high leaf morphological plasticity (Lichtenthaler et al., 2007;
Legner et al., 2014), leaf area that decreases with canopy height
(Koike et al., 2001), and high rates of stomatal conductance
(Thomas, 2010). In addition, while not significant, T. americana
had a trend of higher Topt and greater Topt plasticity, where Topt
declined with increasing canopy position. While canopy gradient
differences in Topt might be influenced by seasonality, we did
not find any differences in Tair between our sample months
(Table S1). This suggests that ambient temperature variation, or
seasonality, likely did not have a strong impact on within-canopy
temperature differences. It is also possible that thermal acclimation is more likely to
occur when experimental warming is applied to entire plants as
opposed to individual leaves or that acclimation is more likely
to occur when the heated treatment is applied to leaves that are
not fully developed. When warming individual leaves as opposed
to entire plants, there is a potential to miss important aspects of
plant physiological acclimation. For example, stomatal closure
is induced through the signaling of the hormone abscisic acid,
a reaction often induced by leaf importation of abscisic acid
produced in roots (Davies and Zhang, 1991; but not always
Sampaio Filho et al., 2018). This suggests a possibility that
warming of leaves might not induce the same amount of stomatal
closure as whole-plant warming. Photosynthetic Acclimation and
Responses to Warming 2017). In addition, our low sample size might have limited
our ability to detect statistical significance. Most warming
studies measure acclimation response after a set time that leaves
are exposed to warmer temperatures, instead of throughout
the warming experiment, making it difficult to pinpoint an
acclimation timeframe. Gunderson et al. (2000) showed that A. saccharum seedlings have the capacity to acclimate to +4◦C Contrary to our hypotheses, neither study species showed
evidence of photosynthetic acclimation (either an increase in
Topt or Aopt) at any canopy position. It is possible that 1 week
of warming was not enough time to allow for acclimation;
although, photosynthetic acclimation has been found to occur
after 1 week of experimental warming (e.g., Smith and Dukes, December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 10 Northern Hardwood Experimental Warming Carter and Cavaleri plant-scale carbon gain. If CO2 release through respiration does
not equally acclimate to the declines in photosynthesis, plant
carbon balance could be negatively affected (Drake et al., 2016). warmer temperatures through a shift in Aopt. While we did
not see any positive shifts in Aopt or Topt with our experiment
(Figure 5), a study conducted on taller trees during a different
growing season that was located about 72 kilometers away from
our study site (that experienced maximum daily temperatures
similar to our study) found A. saccharum Topt to be much higher
than the leaves in our study site (∼27◦C at 12.5 m, compared to
our average of 20.2◦C) (Mau et al., 2018), suggesting that higher
Topt can be found within this species. December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org Drivers of Within-Canopy Photosynthetic
Rates Photosynthetic optimization with height differed between the
two species, and this optimization was likely driven by different
leaf traits. A. saccharum LMA and control leaf Narea were highest
in the upper canopy leaves (Figures 8A–D). High LMA in the
upper canopy is a common trend in canopy gradients and is
due to higher leaf thickness and/or density which can maximize
photosynthetic capacity in high light environment (Niinemets,
1999; Zhang et al., 2011; Coble et al., 2014). Our results are
supported by other studies which have found that A. saccharum
LMA does increase with height (Ellsworth and Reich, 1993; Coble
et al., 2014; Filewod and Thomas, 2014). This is consistent with
other studies where Narea of leaves exposed to high irradiance
is predictive of photosynthetic capacity (Meir et al., 2002), and
agrees with our results which found positive correlations between
Narea and Aopt for both of our study species (Figure S5B). A. saccharum Nmass is distributed evenly throughout the understory
and canopy (Figures 7E,F), a result often found in A. saccharum
canopies (Ellsworth and Reich, 1993; Niinemets and Tenhunen,
1997; Coble and Cavaleri, 2015). T. americana Aopt was higher
than A. saccharum in the sub and upper canopy (Figure 6B),
which can likely be attributed to high Narea, leading to a capacity
for high rates of Rubisco carboxylation ( ˆV cmax), in T. americana
leaves (Figures 6C, 8C,D). This is further supported by the
higher Aopt per Narea found in T. americana sub and upper
canopy leaves (Figure S5B). In contrast to A. saccharum, T. americana photosynthetic rates and Nmass were maximized in
the sub canopy (Figures 6B, 8E,F). In shade tolerant species,
leaf nitrogen is prioritized to more shaded leaves to maximize
the light harvesting capacity (Niinemets, 1997; Schoettle and
Smith, 1999; Koike et al., 2001). Nitrogen is a major component In addition to differences in nitrogen optimization, the two
species also had different patterns of water-associated leaf traits at
different canopy positions. T. americana photosynthesis could be
limited in the upper canopy due to hydraulic constraints, as well
as stomatal and mesophyll conductance restrictions that occur
with increasing canopy height (Niinemets and Tenhunen, 1997;
Bond et al., 1999; Ryan et al., 2006; Duursma and Medlyn, 2012;
Buckley et al., 2013). Lowered mesophyll conductance can limit
Amax through decreased CO2 diffusion through cells and through
alterations in intercellular membrane structure due to tissue
shrinkage (Lawlor and Tezara, 2009). Potential Resilience to Future Warming:
Comparing Species saccharum have found evidence of photosynthetic decline in both
seedlings (Filewod and Thomas, 2014) and saplings (Gunderson
et al., 2000), even in some cases where the trees show positive
Topt acclimation (Gunderson et al., 2000). In addition, the
only other leaf-level canopy warming experiment to study
photosynthesis also found evidence of photosynthetic decline
(Doughty, 2011). In our study, the decline in Aopt is likely due
to a decline in the functioning of photosynthetic machinery
because we did not detect a decline in either ˆV cmax or gs with
experimental warming (Table 2; Figures 5C,D). Leaf scorching
discovered in our heated leaves shows further evidence of
potential photosynthetic apparatus damage. While our study only
investigated photosynthetic acclimation and responses at the leaf
level, declines in Aopt have important implications for larger, December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 11 Northern Hardwood Experimental Warming Carter and Cavaleri el comparing treatment, species, canopy position, and the interactions between all three variables, with individual tree as
eaf area, and leaf water content. TABLE 3 | P-value results for mixed effect model comparing treatment, species, canopy position, and the interactions between all three variables, with individual tree as
the random effect, for LMA, Narea, Nmass, %C, leaf area, and leaf water content. TABLE 3 | P-value results for mixed effect model comparing treatment, species, canopy position, and the interactions between all three variables, with individual tree as
the random effect, for LMA, Narea, Nmass, %C, leaf area, and leaf water content. TABLE 3 | P-value results for mixed effect model comparing treatment, species, canopy position, and the interactions between all three variables, with individual tree as
the random effect, for LMA, Narea, Nmass, %C, leaf area, and leaf water content. LMA
Narea
Nmass
%C
Leaf area
Leaf water content
Treatment
0.516
0.007**
0.336
0.806
0.676
0.420
Species
0.348
<0.001***
<0.001***
0.011*
0.024*
0.001**
Canopy position
<0.001***
<0.001***
<0.001***
0.004**
0.048*
0.001**
Treatment × Species
0.366
0.001**
0.210
0.684
0.301
0.301
Treatment × Canopy position
0.695
0.015*
0.784
0.730
0.386
0.622
Species × Canopy Position
0.722
<0.001***
0.002**
0.548
0.136
0.899
Treatment × Species × Canopy position
0.062
<0.001***
0.444
0.330
0.972
0.113
*Denotes significance at p < 0.05. **Denotes significance at p < 0.01. ***Denotes significance at p < 0.001. The full names for the symbols are provided in the abbreviations list. n = 3. Potential Resilience to Future Warming:
Comparing Species of chlorophyll and photosynthetic enzymes, and the high Nmass
found in the sub canopy likely contributed to high photosynthetic
rates in the sub canopy (Evans, 1989) (Figure S5C). and light availability, this is an interaction that occurs in most
forest ecosystems, as trees existing in the overstory would be
unlikely to develop branches within the understory. In addition,
while there is evidence that suggests differing thermoregulation
between T. americana canopy positions, it should be noted that
T. americana upper canopy leaf area is higher than A. saccharum,
and TLeafMax does not differ between canopy positions for A. saccharum control leaves (Figure S2D). A. saccharum upper canopy heated leaf Narea was higher
than the control leaves, while T. americana had lower Narea
in the upper canopy heated leaves. There were no treatment
differences for T. americana Nmass (Table 3); therefore, this
pattern was largely driven by a reduction in LMA in the upper
canopy heated leaves (Figure 8B). Lowered Narea and LMA with
warming suggests the possibility of lowered substrate availability
in T. americana upper canopy leaves. While we did not measure
respiration, declined LMA in T. americana upper canopy leaves
may be attributed to higher rates of respiration in the heated
leaves. If respiration was higher in the heated leaves, non-
structural carbohydrates could have been used more quickly,
leading to lower leaf mass; although, we had high variability in
T. americana upper canopy heated leaf %C and did not find
differences between treatments in the upper canopy (Figure 8H). A. saccharum had the opposite trend of Narea in the upper
canopy leaves, where Narea was higher in the control leaves
compared to the heated leaves (Figures S4C). Shifts in Narea
suggest possible acclimation of other physiological processes,
such as respiration, which is closely associated with leaf nitrogen
(Turnbull et al., 2003) and has been found occur in A. saccharum
(Gunderson et al., 2000; Reich et al., 2016). Other studies
have found little evidence of Narea acclimation to experimental
warming in temperate trees (Sendall et al., 2015b; Scafaro et al.,
2017; Sharwood et al., 2017); however, a tropical leaf warming
study found that LMA and Narea can increase within 1 week of
experimental warming (Slot et al., 2014). Frontiers in Forests and Global Change | www.frontiersin.org Drivers of Within-Canopy Photosynthetic
Rates Hydraulic restrictions on
gs can limit Amax by decreasing intercellular CO2 concentrations, Frontiers in Forests and Global Change | www.frontiersin.org December 2018 | Volume 1 | Article 11 12 Northern Hardwood Experimental Warming Carter and Cavaleri Heated and control leaf functional traits at different canopy positions. (A) Control leaf mass per area (LMA) of Acer saccharum (open circles) and Tilia
osed circles) leaves by canopy position, (B) heated LMA, (C) control nitrogen per area (Narea), (D) heated Narea, (E) control nitrogen per mass (Nmass),
mass, (G) control % carbon, (H) heated % carbon. Error bars denote SEM. Letters above and below error bars denote results of post-hoc contrasts. e results of post-hoc contrasts between canopy position of A. saccharum is represented by capital letters, T. americana contrasts are represented by
tters. *denotes results of contrast between species at each canopy position. *p < 0.05, ***p < 0.001. n = 3. FIGURE 8 | Heated and control leaf functional traits at different canopy positions. (A) Control leaf mass per area (LMA) of Acer saccharum (open circles) and Tilia
americana (closed circles) leaves by canopy position, (B) heated LMA, (C) control nitrogen per area (Narea), (D) heated Narea, (E) control nitrogen per mass (Nmass),
(F) heated Nmass, (G) control % carbon, (H) heated % carbon. Error bars denote SEM. Letters above and below error bars denote results of post-hoc contrasts. Letters denote results of post-hoc contrasts between canopy position of A. saccharum is represented by capital letters, T. americana contrasts are represented by
lower case letters. *denotes results of contrast between species at each canopy position. *p < 0.05, ***p < 0.001. n = 3. thereby limiting CO2 fixation in the Calvin cycle (Farquhar and
Sharkey, 1982). T. americana gs at Topt and leaf water content
were high in the sub canopy (Figures 7A,D); however, leaf
water content was low in the upper canopy, which could have limited upper canopy photosynthesis. T. americana also had a
more negative WUE relationship with temperature compared to
A. saccharum, which suggests that this species might maintain
higher rates of Eleaf even at a detriment to short-term carbon December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 13 Northern Hardwood Experimental Warming Carter and Cavaleri FUNDING Funding for this project was provided by the National Institute of
Food and Agriculture U.S. Department of Agriculture McIntire-
Stennis Cooperative Forestry Research Program Grant #1001534
and Department of Energy award DE-SC-0011806. Funding
was also provided by the DeVlieg Foundation Fellowship
and Ecosystem Science Center at Michigan Technological
University. Leaf Heating Device Performance Plant acclimation to one type of stress can improve
protection from other stressors (Havaux, 1992). Niinemets et al. (1999) found that electron transport in temperate tree leaves
acclimated to high light environments is more stable under high
temperature conditions. Upper canopy leaves are acclimated to
high light conditions, possibly inducing stress acclimation in the
upper canopy leaves in our study. The single instance of leaf
scorching in the upper canopy occurred in A. saccharum, where
the heated TleafMax was 43.5◦C. This maximum leaf temperature
was at least 5◦C higher than maximum temperatures found at
all canopy positions for both study species (Figure 4). High
temperatures experienced by A. saccharum upper canopy leaves
could have contributed to leaf scorching. • Our study supports our hypothesis that T. americana
will likely have greater resiliency to climate warming
due to a higher thermoregulation ability and higher trait
plasticity between canopy positions of traits associated
thermoregulation. • Canopy
position
photosynthetic
optimization
differed
between our study species and these differences can, in
part, be explained by the species’ leaf traits. Higher rates of
photosynthesis in A. saccharum upper canopy leaves can be
attributed to higher Narea and LMA. Higher T. americana
photosynthetic rates in the sub canopy can be linked to high
Nmass and gs in the sub canopy leaves, as well as hydraulic
limitations on leaf mesophyll experienced by the upper canopy
leaves. Our results suggest that models that predict canopy
photosynthesis based on canopy height or leaf traits, such as
LMA, may incorrectly estimate photosynthesis for species that
do not optimize photosynthesis in their upper canopy. AUTHOR CONTRIBUTIONS KC and MC designed the experiment and wrote the manuscript. KC collected data, performed the data analysis, and drafted the
manuscript. g
In addition, A. saccharum leaves were slightly more affected
by scorching than T. americana. The higher scorch damage
in A. saccharum could have occurred for several reasons. As mentioned previously, higher Eleaf might have allowed
greater thermoregulation in T. americana leaves (Figure 7B). T. americana also had higher leaf water content (Figure 7D), which
might lead to higher tolerance to heat damage. Additionally,
while both species are shade tolerant, T. americana is considered
less so than A. saccharum (Crow, 1990; Baltzer and Thomas,
2007; Thomas, 2010). Species with higher shade tolerance have
an overall lower plasticity to be able to adapt to high stress
environments encountered in upper canopies (Reich et al., 2003). This suggests that A. saccharum could be less tolerant of the
temperature fluctuations associated with the heating device. Another possibility for higher leaf scorching in A. saccharum is an
artifact of the leaf heating device. T. americana petioles position
their leaves so that they are relatively parallel to the ground
compared to A. saccharum leaves. This makes T. americana easier
to consistently heat over the entire surface of the leaf, likely
preventing leaf scorch along leaf margins. Leaf Heating Device Performance Leaf Heating Device Performance
Overall, the novel heating device worked well for both study
species. The leaves were heated successfully +3 ± 0.14◦C above
ambient leaf temperature across canopy positions, times of day,
and sample months (Table S2). Higher heating device efficacy in
the understory may be because the understory leaves were less
exposed to temperature fluctuations due to direct radiation and
sun flecks, allowing fewer spikes in temperature and, therefore,
more consistent heating. While the heaters performed well, there
was evidence of scorch damage to some of the heated leaves, and
slightly more damage in the understory and sub canopy than
the upper canopy. This suggests that shaded leaves may be more
susceptible to damage at supraoptimal temperatures than upper
canopy leaves. Leaves located higher in the canopy are exposed
to more severe environments; i.e., high irradiance, temperatures,
and wind. Plant acclimation to one type of stress can improve
protection from other stressors (Havaux, 1992). Niinemets et al. (1999) found that electron transport in temperate tree leaves
acclimated to high light environments is more stable under high
temperature conditions. Upper canopy leaves are acclimated to
high light conditions, possibly inducing stress acclimation in the
upper canopy leaves in our study. The single instance of leaf
scorching in the upper canopy occurred in A. saccharum, where
the heated TleafMax was 43.5◦C. This maximum leaf temperature
was at least 5◦C higher than maximum temperatures found at
all canopy positions for both study species (Figure 4). High
temperatures experienced by A. saccharum upper canopy leaves
could have contributed to leaf scorching. Leaf Heating Device Performance
Overall, the novel heating device worked well for both study
species. The leaves were heated successfully +3 ± 0.14◦C above
ambient leaf temperature across canopy positions, times of day,
and sample months (Table S2). Higher heating device efficacy in
the understory may be because the understory leaves were less
exposed to temperature fluctuations due to direct radiation and
sun flecks, allowing fewer spikes in temperature and, therefore,
more consistent heating. While the heaters performed well, there
was evidence of scorch damage to some of the heated leaves, and
slightly more damage in the understory and sub canopy than
the upper canopy. This suggests that shaded leaves may be more
susceptible to damage at supraoptimal temperatures than upper
canopy leaves. Leaves located higher in the canopy are exposed
to more severe environments; i.e., high irradiance, temperatures,
and wind. Conclusions gain (Table S4). Stomatal conductance was high in A. saccharum
upper canopy leaves (Figure 7A), likely contributing to high
rates of photosynthesis in the upper canopy. While leaf water
content was lower in the upper canopy than in the understory, A. saccharum photosynthetic rates was highest in the upper canopy
(Figure 6B). This suggests that leaf water is not a limiting factor
for A. saccharum upper canop leaves. • We demonstrated that our novel leaf warming device
successfully heated individual leaves 3.02 ± 0.01◦C above
control leaf temperatures; however, there was evidence of leaf
scorching, suggesting that there is room for improvement with
this method. A simple improvement is to select individual
leaves that are positioned parallel to the ground, which helps
keep the heater in the correct position below the leaf. • We demonstrated that our novel leaf warming device
successfully heated individual leaves 3.02 ± 0.01◦C above
control leaf temperatures; however, there was evidence of leaf
scorching, suggesting that there is room for improvement with
this method. A simple improvement is to select individual
leaves that are positioned parallel to the ground, which helps
keep the heater in the correct position below the leaf. • Our results showed that our two study species were not able
to photosynthetically acclimate to 1 week of leaf warming;
instead, we found declines in photosynthesis. If neither of
these species are able to acclimate to longer-term elevated
temperatures, we could see a decline in CO2 sequestration in
northern hardwood ecosystems. Frontiers in Forests and Global Change | www.frontiersin.org REFERENCES J., Reich, P. B., Barton, C. V., Medlyn,
B. E., et al. (2016). Does physiological acclimation to climate warming stabilize
the ratio of canopy respiration to photosynthesis? New Phytol. 211, 850–863. doi: 10.1111/nph.13978 y
g
g
Buckley, T. N., Cescatti, A., and Farquhar, G. D. (2013). What does optimization
theory actually predict about crown profiles of photosynthetic capacity when
models incorporate greater realism? Plant Cell Environ. 36, 1547–1563. doi: 10.1111/pce.12091 Drake, J. E., Tjoelker, M. G., Vårhammar, A., Medlyn, B. E., Reich, P. B., Leigh, A.,
et al. (2018). Trees tolerate an extreme heatwave via sustained transpirational
cooling and increased leaf thermal tolerance. Glob. Chang. Biol. 24, 2390–2402. doi: 10.1111/gcb.14037 Bukhov, N. G., Wiese, C., Neimanis, S., and Heber, U. (1999). Heat sensitivity
of chloroplasts and leaves: Leakage of protons from thylakoids and
reversible activation of cyclic electron transport. Photosynth. Res. 59, 81–93. doi: 10.1023/A:1006149317411 Duursma, R. A., and Medlyn, B. E. (2012). MAESPA: A model to study interactions
between water limitation, environmental drivers and vegetation function at tree
and stand levels, with an example application to [CO2] drought interactions. Geosci. Model Dev. 5, 919–940. doi: 10.5194/gmd-5-919-2012 Carswell, F. E., Meir, P., Wandelli, E. V., Bonates, L. C., Kruijt, B., Barbosa, E. M.,
et al. (2000). Photosynthetic capacity in a central Amazonian rain forest. Tree
Physiol. 20, 179–186. doi: 10.1093/treephys/20.3.179 y
p y
Carter, K. R. (2017). Effects of in-situ Leaf-Level Canopy Warming in a Northern
Hardwood Forest. Master’s thesis. Houghton, MI: Michigan Technological
University. Ellsworth, D. S., and Reich, P. B. (1993). Canopy structure and vertical patterns
of photosynthesis and related leaf traits in a deciduous forest. Oecologia 96,
169–178. Available online at: http://www.jstor.org/stable/4220518%5Cnhttp://
about.jstor.org/terms Cavaleri, M. A., Reed, S. C., Smith, W. K., and Wood, T. E. (2015). Urgent need
for warming experiments in tropical forests. Glob. Chang. Biol. 21, 2111–2121. doi: 10.1111/gcb.12860 Evans, J. R. (1989). Photosynthesis and nitrogen relationships in leaves of C3
plants. Oecologia 78, 9–19. Farquhar, G. D., and Sharkey, T. D. (1982). Stomatal conductance and
photosynthesis. Annu. Rev. Plant Physiol. 33, 317–345. Cavieres, L. A., Rada, F., Azócar, A., García-Núñez, C., and Cabrera, H. M. (2000). Gas exchange and low temperature resistance in two tropical high
mountain tree species from the Venezuelan Andes. Acta Oecol. 3, 203–211. doi: 10.1016/S1146-609X(00)01077-8 Fauset, S., Freitas, H. C., Galbraith, D. R., Sullivan, M. J. P., Aidar, M. P. M., Joly, C. A., et al. (2018). REFERENCES Crow, T. R. (1990). “Tilia americana L. American basswood,” in Techinical Coord. Silvics North Am. Vol. 2. Hardwoods. Agric. Handb. 654, eds R. M. Burn, B. and
H. Hondala (Washington, DC: U.S. Departent Agric. For. Serv.), 784–791. Baltzer, J. L., and Thomas, S. C. (2007). Physiological and morphological correlates
of whole-plant light compensation point in temperate deciduous tree seedlings. Oecologia 153, 209–223. doi: 10.1007/s00442-007-0722-2 Cunningham, S., and Read, J. (2002). Comparison of temperate and tropical
rainforest tree species: photosynthetic responses to growth temperature. Oecologia 133, 112–119. doi: 10.1007/s00442-002-1034-1 Bastos, A., Gouveia, C. M., Trigo, R. M., and Running, S. W. (2014). Analysing the spatio-temporal impacts of the 2003 and 2010 extreme
heatwaves on plant productivity in Europe. Biogeosciences 11, 3421–3435. doi: 10.5194/bg-11-3421-2014 Davies, W., and Zhang, J. (1991). Drying soil regulation of growth. Annu. Rev. Plant Physiol. 42, 55–76. doi: 10.1146/annurev.pp.42.060191.000415 de Frenne, P., de Schrijver, A., Graae, B. J., Gruwez, R., Tack, W., Vandelook,
F., et al. (2010). The use of open-top chambers in forests for evaluating
warming effects on herbaceous understorey plants. Ecol. Res. 25, 163–171. doi: 10.1007/s11284-009-0640-3 Bauerle, W. L., Bowden, J. D., Wang, G. G., and Shahba, M. A. (2009). Exploring
the importance of within-canopy spatial temperature variation on transpiration
predictions. J. Exp. Bot. 60, 3665–3676. doi: 10.1093/jxb/erp206 Bernacchi, C. J., Singsaas, E. L., Pimentel, C., Portis, A. R., and Long,
S. P. (2001). Improved
temperature
response
functions
for
models
of
Rubisco-limited
photosynthesis. Plant
Cell
Environ. 24,
253–259. doi: 10.1046/j.1365-3040.2001.00668.x De Kauwe, M. G., Lin, Y. S., Wright, I. J., Medlyn, B. E., Crous, K. Y., Ellsworth, D. S., et al. (2016a). A test of the “one-point method” for estimating maximum
carboxylation capacity from field-measured, light-saturated photosynthesis. New Phytol. 210, 1130–1144. doi: 10.1111/nph.13815 Berry, J., and Bjorkman, O. (1980). Photosynthetic response and adaptation
to temperature in higher plants. Annu. Rev. Plant Physiol. 31, 491–543. doi: 10.1146/annurev.pp.31.060180.002423 De Kauwe, M. G., Lin, Y. S., Wright, I. J., Medlyn, B. E., Crous, K. Y.,
Ellsworth, D. S., et al. (2016b). Corrigendum. New Phytol. 210, 1130–1144. doi: 10.1111/nph.14172 Bond, B. J., Farnsworth, B. T., Coulombe, R. A., and Winner, W. E. (1999). Foliage
characteristics and biochemistry in response to radiation gradients in confiers
with varying shade tolerance. Oecologia 120, 183–192. Doughty, C. E. (2011). An in situ leaf and branch warming experiment in the
Amazon. Biotropica 43, 658–665. doi: 10.1111/j.1744-7429.2010.00746.x Drake, J. E., Tjoelker, M. G., Aspinwall, M. SUPPLEMENTARY MATERIAL Jennifer Eikenberry, for use of lab space and leaf nutrient
analyses. We are grateful for excellent field and laboratory
assistance provided by Kaylie Butts, Benjamin Miller, and Elsa
Schwartz. A previous version of this manuscript was included in
a Master’s thesis (Carter, 2017). The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/ffgc.2018. 00011/full#supplementary-material ACKNOWLEDGMENTS The authors would like to thank Mark Sloat and Michigan
Technological University’s Electrical and Computer Engineering
Department for designing the warming device. Thank you, Erik
Lilleskov, Joseph DesRoshers, and the USDA Forest Service
Northern Research Station, for the use of their scaffolding,
climbing equipment, and environmental data. Thank you, December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 14 Carter and Cavaleri Northern Hardwood Experimental Warming REFERENCES Components of leaf dry mass per area – thickness and
density – alter leaf photosynthetic capacity in reverse directions in woody
plants. New Phytol. 144, 35–47. Koike, T., Kitao, M., Maruyama, Y., Mori, S., and Lei, T. T. (2001). Leaf morphology and photosynthetic adjustments among deciduous broad-
leaved trees within the vertical canopy profile. Tree Physiol. 21, 951–958. doi: 10.1093/treephys/21.12-13.951 Niinemets, Ü., Oja, V., and Kull, O. (1999). Shape of leaf photosynthetic electron
transport versus temperature response curve is not constant along canopy light
gradients in temperate deciduous trees. Plant Cell Environ. 22, 1497–1513. doi: 10.1046/j.1365-3040.1999.00510.x Ku, S.-B., and Edwards, G. E. (1978). Oxygen Inhibation of Photosynthesis. Planta
140, 1–6. Niinemets, U., and Tenhunen, J. D. (1997). A model separating leaf structural
and physiological effects on carbon gain along light gradients for the
shade-tolerant species Acer saccharum. Plant Cell Environ. 20, 845–866. doi: 10.1046/j.1365-3040.1997.d01-133.x Lawlor, D. W., and Tezara, W. (2009). Causes of decreased photosynthetic
rate and metabolic capacity in water-deficient leaf cells: a critical evaluation
of mechanisms and integration of processes. Ann. Bot. 103, 561–579. doi: 10.1093/aob/mcn244 NOAA National Centers for Environmental Information (2018). Available online
at: https://www.ncdc.noaa.gov/cdo-web/datatools (Accessed November 12,
2018). Legner, N., Fleck, S., and Leuschner, C. (2014). Within-canopy variation
in
photosynthetic
capacity,
SLA
and
foliar
N
in
temperate
broad-
leaved
trees
with
contrasting
shade
tolerance. Trees
28,
263–280. doi: 10.1007/s00468-013-0947-0 Noh, N. J., Kuribayashi, M., Saitoh, T. M., Nakaji, T., Nakamura, M., Hiura, T.,
et al. (2016). Responses of soil, heterotrophic, and autotrophic respiration to
experimental open-field soil warming in a cool-temperate deciduous forest. Ecosystems 19, 504–520. doi: 10.1007/s10021-015-9948-8 Leigh, A., Sevanto, S., Close, J. D., and Nicotra, A. B. (2017). The influence of leaf
size and shape on leaf thermal dynamics: does theory hold up under natural
conditions? Plant Cell Environ. 40, 237–248. doi: 10.1111/pce.12857 NRCS Soil Survey Staff, Natural Resources Conservation Service, United States
Department of Agriculture. (2018). Official Soil Series Descriptions. NRCS Soil Survey Staff, Natural Resources Conservation Service, United States
Department of Agriculture. (2018). Official Soil Series Descriptions. Ogle, D. H. (2018). FSA: Fisheries Stock Analysis. R package version 0.8.21. Department of Agriculture. (2018). Official Soil Series Descriptions. Ogle, D. H. (2018). FSA: Fisheries Stock Analysis. R package version 0.8.21. p
g
ffi
p
Ogle, D. H. (2018). FSA: Fisheries Stock Analysis. R package version 0.8.21. Lichtenthaler, H. K., Ac, A., Marek, M. V., Kalina, J., and Urban, O. (2007). REFERENCES Thermal plasticity of photosynthesis: the role of acclimation
in forest responses to a warming climate. Glob. Chang. Biol. 16, 2272–2286. doi: 10.1111/j.1365-2486.2009.02090.x Michaletz, S. T., Weiser, M. D., McDowell, N. G., Zhou, J., Kaspari, M., Helliker,
B. R., et al. (2016). The energetic and carbon economic origins of leaf
thermoregulation. Nat. Plants 2, 1–8. doi: 10.1038/nplants.2016.129 Gurevitch, J., and Shuepp, P. H. (1990). Boundary layer properties of highly
dissected leaves: an investigation using an electrochemical fluid tunnel. Plant
Cell Environ. 13, 783–792. Nakamura, M., Makoto, K., Tanaka, M., Inoue, T., Son, Y., and Hiura, T. (2016). Leaf flushing and shedding, bud and flower production, and stem elongation in
tall birch trees subjected to increases in aboveground temperature. Trees Struct. Funct. 30, 1535–1541. doi: 10.1007/s00468-016-1387-4 Havaux, M. (1992). Stress tolerance of photosystem II in vivo: antagonistic effects
of water, heat, and photoinhibition stresses. Plant Physiol. 100, 424–432. Havaux, M., Tardy, F., Ravenel, J., Chanu, D., and Parot, P. (1996). Thylakoid
membrane stability of heat stress studied by flash spectroscopic measurements
of the electrochromic shift in intact potato leaves: influence of the xanthophyll
content. Plant Cell Environ. 19, 1359–1368. Nakamura,
M.,
Muller,
O.,
Tayanagi,
S.,
Nakaji,
T.,
and
Hiura,
T. (2010). Experimental branch warming alters tall tree leaf phenology
and
acorn
production. Agric. For. Meteorol. 150,
1026–1029. doi: 10.1016/j.agrformet.2010.04.001 Huner, N. P. A. (1988). Low-temperature induced alterations in photosynthetic
membranes. CRC Crit. Rev. Plant Sci. 7, 257–278. Nicotra, A. B., Cosgrove, M. J., Cowling, A., Schlichting, C. D., and Jones,
C. S. (2008). Leaf shape linked to photosynthetic rates and temperature
optima in South African Pelargonium species. Oecologia 154, 625–635. doi: 10.1007/s00442-007-0865-1 Iverson, L. R., Prasad, A. M., Matthews, S. N., and Peters, M. (2008). Estimating
potential habitat for 134 eastern US tree species under six climate scenarios. For. Ecol. Manage. 254, 390–406. doi: 10.1016/j.foreco.2007.07.023 Jarvi, M. P., and Burton, A. J. (2013). Acclimation and soil moisture constrain
sugar maple root respiration in experimentally warmed soil. Tree Physiol. 33,
949–959. doi: 10.1093/treephys/tpt068 Niinemets, Ü. (1997). Distribution patterns of foliar carbon and nitrogen as
affected by tree dimensions and relative light conditions in the canopy of Picea
abies. Trees Struct. Funct. 11, 144–154. doi: 10.1007/s004680050070 Jump, A. S., Hunt, J. M., and Peuelas, J. (2006). Rapid climate change-related
growth decline at the southern range edge of Fagus sylvatica. Glob. Chang. Biol. 12, 2163–2174. doi: 10.1111/j.1365-2486.2006.01250.x Niinemets, Ü. (1999). REFERENCES Differences in leaf thermoregulation and water-use strategies
between three co-occurring Atlantic forest tree species. Plant. Cell Environ. 41,
1618–1631. doi: 10.1111/pce.13208 Chung, H., Muraoka, H., Nakamura, M., Han, S., Muller, O., and Son, Y. (2013). Experimental warming studies on tree species and forest ecosystems:
a literature review. J. Plant Res. 126, 447–460. doi: 10.1007/s10265-013-0565-3 Filewod, B., and Thomas, S. C. (2014). Impacts of a spring heat wave on canopy
processes in a northern hardwood forest. Glob. Chang. Biol. 20, 360–371. doi: 10.1111/gcb.12354 Ciais, P., Reichstein, M., Viovy, N., Granier, A., Ogée, J., Allard, V., et al. (2005). Europe-wide reduction in primary productivity caused by the heat and drought
in 2003. Nature 437, 529–533. doi: 10.1038/nature03972 Fu, Y. H., Campioli, M., Deckmyn, G., and Janssens, I. A. (2013). Sensitivity of leaf
unfolding to experimental warming in three temperate tree species. Agric. For. Meteorol. 181, 125–132. doi: 10.1016/j.agrformet.2013.07.016 Coble, A. P., and Cavaleri, M. A. (2015). Light acclimation optimizes leaf functional
traits despite height-related constraints in a canopy shading experiment. Oecologia 177, 1–13. doi: 10.1007/s00442-015-3219-4 Gershunov,
A.,
Cayan,
D. R.,
and
Iacobellis,
S. F. (2009). The
great
2006 heat wave over California and Nevada: signal of an increasing
trend. J. Clim. 22, 6181–6186, 6188, 6190–6192, 6194–6195, 6197–6203. doi: 10.1175/2009JCLI2465.1 Coble, A. P., Cavaleri, M. A., and Niinemets, Ü. (2014). Light drives vertical
gradients of leaf morphology in a sugar maple (Acer saccharum) forest. Tree
Physiol. 34, 146–158. doi: 10.1093/treephys/tpt126 Crous, K. Y., Quentin, A. G., Lin, Y. S., Medlyn, B. E., Williams, D. G., Barton, C. V., et al. (2013). Photosynthesis of temperate Eucalyptus globulus trees outside
their native range has limited adjustment to elevated CO2 and climate warming. Glob. Chang. Biol. 19:3790–3807. doi: 10.1111/gcb.12314 Gunderson, C. A., Norby, R. J., and Wullschleger, S. D. (2000). Acclimation of
photosynthesis and respiration to simulated climatic warming in northern and
southern populations of Acer saccharum: laboratory and field evidence. Tree
Physiol. 20, 87–96. doi: 10.1093/treephys/20.2.87 Frontiers in Forests and Global Change | www.frontiersin.org December 2018 | Volume 1 | Article 11 15 Northern Hardwood Experimental Warming Carter and Cavaleri Melillo, J. M., Butler, S., Johnson, J., Mohan, J., Steudler, P., Lux, H., et al. (2011). Soil warming, carbon-nitrogen interactions, and forest carbon budgets. Proc. Natl. Acad. Sci. U.S.A. 108, 9508–9512. doi: 10.1073/pnas.1018189108 Gunderson, C. A., O’Hara, K. H., Campion, C. M., Walker, A. V., and Edwards,
N. T. (2010). REFERENCES Differences in pigment composition, photosynthetic rates and chlorophyll
fluorescence images of sun and shade leaves of four tree species. Plant Physiol. Biochem. 45, 577–588. doi: 10.1016/j.plaphy.2007.04.006 O’Sullivan, O. S., Heskel, M. A., Reich, P. B., Tjoelker, M. G., Weerasinghe, L. K.,
Penillard, A., et al. (2017). Thermal limits of leaf metabolism across biomes. Glob. Chang. Biol. 23, 209–223. doi: 10.1111/gcb.13477 Lu, Z., Percy, R. G., Qualset, C. O., and Zeiger, E. (1998). Stomatal conductance
predicts yields in irrigated Pima cotton and bread wheat grown at high
temperatures. J. Exp. Bot. 49, 453–460. Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D., and R Core Team (2018). nlme:
Linear and Nonlinear Mixed Effects Models. R package version 3.1-137. Portis, A. R. (2003). Rubisco activase–Rubisco’s catalytic chaperone. Photosynth. Res. 75, 11–27. doi: 10.1023/A:1022458108678 Marchin, R. M., Broadhead, A. A., Bostic, L. E., Dunn, R. R., and Hoffmann, W. A. (2016). Stomatal acclimation to vapour pressure deficit doubles transpiration
of small tree seedlings with warming. Plant Cell Environ. 39, 2221–2234. doi: 10.1111/pce.12790 Potvin, L. R., and Lilleskov, E. A. (2017). Introduced earthworm species exhibited
unique patterns of seasonal activity and vertical distribution, and Lumbricus
terrestris burrows remained usable for at least 7 years in hardwood and pine
stands. Biol. Fertil. Soils 53, 187–198. doi: 10.1007/s00374-016-1173-x Mau, A., Reed, S., Wood, T., and Cavaleri, M. (2018). Temperate and tropical
forest canopies are already functioning beyond their thermal thresholds for
photosynthesis. Forests 9:47. doi: 10.3390/f9010047 R Core Team (2015). R: A Language and Environment for Statistical Computing. Vienna: R Foundation for Statistical Computing. Available online at: https://
www.R-project.org/ Medlyn, B. E., Dreyer, E., Ellsworth, D., Forstreuter, M., Harley, P. C., Kirschbaum,
M. U. F., et al. (2002). Temperature response of parameters of a biochemically
based model of photosynthesis. II. A review of experimental data. Plant Cell
Environ. 25, 1167–1179. doi: 10.1046/j.1365-3040.2002.00891.x Reich, P. B., Sendall, K. M., Stefanski, A., Wei, X., Rich, R. L., and Montgomery, R. A. (2016). Boreal and temperate trees show strong acclimation of respiration to
warming. Nature 531, 633–636. doi: 10.1038/nature17142 Meehl, G. A., and Tebaldi, C. (2004). More intense, more frequent, and
longer lasting heat waves in the 21st century. Science 305, 994–997. doi: 10.1126/science.1098704 Reich, P. B., Wright, I. J., Bares, J. C., Craine, J. M., Oleksyn, J., Westoby, M.,
et al. (2003). The evolution of plant functional variation: traits, spectra , and
strategies. Int. J. Plant Sci. 164, S143–S164. REFERENCES Int. J. Mol. Sci. 19:E2023. doi: 10.3390/ijms19072023 Valladares, F., and Niinemets, Ü. (2008). Shade tolerance, a key plant feature of
complex nature and consequences. Annu. Rev. Ecol. Evol. Syst. 39, 237–257. doi: 10.1146/annurev.ecolsys.39.110707.173506 Vogel, S. (1970). Convective cooling at low airspeeds and the shapes of broad
leaves. J. Exp. Bot. 21, 91–101. Scafaro, A. P., Xiang, S., Long, B. M., Bahar, N. H. A., Weerasinghe, L. K., Creek,
D., et al. (2017). Strong thermal acclimation of photosynthesis in tropical and
temperate wet-forest tree species: the importance of altered Rubisco content. Glob. Chang. Biol. 23, 2783–2800. doi: 10.1111/gcb.13566 Walker, K. V., Davis, M. B., and Sugita, S. (2002). Climate change and shifts in
potential tree species range limits in the Great Lakes Region. J. Great Lakes Res. 28, 555–567. doi: 10.1016/S0380-1330(02)70605-9 Schneider, C. A., Rasband, W. S., and Eliceiri, K. W. (2012). NIH image to ImageJ:
25 years of image analysis. Nat. Methods 9, 671–675. doi: 10.1038/nmeth.2089 Wang,
Z. Y.,
and
Portis,
A. R. (1992). Dissociation
of
ribulose-1,5-
bisphosphate
bound
to
ribulose-1,5-bisphosphate
carboxylase/oxygenase
and its enhancement by ribulose-1,5-bisphosphate carboxylase/oxygenase
activase-mediated
hydrolysis
of
ATP. Plant
Physiol. 99,
1348–1353. doi: 10.1104/pp.99.4.1348 Schoettle, A. W., and Smith, W. K. (1999). Interrelationships among light,
photosynthesis and nitrogen in the crown of mature Pinus contorta ssp. latifolia. Tree Physiol. 19, 13–22. doi: 10.1093/treephys/19.1.13 Sendall, K. M., Lusk, C. H., and Reich, P. B. (2015a). Becoming less tolerant
with age: sugar maple, shade, and ontogeny. Oecologia 179, 1011–1021. doi: 10.1007/s00442-015-3428-x Way, D. A., and Oren, R. (2010). Differential responses to changes in
growth temperature between trees from different functional groups and
biomes: a review and synthesis of data. Tree Physiol. 30, 669–688. doi: 10.1093/treephys/tpq015 Sendall, K. M., Reich, P. B., Zhao, C., Jihua, H., Wei, X., Stefanski, A., et al. (2015b). Acclimation of photosynthetic temperature optima of temperate and boreal
tree species in response to experimental forest warming. Glob. Chang. Biol. 21,
1342–1357. doi: 10.1111/gcb.12781 Way, D. A., and Yamori, W. (2014). Thermal acclimation of photosynthesis: On the
importance of adjusting our definitions and accounting for thermal acclimation
of respiration. Photosynth. Res. 119, 89–100. doi: 10.1007/s11120-013-
9873-7 Sharwood, R. E., Crous, K. Y., Whitney, S. M., Ellsworth, D. S., and Ghannoum,
O. (2017). Linking photosynthesis and leaf N allocation under future elevated
CO2 and climate warming in Eucalyptus globulus. J. Exp. Bot. 68, 1157–1167. doi: 10.1093/jxb/erw484 Yamaguchi, D. P., Nakaji, T., Hiura, T., and Hikosaka, K. (2016). REFERENCES doi: 10.1086/374368 Meir, P., Kruijt, B., Broadmeadow, M., Barbosa, E., Kull, O., Carswell, F.,
et al. (2002). Acclimation of photosynthetic capacity to irradiance in tree
canopies in relation to leaf nitrogen concentration and leaf mass per
unit area. Plant Cell Environ. 25, 343–357. doi: 10.1046/j.0016-8025.2001.0
0811.x Rich, R. L., Stefanski, A., Montgomery, R. A., Hobbie, S. E., Kimball, B. A.,
and Reich, P. B. (2015). Design and performance of combined infrared
canopy and belowground warming in the B4WarmED (Boreal Forest Warming
at an Ecotone in Danger) experiment. Glob. Chang. Biol. 21, 2334–2348. doi: 10.1111/gcb.12855 December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 16 Northern Hardwood Experimental Warming Carter and Cavaleri Rollinson, C. R., and Kaye, M. W. (2012). Experimental warming alters
spring
phenology
of
certain
plant
functional
groups
in
an
early
successional
forest
community. Glob. Chang. Biol. 18,
1108–1116. doi: 10.1111/j.1365-2486.2011.02612.x Australian rainforest tree species. Oecologia 94, 218–228. doi: 10.1007/BF003
41320 Turnbull, M. H., Tissue, D. T., Murthy, R., Wang, X., Sparrow, A. D., and
Griffin, K. L. (2004). Nocturnal warming increases photosynthesis at elevated
CO2 partial pressure in Populus deltoides. New Phytol. 161, 819–826. doi: 10.1111/j.1469-8137.2003.00994.x Ryan,
M. G.,
Phillips,
N.,
and
Bond,
B. J. (2006). The
hydraulic
limitation
hypothesis
revisited. Plant
Cell
Environ. 29,
367–381. doi: 10.1111/j.1365-3040.2005.01478.x Turnbull, M. H., Whitehead, D., Tissue, D. T., Schuster, W. S. F., Brown, K. J., and Griffin, K. L. (2003). Scaling foliar respiration in two contrasting
forest canopies. Funct. Ecol. 17, 101–114. doi: 10.1046/j.1365-2435.2003.0
0713.x Sage,
R. F.,
and
Kubien,
D. S. (2007). The
temperature
response
of
C3
and
C4
photosynthesis. Plant
Cell
Environ. 30,
1086–1106. doi: 10.1111/j.1365-3040.2007.01682.x Salvucci, M. E., Osteryoung, K. W., Crafts-Brandner, S. J., and Vierling, E. (2001). Exceptional sensitivity of Rubisco activase to thermal denaturation in vitro and
in vivo. Plant Physiol. 127, 1053–1064. doi: 10.1104/pp.010357 Urban, J., Ingwers, M. W., McGuire, M. A., and Teskey, R. O. (2017). High
temperature opens stomata and decouples net photosynthesis from stomatal
conductance in Pinus taeda and Populus deltoides × nigra. J. Exp. Bot. 68,
1757–1767. doi: 10.1093/jxb/erx052 1.5 in vivo. Plant Physiol. 127, 1053–1064. doi: 10.1104/pp.010357 Sampaio Filho, I. de J., Jardine, K. J., de Oliveira, R. C. A., Gimenez, B. O., Cobello,
L. O., Piva, L. R. O., et al. (2018). Below versus above ground plant sources of
abscisic acid (ABA) at the heart of tropical forest response to warming. REFERENCES Effects of
seasonal change and experimental warming on the temperature dependence
of photosynthesis in the canopy leaves of Quercus serrata. Tree Physiol. 36,
1283–1295. doi: 10.1093/treephys/tpw021 Slot, M., Garcia, M. A., and Winter, K. (2016). Temperature response of CO2
exchange in three tropical tree species. Funct. Plant Biol. 43, 468–478. doi: 10.1071/FP15320 Zhang, N., Kallis, R. P., Ewy, R. G., and Portis, A. R. (2002). Light modulation
of Rubisco in Arabidopsis requires a capacity for redox regulation of the
larger Rubisco activase isoform. Proc. Natl. Acad. Sci. U.S.A. 99, 3330–3334. doi: 10.1073/pnas.042529999 Slot, M., Rey-Sánchez, C., Gerber, S., Lichstein, J. W., Winter, K., and Kitajima,
K. (2014). Thermal acclimation of leaf respiration of tropical trees and lianas:
Response to experimental canopy warming, and consequences for tropical
forest carbon balance. Glob. Chang. Biol. 20, 2915–2926. doi: 10.1111/gcb.12563 Zhang, R., Cruz, J. A., Kramer, D. M., Magallanes-Lundback, M. E., Dellapenna,
D., and Sharkey, T. D. (2009). Moderate heat stress reduces the pH component
of the transthylakoid proton motive force in light-adapted, intact tobacco
leaves. Plant Cell Environ. 32, 1538–1547. doi: 10.1111/j.1365-3040.2009.02
018.x Smith, N. G., and Dukes, J. S. (2017). Short-term acclimation to warmer
temperatures accelerates leaf carbon exchange processes across plant types. Glob. Chang. Biol., 4840–4853. doi: 10.1111/gcb.13735 Tang, G., and Beckage, B. (2010). Projecting the distribution of forests in
New England in response to climate change. Divers. Distrib. 16, 144–158. doi: 10.1111/j.1472-4642.2009.00628.x Zhang, Y., Equiza, M. A., Zheng, Q., and Tyree, M. T. (2011). Factors
controlling
plasticity
of
leaf
morphology
in
Robinia
pseudoacacia:
III. biophysical constraints on leaf expansion under long-term water
stress. Physiol. Plant. 143,
367–374. doi:
10.1111/j.1399-3054.2011.01
504.x Thomas, C. D., Cameron, A., Green, R. E., Bakkenes, M., Beaumont, L. J.,
Colllingham, Y. C., et al. (2004). Extinction risk from climate change. Nature
427, 145–148. doi: 10.1038/nature02121 Thomas,
S. C. (2010). Photosynthetic
capacity
peaks
at
intermediate
size
in
temperate
deciduous
trees. Tree
Physiol. 30,
555–573. doi: 10.1093/treephys/tpq005 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. Treyger,
A. L.,
and
Nowak,
C. A. (2011). Changes
in
tree
sapling
composition within powerline corridors appear to be consistent with
climatic changes in New York State. Glob. Chang. Biol. 17, 3439–3452. doi: 10.1111/j.1365-2486.2011.02455.x Copyright © 2018 Carter and Cavaleri. REFERENCES This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is permitted, provided the original
author(s) and the copyright owner(s) are credited and that the original publication
in this journal is cited, in accordance with accepted academic practice. No use,
distribution or reproduction is permitted which does not comply with these terms. j
Turgeon, R. (2006). Phloem loading: how leaves gain their independence. Bioscience 56, 15. doi: 10.1641/0006-3568(2006)056[0015:PLHLGT]2.0.CO;2 Turnbull, M. H., Doley, D., and Yates, D. J. (1993). The dynamics of
photosynthetic acclimation to changes in light quantity and quality in three December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 17
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Hubgrade Smart Monitoring Centers: Measuring Resource Consumption and Moving towards a Circular Economy
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Hubgrade Smart Monitoring Centers: Measuring
Resource Consumption and Moving towards a
Circular Economy Electronic version
URL: http://journals.openedition.org/factsreports/4419
ISSN: 1867-8521
Publisher
Institut Veolia
Printed version
Date of publication: 31 December 2017
Number of pages: 32-37
ISSN: 1867-139X Printed version
Date of publication: 31 December 2017
Number of pages: 32-37
ISSN: 1867-139X Field Actions Science Reports
The journal of field actions
Special Issue 17 | 2017
Artificial Intelligence and Robotics in the City Field Actions Science Reports
The journal of field actions
Special Issue 17 | 2017
Artificial Intelligence and Robotics in the City Electronic reference Electronic reference
Antonio Neves Da Silva and Patrice Novo, « Hubgrade Smart Monitoring Centers: Measuring Resource
Consumption and Moving towards a Circular Economy », Field Actions Science Reports [Online], Special
Issue 17 | 2017, Online since 31 December 2017, connection on 21 April 2019. URL : http://
journals.openedition.org/factsreports/4419 Creative Commons Attribution 3.0 License www.factsreports.org How can cities, businesses and industries
boost growth in the face of resource
scarcity? Firstly, they can start by
measuring their consumption in order
to manage it more effectively. Then, all
of us can to move away from a linear
model of consumption. We can accelerate
this transition today, thanks to IoT1,
the digital revolution. At Veolia, we are the fi rst to monitor and
to optimize water, energy and material
fl ows in real-time. We are developing
smart monitoring centers called
Hubgrade relying on connected products
and artifi cial intelligence. With these
centers, we are creating new jobs and
business opportunities to save resources. At Veolia, we are the fi rst to monitor and
to optimize water, energy and material
fl ows in real-time. We are developing
smart monitoring centers called
Hubgrade relying on connected products
and artifi cial intelligence. With these
centers, we are creating new jobs and
business opportunities to save resources. Hubgrade boosts energy effi ciency and
water conservation measures. It optimizes
material recovery and maximizes the use
of renewable energy. However, this is
only possible, with the focus on the
human factor. 1 IoT – Internet of Things Hubgrade in Madrid, Spain 1.1 BOOSTING ECONOMIC GROWTH WHILE FACING
RESOURCE SCARCITY Global urbanization is increasing and cities are putting the planet
under enormous pressure. Since the 1970s, humanity has been
consuming more natural resources than the planet can provide and
renew in a year. Today, humanity actually needs one and a half planets to be
sustainable. By 2050, at current rates, we would need almost three
planets. Our demand for water, energy, food and goods is rapidly
increasing. The Take-Make-Dispose model of the past has also
led to extreme pollution, price volatility and biodiversity collapse. Besides the impact on climate change an on the environment, this
causes evident effects on the economy and society. The challenge
now is to decouple economic growth from resource consumption. We need to move towards a Circular Economy where nothing is
wasted. In practice, this means not only preserving energy and
water, while minimizing waste. We also need to improve energy
efficiency, increase recycling and boost renewable energies. Moreover, we need business opportunities to accelerate this change. End-user End-user ‘Doing more with less’ is conceptually simple but quantifying
resource effi ciency is more complex in practice. INTRODUCTION Hubgrade is the name of Veolia’s smart
monitoring centers for water, energy and waste
management. In these centers, Veolia’s analysts
leverage real-time data to optimize resource
consumption of municipal, commercial and
industrial clients. This innovation relies on a
dedicated organization, disruptive digital tools
and new business models. Hubgrade is a tremendous opportunity to
introduce a cultural change in our organization. We can revolutionize the way we operate and
become more effi cient. At the same time, we are
off ering new services and an enhanced customer
experience to our clients. We can equip each of these centers to manage
data from a multitude of facilities: from municipal
water networks to waste collection systems, to
buildings, to industrial sites, to district energy
systems and more. Today, we have 15 Hubgrades
already accelerating the transition towards a
Circular Economy. They guarantee us that no
precious resources are wasted. 32 AI in the city, the age of
prediction and anticipation
www.factsreports.org AI in the city, the age of
prediction and anticipation
www.factsreports.org 1.2 SMART URBAN METABOLISM: HIGHLIGHTING OPPORTUNITIES
TO SAVE RESOURCES IN REAL-TIME models, using real-time data. Such virtual models
represent the interconnectivity between different
subsystems, which can be urban infrastructures,
offices, schools, hospitals, industries and even
households. The new generation of data solutions allows us a deeper study of
urban metabolism. Urban metabolism is a model representing the
transformation of natural resources in products and services. This
model quantifi es economic value of what we use and what we waste. So, what are the benefits of smart urban
metabolism models? These models make it
is easier to replicate sustainable solutions to
other subsystems. This happens when these
subsystems have similar challenges. For example,
an urban infrastructure needs to become more
resilient, livable or even inclusive. The same
applies to buildings and to industries that must
become greener and more resource effi cient. Now it is possible to have a higher temporal resolution of
consumption. This enables us to build smart urban metabolism Ecological footprint - http://www.footprintnetwork.org Ecological footprint - http://www.footprintnetwork.org The resources we are wasting will end up being
either emissions or discharges to land or to
water sources. In order to avoid such waste, a
holistic approach on resource saving is required. Smart urban metabolism offers exactly that. It highlights the value of a subsystem’s waste
to other economic sectors. This facilitates a
business transaction so that waste can be reused,
recovered or recycled by other subsystems. Emissions and discharges also cost money and
become evident opportunities to save. These predictive models to analyze resource
consumption are something very concrete to us at
Veolia. We convert these models into performance
contracts with our clients, with guaranteed
savings. It may seem that we have always operated
contracts like these. So what has changed? 33 www.factsreports.org Hubgrade, smart monitoring center in Paris, launched 2016 Now, we can commit to save significantly more. We understand the value chain of resources
outside our traditional operations. Sensors, smart
products and other digital technologies extend our
capabilities. Besides implementing and operating
effi cient systems, we engage end-users to play a
major role in the solution. They take responsibility
to change their behaviors because they foresee
the benefits for them. Through smart solutions,
citizens and other end-users receive the insight
they need to save more. Veolia cannot monitor every resource fl ow in a city,
and that is not the point. However, we do commit
to improve resource effi ciency for the perimeter
we operate. 1.2 SMART URBAN METABOLISM: HIGHLIGHTING OPPORTUNITIES
TO SAVE RESOURCES IN REAL-TIME This is why the municipality of Pudong
in Shanghai has chosen Veolia to manage its water
networks. Hubgrade, smart monitoring center in Paris, launched 2016 2.1 WHAT IS HUBGRADE? Energy and water savings, waste minimization
and recycling rates, carbon emission reduction –
Veolia has always tracked these key performance
indicators. Now, we can track them in real-time
and from anywhere we want. However, this
requires a major organizational change. As a result, clients and end users become more aware of how they
can make savings. 2.2 HOW DOES HUBGRADE WORK? More recently in Shanghai, Veolia launched
a Hubgrade for Water, Energy and Waste
management. We can now offer higher level
of commitments to existing customers. We
can also offer these innovative services to new
clients all over China. This way, they too, can save
even more. Clients want control over costs and consumption, so their systems
are fitted with sensors. These sensors transmit data in real time
to Hubgrade. Then, our analysts manage this data to identify
savings. This can result into immediate action or a roadmap for
improvements. We transmit recommendations to clients and our teams, depending
on the type of contract. This helps them identify and prioritize
resource saving measures. From an online dashboard, clients can
monitor their own indicators and compare them to benchmarks. This way, they can clearly measure their progress and see the
reduction in their bills. 1 EMS – Energy Management Systems
2 BMS – Building Management Systems
3 CMMS – Computerized Maintenance Management Systems
4 SCADA – Supervisory control and data acquisition 2.3 DEDICATED TEAM OF EXPERTS Hubgrade relies on a team of experts that share a common goal:
improving resource effi ciency together with operational units on-
site and customers. In order to get the full potential out of Hubgrade,
Veolia developed new engineering profi les: For this reason, Veolia deploys a dedicated
organization, digital tools and new business
models. This is how we created Hubgrade, our
smart monitoring center. Through Hubgrade,
Veolia is bringing operational synergies to all our
water, energy and waste activities. p
g
g p
• Data analyst with expertise in energy, water and waste
management • Auditor-coach who ensures operations on-site follow the analysts’
recommendations • Systems expert setting up the right data from sensors into the
software applications In Hubgrade, we combine data management
with our technical expertise on the field. This
results in signifi cant risk mitigation. At the same
time, we are much closer and more responsive to
customers needs. The capability of the Hubgrade team relies on the ubiquity of digital
technologies. Hubgrade enables Veolia to respond quickly and
in a targeted manner. Whenever there is an alert reported on the
systems, there can be a work order generated. Hubgrade is a real asset for change management
to boost operation performance and to offer new
services. The Hubgrade team can act remotely or dispatch a team on site. If this is necessary, they schedule an intervention and dispatch it
to operational units. The customer can follow up all the process in
parallel through a reporting application. Hubgrades: Dublin, IRELAND; Paris, Marseille,
FRANCE; Brussels, BELGIUM; Bilbao, Madrid,
Barcelona, SPAIN; Birmingham, UK; Milan,
ITALY; Amsterdam, NETHERLANDS; Dubaï, UAE;
Stockholm, SWEDEN; Budapest, HUNGARY;
Shanghai, CHINA; Sydney, AUSTRALIA These new roles ensure the implementation of change management
in our traditional activities. With this team, Veolia closes the loop on
the resource value chain. Thus, Hubgrade guarantees we deliver
more savings. 34 AI in the city, the age of
prediction and anticipation
www.factsreports.org AI in the city, the age of
prediction and anticipation
www.factsreports.org Hubgrade, a new organization to guarantee more savings Hubgrade, a new organization to guarantee more savings 2.4 DIGITAL SOLUTIONS H u b g r a d e re li e s o n s t a tis ti c a l m o d e ls ,
optimization algorithms, geographic information
and forecasting tools. Data mining through
meta-heuristic algorithms allows us to predict
customer needs. We analyze correlations between
consumption patterns and production profi les to
identify improvements. Ensuring different systems communicate with each other is a major
challenge in the digital transformation. At the core of Hubgrade,
various systems are integrated such as EMS1, Waste Management
Platforms, Water Quality Monitoring and Control Systems, BMS2,
CMMS3, Asset Management software, SCADA4 systems, and even
Carbon Footprint, Indoor Air Quality monitoring applications and
more. These systems also cover functionalities such as financial
analysis and benchmarking. In addition, machine learning is making Hubgrade
more powerful and autonomous. It brings new
capabilities to help Veolia’s clients switch to an
“industry 4.0 mindset” by bringing them valuable
information for the resources they need to
operate throughout their entire production chain. In practice, we are not just looking at utilities
anymore. We also developed reporting dashboards to share with our analysts
the most important information from digital systems. This helps
them in decision-making and in communicating performance
indicators to operational teams on the fi eld. Additionally, we provide
the client with an online access to reports, as well as awareness-
raising information to end-users. The way in which these systems work can be broken down into four
stages: • 1. Collection of information from sensors “HUBGRADE IS ALREADY A MAJOR
“ONE VEOLIA” ACHIEVEMENT .”
“CREATING NEW JOBS FOR THE ROLES
OF ANALYSTS, AUDITORS AND SYSTEMS
EXPERTS THAT OPERATE IN HUBGRADE AND
INVESTING IN THEIR CAREER DEVELOPMENT
IS A MAJOR HUMAN RESOURCES
ACCOMPLISHMENT OF VEOLIA.” “HUBGRADE IS ALREADY A MAJOR
“ONE VEOLIA” ACHIEVEMENT .”
“CREATING NEW JOBS FOR THE ROLES
OF ANALYSTS, AUDITORS AND SYSTEMS
EXPERTS THAT OPERATE IN HUBGRADE AND
INVESTING IN THEIR CAREER DEVELOPMENT
IS A MAJOR HUMAN RESOURCES
ACCOMPLISHMENT OF VEOLIA.” AI in the city, the age of
prediction and anticipation
www.factsreports.org 5 Deloitte, Smart Cities How rapid advances in technology are reshaping
our economy and society Version 1.0, November 2015 “HUBGRADE IS ALREADY A MAJOR
“ONE VEOLIA” ACHIEVEMENT .” • 2. Supply of information to databases • 3. Data visualization through dashboards and reports • 4. Reporting to operational teams, client and end-users 35 www.factsreports.org digital businesses. Moving from a TCO6 model to a SaaS7 model has
infl uenced Veolia. We offer Performance as a service on top of our
core activities. Hubgrade in Dubai We are analyzing the consumption at the heart of
our customers’ industrial process. For example,
through machine-learning solutions, we can
monitor the consumption of individual equipment. Moreover, we do not need meters for all of them. We can apply this machine learning technologies
to commercial buildings, too. One single high-
frequency meter powered by machine-learning
algorithms enables us to breakdown electric
consumption per type of equipment: lighting, air
conditioning, computers, appliances and others. In the near future, with a single meter and some
sensors we can even precisely measure the
electricity use by each tenant. Our client will
be able to send his tenants invoices for their
individual consumptions without additional
meters. These solutions will be cost effective
enough for the complete switch from readings. Hubgrade in Dubai Hubgrade in Dubai digital businesses. Moving from a TCO6 model to a SaaS7 model has
infl uenced Veolia. We offer Performance as a service on top of our
core activities. digital businesses. Moving from a TCO6 model to a SaaS7 model has
infl uenced Veolia. We offer Performance as a service on top of our
core activities. Crossing data, from customer activities and from
ours, highlights the direct value we create for
them. We monitor and report indoor air quality
and comfort conditions in real-time. This way,
customers know in transparency that quality
is guaranteed, while consumption is kept to a
minimum. Although sustainability demands a long-term view, product
lifecycles are getting shorter. Anyway, it does not alter our mission. We just need to be continuously innovating as demand varies and
clients’ expectations change. The fi rst main real-time solutions with innovative business models
offered by Veolia are applicable to all our energy, water and waste
management activities: Through an online application, the circular
economy seems more tangible. Clients know
how much money they are saving. They can also
check the emissions they reduced and the waste
diverted from landfi ll. • Monitoring, analysis and optimization • Interactive reporting • End-user apps • Information modeling 6 TCO – Total Cost of Ownership
7 SaaS – Software as a Service 2.5 INNOVATIVE BUSINESS MODELS • Predictive maintenance and condition monitoring “Everything that can be digital, will be digital”5. This influences business models as well. We are
adding a major layer of digital services on top of
our core activities. Consequently, our business
models have to change. 6 TCO – Total Cost of Ownership
7 SaaS – Software as a Service
Hubgrade in Milan Hubgrade in Milan The unique value of the new digital component
of our services has to be unleashed. For that,
we need to integrate new business models in
our offer. These are similar to the ones used in “CHINA IS THE FIRST COUNTRY
TO RECEIVE ALL THIS POTENTIAL
WITH A HUBGRADE THAT DELIVERS
ENERGY, WATER AND WASTE
MANAGEMENT OPTIMIZATION TO
MUNICIPAL, COMMERCIAL AND
INDUSTRIAL CUSTOMERS.” 36 AI in the city, the age of
prediction and anticipation
www.factsreports.org AI in the city, the age of
prediction and anticipation
www.factsreports.org We demonstrate all of these services to clients when visiting a
Hubgrade. Everywhere there are Hubgrades, we offer an enhanced
customer experience. Hubgrade provides all the transparency clients
expect to trust expertise in data and our commitment on results. Citizens of China Let us now discover how it all started. Let us now discover how it all started. Well, we started by optimizing buildings. Human
beings spend 90% of their time in buildings10
- that is why these are the first places where we
need to save resources opportunity11. In our globe,
buildings consume around 40% of energy, 25%
of water and 40% of materials12. Simultaneously,
they account for the biggest share of greenhouse
gas emissions on the planet. This represents
approximately 1/3 of the whole globe. To
address carbon emissions and tackle resource
consumption, Europe established an energy
effi ciency directive in 2012. beings spend 90% of their time in buildings10
- that is why these are the first places where we
need to save resources opportunity11. In our globe,
buildings consume around 40% of energy, 25%
of water and 40% of materials12. Simultaneously,
they account for the biggest share of greenhouse
gas emissions on the planet. This represents
approximately 1/3 of the whole globe. To
address carbon emissions and tackle resource
consumption, Europe established an energy
effi ciency directive in 2012. Back then, to address the challenges of our
customers, Veolia started to develop new digital
solutions for energy management. Shared Value creation8 Although one can argue that artificial intelligence will eradicate
jobs thus harming society, this argument remains vague. We
believe innovation is the best way to create societal value. It has
been through artificial intelligence that we are improving safety
conditions to innumerous workers in the fi eld. This is the case with
the implementation of our waste sorting solution I-Sorter. Workers
received training and new career progression opportunities. This
example is a real value lever for sustainability. In 2014, as part of the group’s reorganization,
Veolia took the opportunity to extent this acquired
expertise in these smart monitoring centers,
to its water and waste management activities,
and created Hubgrade. The fi rst Hubgrades also
optimize, in addition to buildings, the efficiency
of Waste-to-Energy facilities and the electricity
consumption of wastewater treatment plants. Creating new jobs for the roles of analysts, auditors and systems
experts that operate in Hubgrade and investing in their career
development is a major human resources accomplishment of Veolia. The main idea is to combine human and digital capabilities to boost a
social and economic dynamic while preserving the planet. Today, Veolia’s digital solutions apply to all our
activities. We use them for route optimization in
waste collection. With them, we reduce leaks in
water networks. We also use them to improve the
effi ciency of buildings, industries and more. Small and medium IT services companies are developing the
digital tools we use in Hubgrade. With these SMEs9 we establish
partnerships with a long-term view. We commit to create this value
and deliver it to our clients so they can keep sharing it with local
communities. Hubgrade is already a major “One Veolia”
achievement. It enables us to address the needs
of our customers very closely. China is the first country to receive all this
potential with a Hubgrade that delivers energy,
water and waste management optimization to
municipal, commercial and industrial customers. Thanks to Hubgrade, customers and citizens
interact with us in real-time. We can now provide
them with the services and information they
need so that together we can do more for a
sustainable future. 8 M. Porter & M. Kramer – Harvard Business Review – January February 2011
[Note: Creating Shared Value is not included in Corporate Social Responsibility, which is separate
from profi t maximization. CSV is rather a transition and expansion form the concept of CSR.]
9 SME – Small and Medium Enterprises 2.5 INNOVATIVE BUSINESS MODELS With these
solutions, we generated an average 15% savings
in energy consumption. This is the case of Indra
Systems, one of our 160 Energy Performance
Contracts, optimized via a real-time monitoring
center. Indra, which is the IT and Defense
systems leader in Spain reduced by 15% the
energy consumption of its 65 buildings, together
with Veolia. Citizens of China We demonstrate all of these services to clients when visiting a
Hubgrade. Everywhere there are Hubgrades, we offer an enhanced
customer experience. Hubgrade provides all the transparency clients
expect to trust expertise in data and our commitment on results. 10 Source : https://www.buildinggreen.com/blog/we-spend-90-our-
time-indoors-says-who
11 McKinsey – Resource Revolution 2011
12 Source : https://www.euenergycentre.org/images/unep%20
info%20sheet%20-%20ee%20buildings.pdf 8 M. Porter & M. Kramer – Harvard Business Review – January February 2011
[Note: Creating Shared Value is not included in Corporate Social Responsibility, which is separate
from profi t maximization. CSV is rather a transition and expansion form the concept of CSR.]
9 SME – Small and Medium Enterprises 9 SME – Small and Medium Enterprises 3. OVER THREE MILLION DATA POINTS BEING
MONITORED BY VEOLIA Veolia monitors over three million sensors, from which one million
are smart meters. The manner to leverage data from them is far
from optimal. The good news is that Veolia is deploying Hubgrades
worldwide in an industrialized way. We already monitor over
300 thousand data points in our Hubgrades. 37
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FIRE: Fault Injection for Reverse Engineering
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Lecture notes in computer science
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FIRE: fault injection for reverse engineering To cite this version:
Manuel San Pedro, Mate Soos, Sylvain Guilley. FIRE: fault injection for reverse engineering. 5th
Workshop on Information Security Theory and Practices (WISTP), Jun 2011, Heraklion, Crete,
Greece. pp.280-293, 10.1007/978-3-642-21040-2_20. hal-00690868 To cite this version: Manuel San Pedro, Mate Soos, Sylvain Guilley. FIRE: fault injection for reverse engineering. 5th
Workshop on Information Security Theory and Practices (WISTP), Jun 2011, Heraklion, Crete,
Greece. pp.280-293, 10.1007/978-3-642-21040-2_20. hal-00690868 Distributed under a Creative Commons Attribution 4.0 International License FIRE: Fault Injection for Reverse Engineering Manuel San Pedro1, Mate Soos2, and Sylvain Guilley1 1 Institut TELECOM
2 INRIA, Security Research Labs Abstract. In this paper, we propose a new technique that uses fault
injection to reverse-engineer a private block cipher implemented with an
unknown S-box. The private algorithm we wish to retrieve differs from a
known algorithm in the choice of the S-Box, which we find using a novel,
fault-injecting technique. The main idea is to consider the components of
the S-Box as the solutions of a linear boolean system, whose equations
stem from the faults injected, using existing fault models. We focus on
two well-known block ciphers, DES and AES, and prove it to be feasible
to retrieve the the S-Box for both cases. We present the fault models
used, the equations extracted from the faults injected, and analyse the
final results. Given the detailed analysis, the technique can be applied
with ease to most ciphers employing an S-box. HAL Id: hal-00690868
https://inria.hal.science/hal-00690868v1
Submitted on 24 Apr 2012 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
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 1
Introduction According to Kerckhoffs’s principle, a cryptosystem should be secure even if
everything about the system except the secret key is public knowledge [9]. Even
though this became a fundamental principle of modern cryptology, it is moderately
common for companies and sometimes even standards bodies to keep the inner
workings of a system secret [1, 6]. We then talk about security through obscurity,
or black-box cryptography. Under Kerckhoffs’s principle, cryptanalysis consists in retrieving the cipher
key. But when dealing with security through obscurity, the goal is now modified
to also retrieve information on the private algorithm. This is called reverse-
engineering. Nowadays, with the omnipresence of embedded cryptography, it has
become crucial to be able to perform attacks on electronic devices embedding
unknown cryptosystems. Previous attempts at reverse-engineering unknown cryptosystems were either
through (electro-)optical means, such as the discovery of the MIFARE algorithm
[6], or through the use of side-channel analysis [11]. Side-channel analysis was
originally devised to find the secret key through the measurement of physical
characteristics of the chip such as power intake. Guilley et al. [7] employed
this technique to retrieve the internals of black-box ciphers. This is called the
side-channel analysis for reverse-engineering (SCARE) attack. In this paper, we present a new type of attack employing the principle of fault
injection [2] to retrieve the unknown S-box of a black-box cipher. Fault injection
was originally devised to retrieve the secret key through injection of faults into the chip executing the algorithm and observing the modified output. Our attack
injects faults into the chip, collects the output from the chip, performs analysis of
this data and finally converts the data into a set of equations in binary variables,
which are finally solved using Gaussian elimination to retrieve the S-box. This
new type of attack we call fault injection for reverse engineering (FIRE). The rest of the paper is organised as follows. In Sect. 2, we describe the state
of the art, such as physical attacks of cryptosystems and linear systems solving. In Sect. 3, we present a DES-based cryptosystem, and a FIRE attack on it. Then,
in Sect. 4, we describe an AES-based cryptosystem, and its corresponding FIRE
attack. Finally, in Sect. 5 we conclude this paper. 2.2
Solving linear boolean systems 2.2
Solving linear boolean systems If we consider an S-box as a boolean function fn→m (i.e. a boolean function
from {0, 1}n to {0, 1}m), we can split it into m and fn→1, called the components. Each one of the components will be considered as a vector s ∈{0, 1}2n, being
the solution of a linear system in 2n variables. Each one of the faults injected
brings a certain l number of equations (depending on the fault model), that
the component s must satisfy. This means that each component s is one of the
solutions of the system in {0, 1} of l equations: A · X = B. (1) (1) where A is a l ×2n boolean matrix, and both X and B are vectors of 2n elements. The equations are of the form L2n−1
i=0
ai ·xi = bi. Let L be the set of solutions
of the system L = {s ∈{0, 1}2n : A · s = B}. Let us note that where A is a l ×2n boolean matrix, and both X and B are vectors of 2n elements. The equations are of the form L2n−1
i=0
ai ·xi = bi. Let L be the set of solutions
of the system L = {s ∈{0, 1}2n : A · s = B}. Let us note that s ∈L ⇔¯s ∈L
(2) (2) It stems from the fact that if α and β are boolean variables, then α ⊕β = ¯α ⊕¯β. This property will be important for the rest of the study, since the minimum
of candidates returned will be 2. To solve this linear system of equations, we
have used the Sage software [14] to perform the Gaussian elimination, but any
mathematical software is adequate for the job, as the matrices are typically quite
small. 2.1
Physical attacks on cryptographic systems Most of the cryptographic algorithms used in serious applications are supposed
to be secure against algorithmic attacks. However, they are implemented on
physical components, and hence become vulnerable against physicals attacks. Once such algorithms are implemented, either on dedicated hardware or as
software on a micro-controller, the different physical properties of the algorithm
can be observed. Over the years, sophisticated attacks have been developed to
attack cryptographic devices through such observations. Side-channel attack The physical implementation of a cipher may reveal useful
information about the secret key in an indirect way. Kocher in [10] and in
[11] published two novel attack techniques exploiting side channel leakage of
cryptographic devices. Computation requires time, consumes power and causes
electromagnetic radiations: all these are possible sources of information related
to the secret key. These techniques are powerful, as they allow to reduce the
complexity of a brute-force attack by several orders of magnitude. However, they
require physical access to the device to collect the necessary measurements. Fault-injection attack Fault attacks is the active way of attacking the physical
implementation of an algorithm. During the proper functioning of the device, the
attacker perturbs it by injecting hardware faults which produce an erroneous (or
faulted) output. The attacker then exploits this to retrieve secret information. As explained in [8], the most common ways to carry out such an attack are
manipulating the supply voltage or the the external clock, or applying laser or
X-ray beams. The SCARE attack More recently it has been shown ([3, 5, 7]) that side-channel
attacks could be used to retrieve secret parts of private algorithms. This is
called side-channel attack for reverse-engineering, or simply SCARE. when a
side-channel is used to retrieve an S-box on a private block cipher such as DES or
AES, the attacker studies the transition y = SB(x⊕k). In a classical side-channel
attack, SB is known and we wish to retrieve k. In SCARE, we assume to know k
and wish to retrieve SB. 3
The case of DES We first give a description of a FIRE attack on a DES-like cryptosystem. Even if
the attack has already been shown by Biham & Shamir in [2], it gives us a good
foundation to proceed during the more complex case of a SPN such as AES in
Sect. 4. The Data Encryption Standard (DES) was developed in the 1970s by the
National Bureau of Standards with the help of the National Security Agency. Its
purpose was to provide a standard method for protecting sensitive commercial
and unclassified data. IBM created the first draft of the algorithm, calling it
LUCIFER. DES officially became a federal standard in November of 1976 [12]. DES is a symmetric cryptosystem, specifically a 16-round Feistel cipher. It has a
64-bit block size and uses a 56-bit key. From this key, 16 sub-keys are created
and are used at each round. The input is split in two halves. The progression of
the cipher is described in Fig. 1. The round function, applied to a 32 bits register R and a 48 bits round Key
K, F(R, K), consists in the succession of 4 sub-functions: first, E is an expansion
function applied to R which returns a 48 bits output. The key K is then XOR-ed
to E(R). S is the substitution function. It consists in 8 S-Boxes SB0, . . . , SB7
each of which map a 6-bit input to a 4-bit output. A 32-bit permutation P is
finally applied to the output of S. IP
m
L0
R0
F
L1 = R0
R1 = L0 ⊕F(R0, K1)
F
R1 = L0 ⊕F(R0, K1)
R2 = L1 ⊕F(R1, K2)
R14 = L13 ⊕F(R13, K14)
R16 = L15 ⊕F(R15, K16)
L2 = R1
L15 = R14
L16 = R15
F
IP −1
c
Fig. 1. The DES cipher, a 16-round Feistel cipher. IP is a 64 bit permutation. The
round function applies F to the right half of the register, XORs the result to the left
half, and exchanges the roles of the halves. Fig. 1. The DES cipher, a 16-round Feistel cipher. IP is a 64 bit permutation. The
round function applies F to the right half of the register, XORs the result to the left
half, and exchanges the roles of the halves. 3
The case of DES We consider the fault model introduced by Biham and Shamir in [2]: it
assumes that the attacker is able to inject faults at the last round, round no. 15,
on the right register R15. We consider that the substitution function S has been
modified and kept secret. We then wish to retrieve SB0, . . . , SB7, the 8 S-Boxes
which compose it. Let c = (L16, R16) be the correct and c⋆= (L⋆
16, R⋆
16) be the faulty ciphertext,
resulting from the same plaintext m and secret key K. If we consider that the
secret key is not known but fixed to a certain value, we will not retrieve the exact
S-boxes, instead we will retrieve the function x 7→SBi(x ⊕ki), where ki is key
input of the ith S-Box. Without loss of generality, let us consider that the key is known for the attack,
hence we can ignore it for our present discussion. We thus have: R16 = L15 ⊕F(R15) = L15 ⊕F(L16)
and
R⋆
16 = L15 ⊕F(R⋆
15) = L15 ⊕F(L⋆
16). hence we get: R16 ⊕R⋆
16 = F(L16) ⊕F(L⋆
16)
R16 ⊕R⋆
16 = P[S(E(L16))] ⊕P[S(E(L⋆
16))]
P −1[R16 ⊕R⋆
16] = S(E(L16)) ⊕S(E(L⋆
16)). Since c and c⋆are known, the only unknown register, L15, disappears once
R16 is XOR-ed with R⋆
16. The intrinsic design of Feistel block-ciphers allows us to
have the knowledge of the fault injected, and its effect during the cipher, giving
us the difference at the input and output of the S-Boxes. We note ∆in and ∆out,
those differences: ∆in = E(L16) ⊕E(L⋆
16)
∆out = P −1[R16 ⊕R⋆
16]. where ∆in and ∆out are 48 and 32 bits long. However, if we focus on the ith
S-box Sbi for instance, we can consider ∆i
in and ∆i
out as 6 and 4 bits long. We
know x = E(L16)[6 ∗i : 6 ∗(i + 1)], the 6 bits input of SBi during the unaltered
cipher, x⋆= E(L⋆
16)[6 ∗i : 6 ∗(i + 1)], the 6 bits input of SBi during the faulty
cipher. We have the relation: Sbi(x) ⊕Sbi(x⋆) = ∆i
out. Sbi(x) ⊕Sbi(x⋆) = ∆i
out. Our goal is to retrieve Sbi, which is a boolean function from {0, 1}6 to
{0, 1}4. Let’s consider it component-wise, i.e. as 4 functions from {0, 1}6 to {0, 1}:
(s0, s1, s2, s3). 3
The case of DES From each injected fault, we must have: For j = 0, . . . , 3,
sj,x ⊕sj,x⋆= ∆i
out(j),
(3) (3) where ∆i
out(j) is the jth bit of ∆i
out. where ∆i
out(j) is the jth bit of ∆i
out. out( )
out
For each injected fault and for each component j, sj must satisfy the previous
equation. It is then added to the final system. We now have a distinguisher, we
can define Li,j,N as the set of candidates for the jth component of the ith S-box. Considering N fault injections, giving us (xk, x⋆
k, ∆out,k) ( k from 1 to N), we
have: Li,j,N = {s ∈{0, 1}64 such that ∀k, k ≤N : sxk ⊕sx⋆
k = ∆i
out,k(j)}. Simulating an error perturbing randomly one single input bit of an S-box
of DES, we reach the final set of two candidates mentioned at eq. (4) after Fig. 2. Attacking component 0 of the first S-Box of DES: On the x axis, the number of
faults injected, on the y-axis the mean of #L1,0,x after 1000 tries. In the end, we only
have 2 candidates. Fig. 2. Attacking component 0 of the first S-Box of DES: On the x axis, the number of
faults injected, on the y-axis the mean of #L1,0,x after 1000 tries. In the end, we only
have 2 candidates. approximately 130 fault injections. Fig. 2 illustrates the mean progression of
#L1,0,N with 1000 experiments. This attack converges to the expected solution,
meaning that, since we have the property (2), ∃n0 such that ∀n > n0,
Li,j,n = {sj, ¯sj}. (4) (4) Note that in order to fully retrieve the 8 S-boxes, one has to test both candidates
for all the 32 components. This leads to an exhaustive search in 232, which is
trivially feasible. 4
The case of AES AES is a widely used symmetric-key encryption by Daemen and Rijman [4],
adopted as a standard by the National Institute of Standards and Technology of
the US. It is based on a design principle known as a Substitution Permutation
Network (SPN). AES has a fixed block size of 128 bits and a key size of 128, 192,
or 256 bits. It operates on a 4 × 4 array of bytes, termed the state (where 1 byte
= 8 bits). Most calculations carried out by the cipher are done in the finite field
of GF(28). The AES cipher is specified as a number of repetitions of transformation
rounds, each round made up with 4 round transformations: SubBytes, Mix-
Columns, ShiftRows and AddRoundKey. Note that the last round is exempt
from MixColumns. Without loss of generality, since we consider that the cipher key is known, we
set it to 0, and we also discard the final ShiftRows operation since it can trivially
be inverted. Hence we only consider operations MixColumns and SubBytes,
as explained below in detail. MixColumns applies a linear transformation to a column of the state: MixColumns
x
y
z
t
=
02 03 01 01
01 02 03 01
01 01 02 03
03 01 01 02
·
x
y
z
t
, where the operations are performed in GF(28). SubBytes is a non linear transformation which is applied to each byte of the
state. It is traditionally implemented as a S-box, which can be seen as a boolean
function SB from 8 bits to 8 bits. Note that SubBytes is a bijection. SubBytes
x
y
z
t
=
SB(x)
SB(y)
SB(z)
SB(t)
. In our attack, this function is unknown, and the goal is to retrieve it. In our attack, this function is unknown, and the goal is to retrieve it. 4.1
Fault injection Let us assume that we are able to inject a fault on one byte of the block, just
before the last MixColumns, during the 9th round. The attack is column-wise,
meaning that we only care about the column on which the fault is injected. For example, let us look at the first column of a regular cipher, from the last
MixColumns until the end of the cipher. We have:
α
β
γ
δ
MC
−−→
MC
α
β
γ
δ
SB
−→
x
y
z
t
= c. (5) (5) Now, the same data is processed, but with a fault ϵ injected before the last
MixColumns. Fig. 3 illustrates the propagation of the error. Now, the same data is processed, but with a fault ϵ injected before the last
MixColumns. Fig. 3 illustrates the propagation of the error. Round 9
Round 10
MixCol
SubByte
Output
Fig. 3. Propagation of the fault on our simplified AES: we perturb a byte just before
the last MixColumns. The error propagates to the whole column. Fig. 3. Propagation of the fault on our simplified AES: we perturb a byte just before
the last MixColumns. The error propagates to the whole column. We thus have:
α
α ⊕ϵ
α ⊕ϵ
x⋆ We thus have:
α
β
γ
δ
FI
−→
α ⊕ϵ
β
γ
δ
MC
−−→
MC
α ⊕ϵ
β
γ
δ
SB
−→
x⋆
y⋆
z⋆
t⋆
= c⋆. (6) (6) Now that we have a triplet (c, c⋆, ϵ). Let us examine how we could exploit
Fault Injection to extract information on SB. We start the attack from the
ciphertexts, we retrieving SB−1, which is exactly the same since SB is bijective
in SPNs. We have, from eq. (5) and eq. (6): We have, from eq. (5) and eq. (6): ve, from eq. (5) and eq. 4.1
Fault injection (6):
SB−1(c) ⊕SB−1(c⋆) = MC
α
β
γ
δ
⊕MC
α ⊕ϵ
β
γ
δ
= MC
ϵ
0
0
0
=
02 · ϵ
ϵ
ϵ
03 · ϵ
, because MixColumns is linear. It translates into the system because MixColumns is linear. It translates into the system SB−1(x) ⊕SB−1(x⋆) = 02 · ϵ
SB−1(y) ⊕SB−1(y⋆) = ϵ
SB−1(z) ⊕SB−1(z⋆) = ϵ
SB−1(t) ⊕SB−1(t⋆) = 03 · ϵ
. (7) (7) 4.2
Translation of the FI into equations 4.2
Translation of the FI into equations Let us remind ourselves that SB−1 is a boolean function from {0, 1}8 to {0, 1}8. Considering it component-wise, i.e. as 8 independent functions from {0, 1}8 to
{0, 1}: SB−1 = {SB−1
0 , SB−1
1 , . . . , SB−1
7 }
with
SB−1
i
: {0, 1}8 7→{0, 1}}. Now, SB−1
i
can be seen as a set of 256 boolean variables: SB−1
i
= {si,0, si,1, . . . , si,255}. If we consider bit-wise the equations given in (7) then for a fault injected, we
know that, necessarily, for i = 0 . . . 7, SB−1
i
has to satisfy si,x ⊕si,x⋆= (02 · ϵ)i
si,y ⊕si,y⋆= ϵi
si,z ⊕si,z⋆= ϵi
si,t ⊕si,t⋆= (03 · ϵ)i
. (8) (8) (8) These four equations are to be manipulated according to the fault model, and
used to build the final system that is solved with Gaussian elimination to finally
give the solutions. 4.3
Random and unknown faults First, we discuss the fault model that is close to the one presented by Piret and
Quisquater in [13]. The error is injected on the first byte of the state, just before
the last MixColumns. It is random and unknown. By adding lines of the system
(7), without any knowledge of the value of ϵ, we have: SB−1(x) ⊕SB−1(x⋆) ⊕SB−1(y) ⊕SB−1(y⋆) ⊕SB−1(t) ⊕SB−1(t⋆) = 0
SB−1(x) ⊕SB−1(x⋆) ⊕SB−1(z) ⊕SB−1(z⋆) ⊕SB−1(t) ⊕SB−1(t⋆) = 0 , since 03 · ϵ ⊕02 · ϵ ⊕ϵ = 0. The operations are made on GF(28). 1 since 03 · ϵ ⊕02 · ϵ ⊕ϵ = 0. The operations are made on GF(28). Each one of the 8 components of SB−1 has to satisfy these equations. Now that
we have removed ϵ, we can inject them into the system. Once solved, this system
returns all the satisfying candidates, including the eight solutions. Considering N
fault injections, giving us (ck, c⋆
k) (k from 1 to N), we can define the distinguisher
LN for the attack of SB−1: LN =
s ∈{0, 1}256 such that ∀k < N,
sxk ⊕sx⋆
k ⊕syk ⊕sy⋆
k ⊕stk ⊕st⋆
k = 0
sxk ⊕sx⋆
k ⊕szk ⊕sz⋆
k ⊕stk ⊕st⋆
k = 0,
LN =
s ∈{0, 1}256 such that ∀k < N,
sxk ⊕sx⋆
k ⊕syk ⊕sy⋆
k ⊕stk ⊕st⋆
k = 0
sxk ⊕sx⋆
k ⊕szk ⊕sz⋆
k ⊕stk ⊕st⋆
k = 0,
It so happens that after n0 ≈400 faults injected, we have a constant set of
solutions S: ∀n > n0,
Ln = S. More precisely, the attack converges to a set S with 512 candidates. First we
describe in detail this set S, and then we discuss the possible conclusion of the
attack through exhaustive search. To account for the 512 solutions, we consider S as an orbit of the 8 compo-
nents of SB−1: we have always SB−1
0 , SB−1
1 , . . . , SB−1
7
∈S. But we also have
(0, 0, . . . , 0) and (1, 1, . . . , 1) in S (they indeed satisfy all the equations brought
by the distinguisher), we then state that: Proposition 1. u, v ∈S ⇒u ⊕v ∈S Proposition 1. u, v ∈S ⇒u ⊕v ∈S Proposition 1. u, v ∈S ⇒u ⊕v ∈S Proof. Without loss of generality, we shorten the definition of S to a single
boolean equation, which does not change with the real context. For instance: S = {s ∈{0, 1}256 such that
sy ⊕s⋆
y ⊕sz ⊕s⋆
z = 0}. S = {s ∈{0, 1}256 such that
sy ⊕s⋆
y ⊕sz ⊕s⋆
z = 0}. S = {s ∈{0, 1}256 such that
sy ⊕s⋆
y ⊕sz ⊕s⋆
z = 0}. Now let u, v ∈S. uy ⊕uy⋆⊕uz ⊕uz⋆= 0 , and vy ⊕vy⋆⊕vz ⊕vz⋆= 0. Then uy ⊕uy⋆⊕uz ⊕uz⋆⊕vy ⊕vy⋆⊕vz ⊕vz⋆= 0. Then (u ⊕v)y ⊕(u ⊕v)y⋆⊕(u ⊕v)z ⊕(u ⊕v)z⋆= 0. Then uy ⊕uy⋆⊕uz ⊕uz⋆⊕vy ⊕vy⋆⊕vz ⊕vz⋆= 0. Then (u ⊕v)y ⊕(u ⊕v)y⋆⊕(u ⊕v)z ⊕(u ⊕v)z⋆= 0. y
y
y
y
Then (u ⊕v)y ⊕(u ⊕v)y⋆⊕(u ⊕v)z ⊕(u ⊕v)z⋆= 0. Then (u ⊕v)y ⊕(u ⊕v)y⋆⊕(u ⊕v)z ⊕(u ⊕v)z⋆= 0. Finally u ⊕v ∈S. We now can define S such that: S = {a0 · SB−1
0
⊕. . . a7 · SB−1
7
⊕a8 · (1, . . . , 1),
ai ∈{0, 1}}. We can remove from S the trivial solution (1, . . . , 1) and (0, . . . , 0): in fact, it
is mandatory for a SPN S-box to be bijective, and it would not be the case if
(1, . . . , 1) or (0, . . . , 0) was one of the components. From this set, how can the full S-box be efficiently retrieved? We have 510
candidates that must be replaced into the correct position out of 8 possible
choices. A naive exhaustive search would lead to C510
8
× 8! ≈271 possibilities. However, as we have already noticed, ∀s ∈S, ¯s ∈S. We can form 255 groups
of elements of S, each of them including a candidate and its complement. For
an optimal exhaustive search, one has to select 8 of those groups, and then test
the 256 possibilities. This would lead to 28 × C255
8
≈257 possibilities to finish the
attack. This computational complexity is moderately high, but can be achieved
with a large set of modern GPUs and/or FPGAs, and is not out of reach of any
major organisation such as multinational companies or governments. However, we
also propose another solution by finishing the attack using the SCARE method. 4.4
SCARE conclusion of a FIRE attack In this section, we propose a finishing of a FIRE attack when we are in the
context described in Sect. 4.3. We have a set S, of 510 candidates containing the
8 component of SB−1. In order to use side-channel information to finish the attack, we use the curves
of the DPA-Contest [15] to find SB−1. The context is the following. We have – N power traces corresponding of the functioning of the components with
known inputs/outputs/cipher keys. 1 – N power traces corresponding of the functioning of the components with
known inputs/outputs/cipher keys. 1 – The set S of a reduced amount of candidates for the components of SB−1. Here, 510. It is well-known that the power consumption of components strongly depend on
the data processed, and more exactly the number of bit-flips completed. This
number is given by the hamming distance between a register at a time t and
t + 1. We then talk about Hamming distance model. We study here the transition
during the last SubBytes of the AES chiper. For every candidates s ∈S, for every component j of SB−1, we compute
what would be the hamming distance between c (which is known) and the state
at the input of the last SubBytes, if we would have s = SB−1
j . We then use a
distinguisher (Pearson’s correlation) in order to measure the dependence between
those hamming distances and the power traces. On Fig. 4, the correlation traces resulting from the attack of the 7th component
by using SCARE. This means that we are looking for SB−1
7
amongst the 510
members of S. On the figure, by using 10000 traces, we clearly can identify SB−1
7
in red and bold,
¯
SB−1
7 , the symmetric below, and the 508 bad candidates, giving
a correlation close to zero. An adversary able to perform fault injections on a Fig. 4. SCARE on the 7th component of SB−1, with N = 10000 power traces. We have
the 510 correlation traces: on the x-axis the time samples of the power traces, on the
y-axis, the value of the correlation. We clearly identify here the solution (on the top)
and its complementary (on the bottom) Fig. 4. SCARE on the 7th component of SB−1, with N = 10000 power traces. 4.4
SCARE conclusion of a FIRE attack We have
the 510 correlation traces: on the x-axis the time samples of the power traces, on the
y-axis, the value of the correlation. We clearly identify here the solution (on the top)
and its complementary (on the bottom) component is very likely to be able to get a campaign of acquisition of power
traces in order to conclude the attack this way. Hence it alleviates the burden on
the attacker of making the exhaustive search in 257 as munitioned at the end of
Sec. 4.3. Note that the SCARE attack is feasible here since we have a very restricted
number of candidate for the solutions. When dealing with SCA, the number
of candidates to test is very important: 256 hypothesis to test when we want
to retrieve a key byte, but 228 hypothesis to test when we are looking for a
single component of SB−1. Here the FIRE attack carried out most of the job by
reducing the 228 to 510. On Fig. 4, we have the results with N = 10000 power curves. However, from
N = 5000 curves (taken randomly from the ones available for the DPA Contest),
the attack is feasible, meaning that we are able to extract the solutions. 4.5
Results with various fault models and contexts In this section, we present several other realistic fault models, or context allowing
us to perform a FIRE attacks. Random and known faults Let us consider the strongest fault model: we are
able to inject a random and known fault during the cipher execution. 100
110
120
130
140
150
160
170
180
190
200
0
10
20
30
40
50
60
70
80
90
100
Number of faults injected
Number of Solutions returned
Case of AES
Fig. 5. Attacking component 0 of AES S-Box inverse. On the x-axis, the number of
faults injected, on the y-axis the mean of #L0,x after 100 tries. In the end, we only
have 2 candidates. 100
110
120
130
140
150
160
170
180
190
200
0
10
20
30
40
50
60
70
80
90
100
Number of faults injected
Number of Solutions returned
Case of AES Fig. 5. Attacking component 0 of AES S-Box inverse. On the x-axis, the number of
faults injected, on the y-axis the mean of #L0,x after 100 tries. In the end, we only
have 2 candidates. The advantage with this model, is that, since we know ϵ, we are able to target
which one of the components of SB−1 we are attacking. k Considering N fault injections, giving us (ck, c⋆
k, ϵk), k = 1, . . . N, we can
define the distinguisher Li,N for the attack of the ith component of SB−1: Li,N =
s ∈{0, 1}256 such that ∀k < N,
sxk ⊕sx⋆
k = (02 · ϵk)i
syk ⊕sy⋆
k = ϵk
i
szk ⊕sz⋆
k = ϵk
i
stk ⊕st⋆
k = (03 · ϵk)i
. This model, combined with the technique described in Sect. 4.2, allows us
to retrieve the full SB−1 in less than 180 faults injected. Fig. 5 illustrates the
progression of L0,N, simulating an error occurring randomly on the first byte of
the state just before the last MixColumns. Stuck-at model It has been shown that it is possible for an attacker to force a
byte to a certain value, that it can choose. 5
Conclusion In this paper, we have introduced a new tool to reverse-engineer a private
algorithm. This new FIRE attack allows us to retrieve the S-Box of private
block-ciphers in a reasonable number of faults injected and under plausible and
existing fault models. For the sake of practical demonstration, we have carried
out the attack on two major ciphers, AES and DES, but the attack can be made
to work on almost any cipher containing and unknown S-Box. In the case of the
DES S-boxes, around 1000 Fault Injections are needed and a final exhaustive
search in 232 is necessary to fully retrieve all the 8 S-Boxes. For AES, under
the most plausible model, around 400 fault injections suffice and lead to a finite
set of 510 candidates. We can then either conclude the attack using exhaustive
search in 257, or perform a data acquisition campaign and finish the attack using
SCARE. ∃ϵ ∈GF(28) such that τ = α ⊕ϵ. ∃ϵ ∈GF(28) such that τ = α ⊕ϵ. It hence leads to the fault model presented at Sec. 4.3, just as if we would have
injected an unknown and random ϵ. 3. Clavier, C. An improved scare cryptanalysis against a secret a3/a8 gsm algorithm.
In ICISS (2007), P. D. McDaniel and S. K. Gupta, Eds., vol. 4812 of Lecture Notes
in Computer Science, Springer, pp. 143–155. 2. Biham, E., and Shamir, A. Differential fault analysis of secret key cryptosystems.
In CRYPTO (1997), B. S. K. Jr., Ed., vol. 1294 of Lecture Notes in Computer
Science, Springer, pp. 513–525. 1. Anderson, R. A5 (was: Hacking digital phones). Newsgroup Communication,
1994. 4.5
Results with various fault models and contexts If we suppose that, just before the
last SubBytes, one can force the first byte to a given value τ:
α
β
γ
δ
FI
−→
τ
β
γ
δ
SB
−→
x⋆
y⋆
z⋆
t⋆
= c⋆. Hence he has access to x⋆which is equal to SB(τ). It leads to a trivial attack,
since with 256 accurate stuck-at injections, one can retrieve the full S-Box. Note that even one single stuck-at injection, we get a lot of information to
bring into the system, if we decide do use different models during the attack. In the case where it is not possible to inject a stuck-at fault at the input of
the last SubBytes, but that it can be done just before the last MixColumns:
α
β
γ
δ
FI
−→
τ
β
γ
δ
MC
−−→
MC
τ
β
γ
δ
SB
−→
x⋆
y⋆
z⋆
t⋆
= c⋆. In that case, even if we know the value of τ, α is assumed to be random. But we
have: 4. Daemen, J., and Rijmen, V. The block cipher rijndael. In CARDIS (1998), J.-J.
Quisquater and B. Schneier, Eds., vol. 1820 of Lecture Notes in Computer Science,
Springer, pp. 277–284. References 2. Biham, E., and Shamir, A. Differential fault analysis of secret key cryptosystems. In CRYPTO (1997), B. S. K. Jr., Ed., vol. 1294 of Lecture Notes in Computer
Science, Springer, pp. 513–525. 3. Clavier, C. An improved scare cryptanalysis against a secret a3/a8 gsm algorithm. In ICISS (2007), P. D. McDaniel and S. K. Gupta, Eds., vol. 4812 of Lecture Notes
in Computer Science, Springer, pp. 143–155. 4. Daemen, J., and Rijmen, V. The block cipher rijndael. In CARDIS (1998), J.-J. Quisquater and B. Schneier, Eds., vol. 1820 of Lecture Notes in Computer Science,
Springer, pp. 277–284. 13 5. Daudigny, R., Ledig, H., Muller, F., and Valette, F. Scare of the des. In
ACNS (2005), pp. 393–406. 6. Garcia, F. D., and et al. Dismantling MIFARE Classic. In ESORICS (2008),
S. Jajodia and J. L´opez, Eds., vol. 5283 of LNCS, Springer, pp. 97–114. 7. Guilley, S., Sauvage, L., Micolod, J., R´eal, D., and Valette, F. Defeating
any secret cryptography with scare attacks. In LATINCRYPT (2010), M. Abdalla
and P. S. L. M. Barreto, Eds., vol. 6212 of Lecture Notes in Computer Science,
Springer, pp. 273–293. 8. Hamid, H. B.-E., Choukri, H., Tunstall, D. N. M., and Whelan, C. The
sorcerer’s apprentice guide to fault attacks. 9. Kerckhoffs, A. La cryptographie militaire. Journal des sciences militaires IX
(January 1883), 5–83. 10. Kocher, P. C. Timing attacks on implementations of diffie-hellman, rsa, dss, and
other systems. 104–113. 11. Kocher, P. C., Jaffe, J., and Jun, B. Differential power analysis. In CRYPTO
(1999), M. J. Wiener, Ed., vol. 1666 of Lecture Notes in Computer Science, Springer,
pp. 388–397. 12. National Bureau of Standards. Data Encryption Standard, 1977. 13. Piret, G., and Quisquater, J.-J. A differential fault attack technique against
spn structures, with application to the aes and khazad. In Cryptographic Hardware
and Embedded Systems - CHES 2003, vol. 2779 of Lecture Notes in Computer
Science. Springer Berlin / Heidelberg, 2003, pp. 77–88. 14. Stein, W., et al. Sage Mathematics Software. http://www.sagemath.org. 15. VLSI Research Group TELECOM ParisTech. The DPA contest, 2008/2009. http://www.dpacontest.org/. 14
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Flight State Identification of a Self-Sensing Wing via an Improved Feature Selection Method and Machine Learning Approaches
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Sensors
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Received: 31 March 2018; Accepted: 24 April 2018; Published: 29 April 2018 Abstract: In this work, a data-driven approach for identifying the flight state of a self-sensing
wing structure with an embedded multi-functional sensing network is proposed. The flight state is
characterized by the structural vibration signals recorded from a series of wind tunnel experiments
under varying angles of attack and airspeeds. A large feature pool is created by extracting potential
features from the signals covering the time domain, the frequency domain as well as the information
domain. Special emphasis is given to feature selection in which a novel filter method is developed
based on the combination of a modified distance evaluation algorithm and a variance inflation
factor. Machine learning algorithms are then employed to establish the mapping relationship from
the feature space to the practical state space. Results from two case studies demonstrate the high
identification accuracy and the effectiveness of the model complexity reduction via the proposed
method, thus providing new perspectives of self-awareness towards the next generation of intelligent
air vehicles. Keywords: self-sensing wing; feature extraction; feature selection; flight state identification;
machine learning Xi Chen 1,*, Fotis Kopsaftopoulos 2 ID , Qi Wu 3, He Ren 1 and Fu-Kuo Chang 4 1
Shanghai Engineering Research Center of Civil Aircraft Health Monitoring, Shanghai Aircraft Customer
Service Co., Ltd., Shanghai 200241, China; renhe@comac.cc
2
Department of Mechanical, Aerospace, and Nuclear Engineering, Rensselaer Polytechnic Institute,
Troy, NY 12180, USA; kopsaf@rpi.edu
3
School of Electronic, Information and Electrical Engineering, Shanghai Jiao Tong University,
Shanghai 200240, China; wuqi7812@sjtu.edu.cn 3
School of Electronic, Information and Electrical Engineering, Shanghai Jiao Tong University,
Shanghai 200240, China; wuqi7812@sjtu.edu.cn 4
Department of Aeronautics and Astronautics, Stanford University, Stanford, CA 94305, USA;
fkchang@stanford.edu g
*
Correspondence: chenxi1@comac.cc or aero.x.chen@gmail.com; Tel.: +86-21-20875861 sensors sensors sensors rticle
Flight State Identification of a Self-Sensing Wing via
an Improved Feature Selection Method and Machine
Learning Approaches Xi Chen 1,*, Fotis Kopsaftopoulos 2 ID , Qi Wu 3, He Ren 1 and Fu-Kuo Chang 4 1. Introduction In the active mode, they can be used for damage detection and structural
health monitoring while in passive mode, the wing structural vibration during flying can be captured
to reflect the air dynamic characteristics [11]. The wing configuration is shown in Figure 1. the current structural design and data processing methods with a departure from the existing
technologies. Recent years have seen the development of different sensing network architectures and
simulations [3–6], among which, an expandable network made of polymer-based substrates was
designed by the Structure and Composites Lab (SACL) at Stanford University. This network contains
many micro-nodes which have the potential to integrate micro-sensors, actuators and electronics for
different applications [7]. Based on the development of integration and fabrication techniques [8–10],
a smart structure with the sensor network monolithically embedded in the layup of a composite UAV
wing was successfully fabricated [11]. This smart wing consists of four sensor networks and each
network is integrated with strain gauges, resistive temperature detectors (RTD) and piezoelectric lead
zirconate titanate (PZT) transducers. Specifically, the strain gauge is used to measure the wing strain
distribution and identify any potentially dangerous areas. RTD detects the temperature distribution
in order to provide the temperature compensation [12]. PZT transducers can be used for both active
and passive measurements. In the active mode, they can be used for damage detection and structural
health monitoring while in passive mode, the wing structural vibration during flying can be captured
to reflect the air dynamic characteristics [11]. The wing configuration is shown in Figure 1. Figure 1. The self-sensing composite wing design [2]. Figure 1. The self-sensing composite wing design [2]. Figure 1. The self-sensing composite wing design [2]. Figure 1. The self-sensing composite wing design [2]. After realizing sensing ability through multi functional structures development, the next step is
to equip the smart wing with thinking and judging capability, i.e., the structure is expected to be
aware of surroundings and identify its current flying state. There have been studies devoted to
addressing the related identification problem based on either strain or vibration signals obtained
from experiments. Huang et al. studied the active flutter control and closed-loop flutter identification
and a fast-recursive subspace method was applied in high-dimensional aero-servo-elastic system. The wind tunnel test showed that the natural frequency and modal damping ratios of the flutter
modes can be precisely tracked [13]. 1. Introduction Pang and Cesnik employed non-linear least squares fit and
Kalman filtering to obtain wing shape information and rigid body attitude. Results revealed that the
Kalman filter has good performance in the presence of sensor noise [14]. For elastic deformation,
Sodja et al. conducted a dynamic aeroelastic wind tunnel experiment under harmonic pitching
excitations, experimental data including the bending and torsion deformation were consistent with
the elastic analysis model developed by the Delft University of Technology [15]. For more general
flight states, Kopsaftopoulos and Chang established a stochastic global identification method using
PZT signals from both time and frequency domain based on developed Vector-dependent
Functionally Pooled (VFP) model [2,16,17]. A large range of airspeeds and angles of attack were
considered in the VFP-based identification framework and the structural dynamics of the composite
wing could be captured and predicted. Overall, the above data processing approaches mainly belong to state space methods and
improved time series analysis Based on the previous study yet from another perspective if we can
After realizing sensing ability through multi-functional structures development, the next step is to
equip the smart wing with thinking and judging capability, i.e., the structure is expected to be aware of
surroundings and identify its current flying state. There have been studies devoted to addressing the
related identification problem based on either strain or vibration signals obtained from experiments. Huang et al. studied the active flutter control and closed-loop flutter identification and a fast-recursive
subspace method was applied in high-dimensional aero-servo-elastic system. The wind tunnel test
showed that the natural frequency and modal damping ratios of the flutter modes can be precisely
tracked [13]. Pang and Cesnik employed non-linear least squares fit and Kalman filtering to obtain
wing shape information and rigid body attitude. Results revealed that the Kalman filter has good
performance in the presence of sensor noise [14]. For elastic deformation, Sodja et al. conducted
a dynamic aeroelastic wind tunnel experiment under harmonic pitching excitations, experimental
data including the bending and torsion deformation were consistent with the elastic analysis model
developed by the Delft University of Technology [15]. For more general flight states, Kopsaftopoulos
and Chang established a stochastic global identification method using PZT signals from both time and
frequency domain based on developed Vector-dependent Functionally Pooled (VFP) model [2,16,17]. 1. Introduction The current state sensing and awareness of flight vehicles relies on traditional sensors and
detection devices mounted on different locations of the vehicle, e.g., Pitot tubes installed in front of the
nose for airspeed measurement, transducers located on each side of the fuselage for angle of attack
detection. Inspired by the unsurpassed flight capabilities of birds, a novel “fly-by-feel” (FBF) concept
has been recently proposed for the development of the next generation of intelligent air vehicles that
can “feel”, “think”, and “react” [1,2]. Such bio-inspired systems will not only be able to sense the
environment (temperature, pressure, aerodynamic forces, etc.), but also be able to think in real-time and
be aware of their current flight state and structural health condition. Further, such systems will react
intelligently under various situations and achieve superior performance and agility. Compared with
the traditional approaches, this FBF concept has the following advantages: (1) structural complexity
reduction by integrated structures with self-sensing ability, (2) structural health on-line monitoring
through embedded multi-functional materials, (3) autonomous flight control and decision-making Sensors 2018, 18, 1379; doi:10.3390/s18051379 www.mdpi.com/journal/sensors 2 of 21 Sensors 2018, 18, 1379 based on self-awareness [2]. Towards this end, great challenges have been posed to the current
structural design and data processing methods with a departure from the existing technologies. Sensors 2018, 18, x
2 of 21
decision-making based on self-awareness [2]. Towards this end, great challenges have been posed to Recent years have seen the development of different sensing network architectures and
simulations [3–6], among which, an expandable network made of polymer-based substrates was
designed by the Structure and Composites Lab (SACL) at Stanford University. This network contains
many micro-nodes which have the potential to integrate micro-sensors, actuators and electronics for
different applications [7]. Based on the development of integration and fabrication techniques [8–10],
a smart structure with the sensor network monolithically embedded in the layup of a composite UAV
wing was successfully fabricated [11]. This smart wing consists of four sensor networks and each
network is integrated with strain gauges, resistive temperature detectors (RTD) and piezoelectric lead
zirconate titanate (PZT) transducers. Specifically, the strain gauge is used to measure the wing strain
distribution and identify any potentially dangerous areas. RTD detects the temperature distribution
in order to provide the temperature compensation [12]. PZT transducers can be used for both active
and passive measurements. 1. Introduction A large range of airspeeds and angles of attack were considered in the VFP-based identification
framework and the structural dynamics of the composite wing could be captured and predicted. improved time series analysis. Based on the previous study yet from another perspective, if we can
Overall, the above data processing approaches mainly belong to state space methods and
improved time series analysis. Based on the previous study yet from another perspective, if we can Sensors 2018, 18, 1379 3 of 21 extract distinguished features from the continuous coupled structural aerodynamic behavior, it is
possible to identify the flight state directly using the limited features instead of detailed characterization
of the structural responses. Machine learning techniques can be employed to establish the mapping
relationship from the feature space to the practical state space. Facing a series of signals generated from the embedded sensor network, one of the main challenges
is what kind of features should be extracted and whether these features are useful for classification. A set of features without careful selection and evaluation may lead to poor results whatever superior
machine learning models are applied. Feature engineering is such a research field including feature
extraction and selection. For a period of time series signals with noise, various statistical features
can be calculated such as the mean value, standard deviation, peak value, kurtosis, etc. from both
time domain and frequency domain [18], a feature pool is then created with different number of
features depending on the characteristics of the signals [19–21]. More features are encouraged to
avoid missing important candidates with superior classification performance. The next step is feature
selection in which a limited subset is obtained by eliminating less effective features. It reduces
model dimension and computational time [22]. Generally, feature selection can be divided into three
categories as filter, wrapper and embedded. Filter methods rank the variables completely separate
to the model used for classification. The assignment of feature importance is based on information
generated by some statistical algorithms. Filter methods are computationally simple and fast without
the interaction with the classifier and feature dependencies [23]. Embedded solutions select salient
features as part of the learning process of the model, which can be linear regression, support vector
machine, decision tree, random forest, etc. These methods integrate the subset selection into the model
construction but are difficult to adjust for the optimal search [24]. 1. Introduction The third category is wrapper,
in which features are selected based on the performance of a given model by searching the possible
subsets space and assessing the performance of the given model on each subset, models can be various
learning machines [25]. Although wrapper methods often achieve sound classification performance by
considering the feature dependencies, the frequent interactions between feature subset search and the
classifier cause high computational costs [26]. We have demonstrated the effectiveness of establishing the mapping relationship from the feature
space to the flight state space through neural networks modelling [27]. This paper significantly
improves the previous work by creating a much larger feature pool and considering the co-linearity
among various features. To sum up, the objective of this paper is the introduction and evaluation
of a novel feature selection method for accurate flight state identification of a self-sensing wing
structure based on experimental vibration data recorded by piezoelectric sensors under multiple flight
states. The developed method belongs to the filter family and is capable of obtaining a group of
most important features for classification with low mutual dependency. The framework of the data
acquisition, methodology development, evaluation and application is shown in Figure 2. The rest of the paper is organized as follows: Section 2 presents the problem statement. Section 3
focuses on the feature extraction and feature selection in which the novel filter algorithm is introduced. Two case studies including the general flight state identification and the stall detection and alerting are
conducted in Section 4 followed by their results and discussions in Section 5. Concluding marks are
made in the last section. 4 of 21
loyed to Sensors 2018, 18, 1379
haracterization of Data Acquisition
Self-sensing wing
PZT signal preparation
Methodology Development
Feature extraction
Feature selection
Application: Stall Alerting
2
1
VIF
1
j
j
R
Modified distance evaluation
(
)
,
1
1
K
w
j
k j
k
d
d
K
( )
(
)
b
j
j
j
w
j
d
d
Variance inflation factor
( )
,
,
,
1
1
(
1)
K
b
j
e j
k j
k e
d
u
u
K K
Feature pool
Time domain
Frequency
domain
Information
domain
Evaluation: Flight State Identification
3D visulizaiton
Correlation Analysis
Accuracy comparison
Stall phenomenon
Accuracy comparison
Figure 2. Framework of the proposed methodology. 1. Introduction Facing a series of signals generated from the embedded sensor network, one of t
enges is what kind of features should be extracted and whether these features are u
ification. A set of features without careful selection and evaluation may lead to poo
tever superior machine learning models are applied. Feature engineering is such a resea
di
f
i
d
l
i
F
i d
f i
i
i
l
i h
i
Figure 2. Framework of the proposed methodology. Problem Statement
The problem statement of this work is as follows: based on signals collected from th
nsors embedded in the self-sensing wing through a series of experiments under varying
ates develop a feature selection method that is capable of obtaining limited useful featur Data Acquisition
Self-sensing wing
PZT signal preparation
Methodology Development
Feature extraction
Feature selection
2
1
VIF
1
j
j
R
Modified distance evaluation
(
)
,
1
1
K
w
j
k j
k
d
d
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( )
(
)
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,
,
,
1
1
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Time domain
Frequency
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Information
domain Data Acquisition Methodology Development Methodology Development Evaluation: Flight State Identification
3D visulizaiton
Correlation Analysis
Accuracy comparison Application: Stall Alerting
Evaluation: Flight State Identification
3D visulizaiton
Correlation Analysis
Accuracy comparison
Stall phenomenon
Accuracy comparison
Figure 2. Framework of the proposed methodology. Figure 2. Framework of the proposed methodology. Evaluation: Flight State Identification Application: Stall Alerting Figure 2. Framework of the proposed methodology
Figure 2. Framework of the proposed methodology. Facing a series of
hallenges is what kind
2. Problem Statement hallenges is what kind of features should be extracted and whether these features are useful fo
lassification. A set of features without careful selection and evaluation may lead to poor result
whatever superior machine learning models are applied. Feature engineering is such a research field
ncluding feature extraction and selection. For a period of time series signals with noise, variou
The problem statement of this work is as follows: based on signals collected from the PZT
sensors embedded in the self-sensing wing through a series of experiments under varying flight
states, develop a feature selection method that is capable of obtaining limited useful features for
flight state identification with high accuracy and low model complexity. Specifically, the coupled 5 of 21 Sensors 2018, 18, 1379 aerodynamic-mechanical responses represent different flight states, with each state characterized by
a specific angle of attack (AoA) and airspeed and kept constant during the data collection. The first
problem is that whether a few salient features can be extracted from a period of vibrational time series
(e.g., thousands of data points) as a representation of the corresponding flight state. In this way, we can
skip the investigation into the detailed aeroelastic behavior and use the limited features to identify
the specific flight state directly instead of using the entire lengthy signal. This would significantly
reduce the complexity of the flight state characterization. The second problem is how to guarantee the
effectiveness of selected features. If the selected strong features are highly correlated with each other,
they will exhibit similar identification ability which are still away from the optimal subset. The above two problems constitute the motivation of this study and are addressed in the following
approaches: firstly, a large number of features is extracted to cover a wide range of descriptions of
the flight state. Then, a modified distance evaluation algorithm is conducted to obtain a subset of
individually powerful features followed by the combination of a variance inflation factor algorithm
to reduce high dependency among features in the subset. Machine learning models are employed to
evaluate the above method for multiple flight states identification as well as a specific case of stall
detection and alerting. The main novel aspects of this study include: The main novel aspects of this study include: (1)
A large feature pool is created covering up to 47 different features from the time, frequency and
information domains. Facing a series of
hallenges is what kind
2. Problem Statement (1)
A large feature pool is created covering up to 47 different features from the time, frequency and
information domains. (2)
A novel filter feature selection method is developed by combining a modified distance evaluation
algorithm and a variance inflation factor. (2)
A novel filter feature selection method is developed by combining a modified distance evaluation
algorithm and a variance inflation factor. (3)
The flight state identification is treated as a classification problem by establishing the mapping
relationship from the feature space to the physical space characterized by varying angle of attack
and airspeed of the self-sensing wing structure in wind tunnel experiments. (3)
The flight state identification is treated as a classification problem by establishing the mapping
relationship from the feature space to the physical space characterized by varying angle of attack
and airspeed of the self-sensing wing structure in wind tunnel experiments. (4)
The application on stall detection and alerting with high identification accuracy provides new
perspectives for autonomous flight control with real-time flight state monitoring. (4)
The application on stall detection and alerting with high identification accuracy provides new
perspectives for autonomous flight control with real-time flight state monitoring. 3. Methodology Development In this section, a novel filter feature selection method is proposed via the combination of
a modified distance evaluation algorithm and a variance inflation factor. In order to obtain sufficient
feature candidates, a large feature pool is firstly created by extracting features covering a wide range. The output of this method is a feature subset consisting of most salient features with low correlation,
which is able to represent a lengthy time-series signal of the wing structural response under certain
flight state. 3.1. Feature Extraction t4 = ∑N
n=1 (x(n)−t1)4
N
t10 = min(x(n))
t16 = t9
t7
t5 = ∑N
n=1(x(n)−t1)2
N
t11 = t9 −t10
t17 = t3
t63
t6 =
q
∑N
n=1 (x(n))2
N
t12 = ∑N
n=1|x(n)−t1|
N
t18 = t4
t64
t25 = ∑N
n=1 (x(n)−t1)9
N·t29
Note: x(n) is a signal series for n = 1, 2, . . . , N, N is the number of data points. Table 1. Features in time domain. Time Domain Feature Parameters
Un-Dimensional
t1 = ∑N
n=1 x(n)
N
t7 =
∑N
n=1
√
|x(n)|
N
2
t13 = t9
t6
t19 = ∑N
n=1 (x(n)−t1)3
N·t23
t2 =
q
∑N
n=1(x(n)−t1)2
N
t8 = ∑N
n=1|x(n)|
N
t14 = t6
t8
t20 = ∑N
n=1 (x(n)−t1)4
N·t24
t3 = ∑N
n=1 (x(n)−t1)3
N
t9 = max(x(n))
t15 = t9
t8
. . . t4 = ∑N
n=1 (x(n)−t1)4
N
t10 = min(x(n))
t16 = t9
t7
t5 = ∑N
n=1(x(n)−t1)2
N
t11 = t9 −t10
t17 = t3
t63
t6 =
q
∑N
n=1 (x(n))2
N
t12 = ∑N
n=1|x(n)−t1|
N
t18 = t4
t64
t25 = ∑N
n=1 (x(n)−t1)9
N·t29
Note: x(n) is a signal series for n = 1, 2, . . . , N, N is the number of data points. Table 2. Features in the frequency domain. Table 2. Features in the frequency domain. Frequency Domain Feature Parameters
f1 = ∑K
k=1 y(k)
N
f6 =
q
∑K
k=1 ( f rk−f5)2y(k)
K
f10 = f6
f5
f2 = ∑K
k=1 (y(k)−f1)2
K
f7 =
r
∑K
k=1 f rk2y(k)
∑K
k=1 y(k)
f11 = ∑K
k=1 ( f rk−f5)3y(k)
K· f63
f3 = ∑K
k=1 (y(k)−f1)3
K(√
f2)
3
f8 =
r
∑K
k=1 f rk4y(k)
∑K
k=1 f rk2y(k)
f12 = ∑K
k=1 ( f rk−f5)4y(k)
K· f64
f4 = ∑K
k=1 (y(k)−f1)4
K· f22
f9 =
∑K
k=1 f rk2y(k)
√
∑K
k=1 y(k)∑K
k=1 f rk4y(k)
f13 = ∑K
k=1
√
| f rk−f5|y(k)
K√
f6
f5 = ∑K
k=1 ( frk·y(k))
∑K
k=1 y(k)
Note: y(k) is a spectrum for k = 1, 2, . . . , K, K is the number of spectrum components;f rk is the frequency value of
the kth spectrum line. 3.1. Feature Extraction Feature extraction relies heavily on experts’ knowledge, it is encouraged to extract different kinds
of features, as many as possible in case of missing useful ones. In this study, we intend to create a large
feature pool from three main sources, namely the time, frequency and information domains. In time domain, 25 statistical features are calculated including 12 commonly used features such
as mean, standard deviation, variance, peak, mean absolute deviation, etc. and 13 un-dimensional
features such as crest factor, shape factor and a series of normalized central moments. The expressions
of all time domain features are listed in Table 1. In terms of their physical insights, t1–t12 may reflect
the vibration amplitude and energy while t13–t25 may represent the series distribution of the signal in
time domain. Previous studies employed Fast Fourier Transform (FFT) to convert the time series into frequency
spectrum [19,20]. However, the signal instances from the wind tunnel experiments are samples of
a stochastic process with considerable noise. Welch’s method improves FFT by shortening the signals
and averaging, and thus the peaks are smoothed for noise reduction [28]. Herein, a sample-long 6 of 21 Sensors 2018, 18, 1379 Hamming data window with 90% overlap is used for the Welch-based spectral estimation. A series of
power spectrum y(k) without log transformation is then used for frequency domain feature extraction. Thirteen statistical features such as mean spectrum, spectrum center, root mean square spectrum, etc. and their mathematical expressions are shown in Table 2. f 1 may indicate the vibration energy in the
frequency domain. f 2–4, f 6, f 10–13 may describe the convergence of the spectrum power. f 5, f 7–9 may
show the position change of the main frequency. Hamming data window with 90% overlap is used for the Welch-based spectral estimation. A series of
power spectrum y(k) without log transformation is then used for frequency domain feature extraction. Thirteen statistical features such as mean spectrum, spectrum center, root mean square spectrum, etc. and their mathematical expressions are shown in Table 2. f 1 may indicate the vibration energy in the
frequency domain. f 2–4, f 6, f 10–13 may describe the convergence of the spectrum power. f 5, f 7–9 may
show the position change of the main frequency. Table 1. Features in time domain. 3.1. Feature Extraction Time Domain Feature Parameters
Un-Dimensional
t1 = ∑N
n=1 x(n)
N
t7 =
∑N
n=1
√
|x(n)|
N
2
t13 = t9
t6
t19 = ∑N
n=1 (x(n)−t1)3
N·t23
t2 =
q
∑N
n=1(x(n)−t1)2
N
t8 = ∑N
n=1|x(n)|
N
t14 = t6
t8
t20 = ∑N
n=1 (x(n)−t1)4
N·t24
t3 = ∑N
n=1 (x(n)−t1)3
N
t9 = max(x(n))
t15 = t9
t8
. . . t4 = ∑N
n=1 (x(n)−t1)4
N
t10 = min(x(n))
t16 = t9
t7
t5 = ∑N
n=1(x(n)−t1)2
N
t11 = t9 −t10
t17 = t3
t63
t6 =
q
∑N
n=1 (x(n))2
N
t12 = ∑N
n=1|x(n)−t1|
N
t18 = t4
t64
t25 = ∑N
n=1 (x(n)−t1)9
N·t29
Note: x(n) is a signal series for n = 1, 2, . . . , N, N is the number of data points. Table 2. Features in the frequency domain. Frequency Domain Feature Parameters
f1 = ∑K
k=1 y(k)
N
f6 =
q
∑K
k=1 ( f rk−f5)2y(k)
K
f10 = f6
f5
f2 = ∑K
k=1 (y(k)−f1)2
K
f7 =
r
∑K
k=1 f rk2y(k)
∑K
k=1 y(k)
f11 = ∑K
k=1 ( f rk−f5)3y(k)
K· f63
f3 = ∑K
k=1 (y(k)−f1)3
K(√
f2)
3
f8 =
r
∑K
k=1 f rk4y(k)
∑K
k=1 f rk2y(k)
f12 = ∑K
k=1 ( f rk−f5)4y(k)
K· f64
f4 = ∑K
k=1 (y(k)−f1)4
K· f22
f9 =
∑K
k=1 f rk2y(k)
√
∑K
k=1 y(k)∑K
k=1 f rk4y(k)
f13 = ∑K
k=1
√
| f rk−f5|y(k)
K√
f6
f5 = ∑K
k=1 ( frk·y(k))
∑K
k=1 y(k)
Note: y(k) is a spectrum for k = 1, 2, . . . , K, K is the number of spectrum components;f rk is the frequency value of
he kth spectrum line. Table 1. Features in time domain. Table 1. Features in time domain. Time Domain Feature Parameters
Un-Dimensional
t1 = ∑N
n=1 x(n)
N
t7 =
∑N
n=1
√
|x(n)|
N
2
t13 = t9
t6
t19 = ∑N
n=1 (x(n)−t1)3
N·t23
t2 =
q
∑N
n=1(x(n)−t1)2
N
t8 = ∑N
n=1|x(n)|
N
t14 = t6
t8
t20 = ∑N
n=1 (x(n)−t1)4
N·t24
t3 = ∑N
n=1 (x(n)−t1)3
N
t9 = max(x(n))
t15 = t9
t8
. . . 3.1. Feature Extraction Its
mathematical expression using normalized singular spectrum is: FI = ∑
M−1
i=1
(σi+1 −σi)2
σi
(5) (5) where σi is the normalized value through σi = σi/∑M
j=1 σj, and M is the number of singular value j
j
Approximate Entropy (ApEn) quantifies the amount of regularity and the unpredictability of
fluctuations of a signal [36], which is computed in the following procedures: j
j
Approximate Entropy (ApEn) quantifies the amount of regularity and the unpredictability of
fluctuations of a signal [36], which is computed in the following procedures: (1)
Set the input as [x1, x2, . . . , xN]. (1)
Set the input as [x1, x2, . . . , xN]. (2)
Construct the subsequence x(i, m) = [xi, xi+1, . . . , xi+m−1] for 1 ≤i ≤N −m, where m is the
subsequence length. (3)
C
t
t
t f
b
{x(j m)}
{x(j m)|j ∈[1
N
m]}
h
x(j m) i d fi
d ( )
p
[ 1
N]
(2)
Construct the subsequence x(i, m) = [xi, xi+1, . . . , xi+m−1] for 1 ≤i ≤N −m, where m is the
subsequence length. (2)
Construct the subsequence x(i, m) = [xi, xi+1, . . . , xi+m−1] for 1 ≤i ≤N −m, where m is the
subsequence length. (2)
Construct the subsequence x(i, m) = [xi, xi+1, . . . , xi+m−1] for 1 ≤i ≤N
m, where m is the
subsequence length. (3)
Construct a set of subsequences {x(j, m)} = {x(j, m)|j ∈[1, . . . N −m]}, where x(j, m) is defined
in Step (2). q
g
(3)
Construct a set of subsequences {x(j, m)} = {x(j, m)|j ∈[1, . . . N −m]}, where x(j, m) is defined
in Step (2). (3)
Construct a set of subsequences {x(j, m)} = {x(j, m)|j ∈[1, . . . N −m]}, where x(j, m) is define
in Step (2). (4)
For each x(i, m) ∈{x(j, m)}, C(i, m) =
∑N−m
j=1
kj
N−m , where kj =
(
1
if |x(i, m) −x(j, m)|< r
0
otherwise (4)
For each x(i, m) ∈{x(j, m)}, C(i, m) =
∑N−m
j=1
kj
N−m , where kj =
(
1
if |x(i, m) −x(j, m)|< r
0
otherwise
. 3.1. Feature Extraction . . , MSE(12)]. where Ske = 3(MSEa −MSEb)/MSEc, MSEa, MSEb, MSEc represent mean, median and standard
deviation of MSE(τ) = [MSE(1), MSE(2), . . . , MSE(12)]. Fractal dimension characterizes the space filling capacity of a pattern that changes with the scale
at which it is measured [33]. Herein, two approaches are used as Petrosian Fractal Dimension (PFD)
and Higuchi Fractal Dimension (HFD). PFD is calculated as: PFD =
log10 N
log10 N + log10(N/(N + 0.4Nδ))
(3) (3) where N is the length of the signal and Nδ is the number of sign changes in the signal derivative [30]. In terms of HFD, firstly k new series are constructed from the original signal [x1, x2, . . . , xN] by
[xm, xm+k, xm+2k, . . . , xm+⌊(N−m)/k⌋k], where m = 1, 2, . . . , k. Secondly the length L(m, k) for each new
series is calculated as: where N is the length of the signal and Nδ is the number of sign changes in the signal derivative [30]. In terms of HFD, firstly k new series are constructed from the original signal [x1, x2, . . . , xN] by
[xm, xm+k, xm+2k, . . . , xm+⌊(N−m)/k⌋k], where m = 1, 2, . . . , k. Secondly the length L(m, k) for each new
series is calculated as: L(m, k) =
∑⌊(N−m)/k⌋
i=2
xm+ik −xm+(i−1)k
(N −1)
⌊(N −m)/k⌋k
(4) (4) and the average length L(k) =
j
∑k
i=1 L(i, k)
k
/k. After kmax repetitions, a least-squares method is used
to obtain the best slope that fits the curve of ln(L(k)) versus ln(1/k), which is defined as the Higuchi
Fractal Dimension. For details, please refer to [34]. and the average length L(k) =
j
∑k
i=1 L(i, k)
k
/k. After kmax repetitions, a least-squares method is used
to obtain the best slope that fits the curve of ln(L(k)) versus ln(1/k), which is defined as the Higuchi
Fractal Dimension. For details, please refer to [34]. Fisher Information (FI) measures the expected value of the observed information [35]. 3.1. Feature Extraction Frequency Domain Feature Parameters
f1 = ∑K
k=1 y(k)
N
f6 =
q
∑K
k=1 ( f rk−f5)2y(k)
K
f10 = f6
f5
f2 = ∑K
k=1 (y(k)−f1)2
K
f7 =
r
∑K
k=1 f rk2y(k)
∑K
k=1 y(k)
f11 = ∑K
k=1 ( f rk−f5)3y(k)
K· f63
f3 = ∑K
k=1 (y(k)−f1)3
K(√
f2)
3
f8 =
r
∑K
k=1 f rk4y(k)
∑K
k=1 f rk2y(k)
f12 = ∑K
k=1 ( f rk−f5)4y(k)
K· f64
f4 = ∑K
k=1 (y(k)−f1)4
K· f22
f9 =
∑K
k=1 f rk2y(k)
√
∑K
k=1 y(k)∑K
k=1 f rk4y(k)
f13 = ∑K
k=1
√
| f rk−f5|y(k)
K√
f6
f5 = ∑K
k=1 ( frk·y(k))
∑K
k=1 y(k)
Note: y(k) is a spectrum for k = 1, 2, . . . , K, K is the number of spectrum components;f rk is the frequency value of
the kth spectrum line. Frequency Domain Feature Parameters Note: y(k) is a spectrum for k = 1, 2, . . . , K, K is the number of spectrum components;f rk is the frequency value of
the kth spectrum line. In electroencephalograph (EEG) analysis for neural diseases diagnosis and vibration analysis for
mechanical defects, fractal dimensions from computational geometry and entropies from information
theory have demonstrated effectiveness in early diseases/fault diagnosis [29,30]. Inspired by that,
a group of complex features are employed and their terminologies are Multi-Scale Entropy, Partial Mean
of Multi-Scale Entropy, Petrosian Fractal Dimension, Higuchi Fractal Dimension, Fisher Information,
Approximate Entropy, and Hurst Exponent, respectively. pp
py
p
p
y
Multi-Scale Entropy (MSE) introduces the scale factor based on the sample entropy to measure
the complexity of signal under different scale factors [31]. It is calculated as: MSE = {τ
SampEn(τ, m, r) = −ln[Cr,m+1(r)/Cr,m(r)]}
(1) (1) where τ is the scale factor, m is the embedding dimension and r is the threshold. Here m = 2,
r = 0.2 * standard deviation, τ = 12. Sensors 2018, 18, 1379 7 of 21 The first three values are selected due to the relatively high distinction among different classes. Also, an integrated non-linear index called Partial Mean of Multi-Scale Entropy (PMMSE) is used to
simultaneously reflect the mean value and variation trend of MSE [32], which is expressed as: PMMSE = (1+|Ske|/3) · MSEa
(2) (2) where Ske = 3(MSEa −MSEb)/MSEc, MSEa, MSEb, MSEc represent mean, median and standard
deviation of MSE(τ) = [MSE(1), MSE(2), . 3.1. Feature Extraction (5)
ApEn is calculated as: (5)
ApEn is calculated as: ApEn(m, r, N) =
1
N −M
"
N−m
∑
i=1
ln
C(i, m)
C(i, m + 1)
#
(6) (6) Hurst Exponent (HST) measures the long-term memory of a signal. It is used to quantify the relative
tendency of the signal either to regress to the mean or to cluster in a direction [37]. For time series 8 of 21 Sensors 2018, 18, 1379 X = [x1, x2, . . . , xN], its accumulated deviation within range T is calculated as X(t, T) = ∑t
i=1 (xi −x),
where x = 1
T ∑T
i=1 xi, t ∈[1, 2, . . . , N]. Then: R(T)
S(T) = max(X(t, T)) −min(X(t, T))
q
(1/T)∑T
t=1 [x(t) −x]2
(7) (7) The slope of ln(R(n)/S(n)) versus ln(n) for n ∈[2, 3, . . . , N] is defined as the Hurst Ex The slope of ln(R(n)/S(n)) versus ln(n) for n ∈[2, 3, . . . , N] is defined as the Hurst Exponent. The slope of ln(R(n)/S(n)) versus ln(n) for n ∈[2, 3, . . . , N] is defined as the Hurst Exponent. In summary, abbreviations of the complex features extracted from information domain are listed
in Table 3. The slope of ln(R(n)/S(n)) versus ln(n) for n ∈[2, 3, . . . , N] is defined as the Hurst Exponent. In summary, abbreviations of the complex features extracted from information domain are listed
in Table 3. In summary, abbreviations of the complex features extracted from information domain are listed
in Table 3. In summary, abbreviations of the complex features extracted from information domain are listed
able 3. Table 3. Features in information domain. Table 3. Features in information domain. Table 3. Features in information domain. Table 3. Features in information domain. Information Domain Feature Parameters
I1 = MSE [1]
I4 = PMMSE
I7 = FI
I2 = MSE [2]
I5 = PFD
I8 = ApEn
I3 = MSE [3]
I6 = HFD
I9 = HST
3 2 Feature Selection Information Domain Feature Parameters
I1 = MSE [1]
I4 = PMMSE
I7 = FI
I2 = MSE [2]
I5 = PFD
I8 = ApEn
I3 = MSE [3]
I6 = HFD
I9 = HST Information Domain Feature Parameters 3.2. Feature Selection 3.2. Feature Selection , K, k ̸= e
(11) (11) 9 of 21 Sensors 2018, 18, 1379 (3)
Calculate the variance factor of d(b)
j
as: v(b)
j
=
sum(
ue,j −uk,j
)
min(
ue,j −uk,j
)
(12) v(b)
j
=
sum(
ue,j −uk,j
)
min(
ue,j −uk,j
)
(12) (12) (4)
Calculate the compensation factor as: (4)
Calculate the compensation factor as: (4)
Calculate the compensation factor as: δj =
sum(vb
j )
v(b)
j
(13) δj =
sum(vb
j )
v(b)
j δj =
sum(vb
j )
v(b)
j
(13) (13) (5)
Calculate the ratio d(b)
j
and d(w)
j
considering the compensation factor: αj = δj
d(b)
j
d(w)
j
(14) (14) then normalize αj and obtaining the feature importance criteria: then normalize αj and obtaining the feature importance criteria: then normalize αj and obtaining the feature importance criteria: then normalize αj and obtaining the feature importance criteria: αj =
αj
sum(αj)
(15) (15) A higher αj indicates that the corresponding feature j has greater importance. Features can be
ranked in terms of the αj values in Equation (15) in descending order. This algorithm is referred
to as Modified Distance Evaluation algorithm (MDE). Although the top ranked features have
superior discriminative capability, they may suffer from high multi-collinearity, which refers to the
non-independence among features [39]. Herein, the variance inflation factor (VIF) is used to avoid
high collinearity. Assuming a training sample set X with J features X1, X2, . . . , XJ and class Y, the VIF
of feature j is calculated as: VIFj =
1
1 −R2
j
(16) (16) where R2
j is the R-squared value of the regression equation Xj = β0 + βX′, in which X′ contains all
features except Xj. An improved algorithm combining MDE and VIF is presented in Algorithm 1 and
is abbreviated as MDV (Modified Distance evaluation and variance inflation Factor). Set the selected future subset Fsub = ∅, j = 1; (1)
Set the selected future subset Fsub = ∅, j = 1; (1)
Set the selected future subset Fsub = ∅, j = 1; (2)
Rank the J features in terms of the αj defined in Equation (15) in descending order. Set Fr to represent t
index list of the ranked features. (1)
Set the selected future subset Fsub = ∅, j = 1; Algorithm 1: MDV Algorithm. 3.2. Feature Selection 3.2. Feature Selection Feature extraction guarantees a wide coverage of the object descriptions from various aspects
while feature selection ensures that a set of most salient descriptions can be utilized. For large-scale
models, feature selection is of utter importance in computation reduction and efficiency improvement. The distance evaluation technique ranks the feature importance independent of the model used
for classification, which belongs to the filter category as mentioned in the Introduction. Salient
features result in minimum inner-class distances of the same class while have maximum margins for
different classes. It has been widely used in fault diagnosis of rotating machinery [20,21,38]. Suppose
a feature set has K conditions,
qi,k,j, i = 1, 2, . . . , Ik; k = 1, 2, . . . , K; j = 1, 2, . . . , J
, where qi,k,j is the
jth eigenvalue of the ith sample under the kth condition, Ik is the sample number of the kth condition,
and J is the feature number of each sample. Totally Ik × K × J features are obtained in the feature set
qi,k,j
. Herein, a modified distance evaluation algorithm is presented as follows: e average distance of the same condition samples: (1)
Calculate the average distance of the same condition samples: dk,j =
1
Ik × (Ik −1)∑
Ik
l,i=1
qi,k,j −ql,k,j
,
l, i = 1, 2, . . . , Ik, l ̸= i
(8) (8) then obtain the average distance of K conditions: then obtain the average distance of K conditions: d(w)
j
= 1
K∑
K
k=1 dk,j
(9) d(w)
j
= 1
K∑
K
k=1 dk,j (9) (2)
Calculate the average eigenvalue of all samples under the same condition: (2)
Calculate the average eigenvalue of all samples under the same condition: (2)
Calculate the average eigenvalue of all samples under the same condition: (2) uk,j = 1
Ik∑
Ik
i=1 qi,k,j
(10) (10) then obtain the average distance between condition samples: then obtain the average distance between condition samples: then obtain the average distance between condition samples: d(b)
j
=
1
K(K −1)∑
K
k,e=1
ue,j −uk,j
,
k, e = 1, 2, . . . 3.2. Feature Selection Add the first feature in Fr to Fsub, j = j + 1; (3)
while j < J :
calculate the VIFj of the jth feature in Fr with the features in Fsub;
if VIFj < 10: add the jth feature in Fr to Fsub; The MDV algorithm describes the feature-subset selection for multi-class classification based on
the filter method with the MDE and VIF. The threshold of 10 in MDV is an empirical value. A larger 10 of 21 Sensors 2018, 18, 1379 threshold will result in a higher correlation of the selected feature in Fr with the existing features in
Fsub [23]. threshold will result in a higher correlation of the selected feature in Fr with the existing features in
Fsub [23]. 4.1. Data Prepraration A series of wind tunnel experiments of the self-sensing composite wing were conducted under
various angles of attack (AoAs) and freestream velocities at Stanford University. The open-loop wind
tunnel with a square test section of 0.76 m by 76 m was used and a basis was designed to supported
the composite wing allowing adjustments in the angle of attack (AoA). The composite wing dimension
is outlined in Table 4. Table 4. Wing Dimension. Wing Geometry
Chord
0.235 m
Span
0.86 m
Area
0.2 m2
Aspect ratio
3.66 Table 4. Wing Dimension. Compared with the size of the wind tunnel test section, the additional 0.1 m extension of the
wing span was attached to the wing fixture. The AoAs range from 0 degree up to 18 degrees with an
incremental step of 1 degree. At each degree, data were collected for all velocities ranging from 9 to
22 m/s (incremental step of 1 m/s). For experimental details, please refer to [2]. PZT signals reflect the coupled airflow-structural dynamics through the wing structural vibration
and each time series contains coupled behavior with repeated patterns of a certain flight state. This
study focuses on the usage of PZT sensor signals for flight state identification. In each experiment,
the structural vibration responses (60,000 data points) were recorded from the PZT located near the
wing root at 1000 Hz sampling frequency. For each flight state, data are prepared in two steps: (1) the
entire signal of 60,000 data points is divided into 60 segments (1000 data points for each segment) to
ensure enough samples for training while each segment has sufficient data points for feature extraction;
(2) the first order difference and zero-mean are conducted for each sample sequence in order to
eliminate the influence of zero drift. To evaluate the effectiveness of the proposed method and apply it
for dangerous state pre-warning, two sets of data are collected for general flight state identification
and stall detection and alerting. 4.2. General Flight State Identification To evaluate the effectiveness of the proposed
method and apply it for dangerous state pre-warning, two sets of data are collected for general flight
state identification and stall detection and alerting. (4)
Stability selection is a kind of wrapper method, in which features are selected based on the
established models using different subsets, model could be of various types and structures such
as logistic regression, SVM, etc. By calculating the frequency of a feature ended up being selected
as important from a feature subset being tested, powerful features are expected to have high
scores close to 100%, weaker features will have lower score and the least useful ones will close to
zero [44]. Herein, a randomized logistic regression is used as the selection model. state identification and stall detection and alerting. 4.2. General Flight State Identification
The first data set includes PZT signals with a coarse resolution covering the range of 16 flight
states corresponding to combinations of four AoAs (1, 5, 9, 13 degrees) and four airspeeds (10, 13, 16,
19 m/s). Four signal segments are shown in Figure 3 under a series of AoAs and a fixed airspeed of
10 m/s as an example Figure 3. Indicative signals under a set of AoAs and a constant velocity of 10 m/s. Figure 3. Indicative signals under a set of AoAs and a constant velocity of 10 m/s. Figure 3. Indicative signals under a set of AoAs and a constant velocity of 10 m/s. Figure 3. Indicative signals under a set of AoAs and a constant velocity of 10 m/s. 4.2. General Flight State Identification For experimental details, please refer to [2]. PZT signals reflect the coupled airflow-structural dynamics through the wing structural It measures the mutual dependence between variable X and Y. Features with low rankings
are removed. incremental step of 1 degree. At each degree, data were collected for all velocities ranging from 9 to
22 m/s (incremental step of 1 m/s). For experimental details, please refer to [2]. PZT signals reflect the coupled airflow structural dynamics through the wing structural (2)
SVM_L1 is one of the embedded methods, which selects salient features as part of the learning
system [18]. Support Vector Machine (SVM) is a popular machine learning method based on
structural risk minimization principle. It constructs a hyperplane that has the largest distance to
the nearest training data points, which are so called support vectors. An appropriate separation
can reduce the generalization error of the classifier [41]. L1 is a regularization item added to the
loss function as |W|, where W standards for the parameter matrix of the learning model [42]. This is a penalty item to make the model sparse with fewer useful input dimensions. PZT signals reflect the coupled airflow-structural dynamics through the wing structural
vibration and each time series contains coupled behavior with repeated patterns of a certain flight
state. This study focuses on the usage of PZT sensor signals for flight state identification. In each
experiment, the structural vibration responses (60,000 data points) were recorded from the PZT
located near the wing root at 1000 Hz sampling frequency. For each flight state, data are prepared in
two steps: (1) the entire signal of 60,000 data points is divided into 60 segments (1000 data points for
each segment) to ensure enough samples for training while each segment has sufficient data points
for feature extraction; (2) the first order difference and zero-mean are conducted for each sample (3)
GBDT is a tree-based model belonging to the embedded category. It combines weak decision
trees in an iterative manner based on gradient descent through additive training. Trees are added
at each iteration with modified parameters learned in the direction of residual loss reduction [43]. for feature extraction; (2) the first order difference and zero mean are conducted for each sample
sequence in order to eliminate the influence of zero drift. 4.2. General Flight State Identification The first data set includes PZT signals with a coarse resolution covering the range of 16 flight
states corresponding to combinations of four AoAs (1, 5, 9, 13 degrees) and four airspeeds (10, 13, 16,
19 m/s). Four signal segments are shown in Figure 3 under a series of AoAs and a fixed airspeed of
10 m/s as an example. It is noticed that the flight state with AoA of 13 degrees and velocity of 10 m/s can be obviously
identified since the amplitude of the voltage distinguishes it from other signals (it is because this
flight state is close to the stall condition which will be discussed later). The second largest amplitude
comes with 9 degrees which can be separated to a certain extent but already has overlaps with the
rest two. In the study, the identification of the different flight states relies on the features selected by
the developed method in Section 3. To compare the feature selection effectiveness, four other feature
selection methods are employed including Univariate Feature Selection based on mutual information
(UFS_m), Support Vector Machine with L1 regularization (SVM_L1), Gradient Boosted Decision Tree
(GBDT) and Stability selection (STAB). These methods cover three main feature selection categories. A brief introduction is presented as follows: rs 2018, 18, 1379
11 of 2 11 of 21 Sensors 2018, 18, 1379 (1)
UFS_m is a commonly used filter method. It performs test on each feature by evaluating the
relationship between the feature and the response variable based on mutual information [40],
which is defined as
p(x y)
Sensors 2018, 18, x
10 of 21 (1)
UFS_m is a commonly used filter method. It performs test on each feature by evaluating the
relationship between the feature and the response variable based on mutual information [40],
which is defined as
p(x y)
Sensors 2018, 18, x
10 of 21 I(X, Y) = ∑y∈Y ∑x∈X p(x, y) log( p(x, y)
p(x)p(y))
(17)
e of the wind tunnel test section, the additional 0.1 m extension of the
he wing fixture. The AoAs range from 0 degree up to 18 degrees with an (17)
f the
th an It measures the mutual dependence between variable X and Y. Features with low rankings
are removed. incremental step of 1 degree. At each degree, data were collected for all velocities ranging from 9 to
22 m/s (incremental step of 1 m/s). It is noticed that the flight state with AoA of
identified since the amplitude of the voltage dis
4.3. Application to Stall Detection and Identification From the wind tunnel experiments, the
mean values of the signal energy for a series of AoAs (from 0 to 17 degrees) under four airspeeds (10,
13, 16, 19 m/s) are obtained and shown in Figure 4. Figure 4. Signal energy under various flight states. Figure 4. Signal energy under various flight states. Figure 4. Signal energy under various flight states. Figure 4. Signal energy under various flight states. It is noticed that the flight state with AoA of
identified since the amplitude of the voltage dis
4.3. Application to Stall Detection and Identification Moreover, it is observed that at 12 degrees, the signal energy for
some flight states has certain increase compared with the rest small angles. This degree is defined as
the alert region as the transition between the safe region marked in light green and the critical stall
region. When the self-sensing wing comes to this region, warnings should be provided to the flight
control for angle reduction. The second data set covers a higher resolution of flight states (AoAs: 11, 12, 13 degrees,
airspeeds: 10, 13, 16, 19 m/s) for critical states alerting. In aerodynamics, stall phenomenon is one of
the dangerous conditions wherein a sudden reduction of the lift coefficient occurs as the angle of
attack increases beyond a critical point. According to previous analysis [2], the signal energy can be
used as an indicator of the lift loss of the self-sensing wing. From the wind tunnel experiments, the
mean values of the signal energy for a series of AoAs (from 0 to 17 degrees) under four airspeeds (10,
13, 16, 19 m/s) are obtained and shown in Figure 4. 13 degrees with the high speed of 19 m/s, which is reflected in the red line with zero energy starting
from 14 degrees. Therefore, we define the orange shaded area starting from 13 degrees as the stall
region which should be avoided. Moreover, it is observed that at 12 degrees, the signal energy for
some flight states has certain increase compared with the rest small angles. This degree is defined as
the alert region as the transition between the safe region marked in light green and the critical stall
region. When the self-sensing wing comes to this region, warnings should be provided to the flight
control for angle reduction. The second data set covers a higher resolution of flight states (AoAs: 11, 12, 13 degrees,
airspeeds: 10, 13, 16, 19 m/s) for critical states alerting. In aerodynamics, stall phenomenon is one of
the dangerous conditions wherein a sudden reduction of the lift coefficient occurs as the angle of
attack increases beyond a critical point. According to previous analysis [2], the signal energy can be
used as an indicator of the lift loss of the self-sensing wing. It is noticed that the flight state with AoA of
identified since the amplitude of the voltage dis
4.3. Application to Stall Detection and Identification p
g
g
g
(
flight state is close to the stall condition which will be discussed later). The second largest amplitude
comes with 9 degrees which can be separated to a certain extent but already has overlaps with the
rest two. In the study, the identification of the different flight states relies on the features selected by
the developed method in Section 3. To compare the feature selection effectiveness, four other feature
selection methods are employed including Univariate Feature Selection based on mutual information
(UFS_m), Support Vector Machine with L1 regularization (SVM_L1), Gradient Boosted Decision Tree
(GBDT) and Stability selection (STAB). These methods cover three main feature selection categories. The second data set covers a higher resolution of flight states (AoAs: 11, 12, 13 degrees, airspeeds:
10, 13, 16, 19 m/s) for critical states alerting. In aerodynamics, stall phenomenon is one of the dangerous
conditions wherein a sudden reduction of the lift coefficient occurs as the angle of attack increases
beyond a critical point. According to previous analysis [2], the signal energy can be used as an indicator
of the lift loss of the self-sensing wing. From the wind tunnel experiments, the mean values of the
signal energy for a series of AoAs (from 0 to 17 degrees) under four airspeeds (10, 13, 16, 19 m/s) are
obtained and shown in Figure 4. A brief introduction is presented as follows:
(1) UFS_m is a commonly used filter method. It performs test on each feature by evaluating the
relationship between the feature and the response variable based on mutual information [40],
which is defined as
The signal energy variation with respect to the angle of attack is similar under four different
airspeeds. It is noticed that for relatively low velocities (10 m/s, 13 m/s & 16 m/s), the significant
increase occurs approximately after 14 degrees while for the relatively high speed (19 m/s), stall
happens much early at 13 degrees. It should be noted that the data were stopped recording after 12 of 21 Sensors 2018, 18, 1379
[
] 13 degrees with the high speed of 19 m/s, which is reflected in the red line with zero energy starting
from 14 degrees. Therefore, we define the orange shaded area starting from 13 degrees as the stall
region which should be avoided. ai
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increase occurs approximately afte
5.1. General Flight State Identification happens much early at 13 degrees. It should be noted that the data were stopped recording after 13
degrees with the high speed of 19 m/s, which is reflected in the red line with zero energy starting
The first data set with a relatively low resolution of 16 flight states is used to evaluate the
performance of six feature selection methods, which include Univariate Feature Selection based on
mutual information (UFS_m), Support Vector Machine with L1 regularization (SVM_L1), Gradient
Boosted Decision Tree (GBDT) and Stability selection (STAB), Modified Distance Evaluation (MDE),
and our proposed filter method Modified Distance Evaluation with Variance Inflation Factor (MDV). Feature rankings are obtained and the top 10 features for different methods are listed in Table 5 and
their detailed expressions are listed in Appendix A. Table 5. Top 10 ranking matrix. Ranking
UFS_m
SVM_L1
GBDT
STAB
MDE
MDV
1
F25
F41
F47
F47
F35
F35
2
F34
F43
F40
F12
F26
F30
3
F6
F39
F46
F21
F2
F5
4
F2
F25
F14
F20
F6
F28
5
F5
F46
F39
F19
F31
F42
6
F4
F19
F44
F18
F30
F45
7
F40
F33
F41
F17
F12
F41
8
F23
F13
F1
F16
F8
F46
9
F42
F44
F21
F15
F36
F14
10
F17
F10
F45
F14
F10
F23 Table 5. Top 10 ranking matrix. Sensors 2018, 18, 1379 13 of 21 13 of 21 It is observed from the table that the ranking results vary with the different methods. An intuitive
evaluation is to simply visualize the features distribution under various flight states. For example, four
features are plotted in Figure 5 including: F1 (mean value in time domain), F29 (spectrum kurtosis in
frequency domain), F35 (spectrum power convergence in frequency domain), and F47 (Hurst Exponent
in information domain). The x axis denotes the 16 flight states while the y axis is the feature value before
normalization. The shaded area along each vertical line segment represents the feature distribution
in a single flight state and each subplot of Figure 5 describes a feature distribution on 16 flight
states. As mentioned in Section 3, F1 (mean value) has no effects in classification. Correspondingly,
F1 has the highest overlap among flight states. Similarly, F47 has large overlaps which exhibits pool
classification capability. Theoretically, the ranking of F1 and F47 should be low but they are ranked
high in GBDT and STAB. ai
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increase occurs approximately afte
5.1. General Flight State Identification Correlation between features by MDV (a) and MDE (b). can be seen that the feature subset through MDV sel
mpared to the entire feature pool p
y
g
y
mparison, the overall collinearity of the features in MDV is much lower except for the small regio
the top three. To visualize the feature selection performance by MDV, t-Distributed Stochastic Neighb
mbedding (t-SNE) is employed which is a relatively new method of dimension reductio
rticularly suitable for non-linear and high-dimensional datasets. It is a kind of manifold learnin
hnique by mapping to probability distributions through affine transformation. For detaile
gorithm, please refer to [45]. The 3D visualization by t-SNE is shown in Figure 7. The left figure
e visualization using the entire feature pool while the right figure uses only top six featur
(a)
(b)
gure 7. 3D visualization by t-SNE: (a) t-SNE using original features; (b) t-SNE using selecte
atures
Figure 7. 3D visualization by t-SNE: (a) t-SNE using original features; (b) t-SNE using selected features. p
mparison, the overall collinearity of the fea
he top three. To visualize the feature selection perf
bedding (t-SNE) is employed which is
ticularly suitable for non-linear and high-
hnique by mapping to probability distr
orithm, please refer to [45]. The 3D visual
visualization using the entire feature p
(a) much lower except for the sm DV is much lower except for the small regi
by MDV, t-Distributed Stochastic Neighb
vely new method of dimension reducti
nal datasets. It is a kind of manifold learni
hrough affine transformation. For detail
t-SNE is shown in Figure 7. The left figure
the right figure uses only top six featur
(b) (b) (a) ure 7. 3D visualization by t-SNE: (a) t-SNE using original features; (b) t-SNE using selec
Figure 7. 3D visualization by t-SNE: (a) t-SNE using original features; (b) t-SNE using selected features. Further, machine learning techniques are used to quantify the flight state identification proce
each feature selection method, the most salient 6 features are obtained as model inputs and
light states are set as model outputs. Five supervised learning models are employed includ
istic Regression (LR), Support Vector Machine (SVM), Naïve Bayes (NB), Random Forest (R
Neural Network (NN). ai
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a
o
e a
increase occurs approximately afte
5.1. General Flight State Identification In comparison, F30 and F35 show smaller overlap and thus have better
classification performance. This may provide some physical insights of the effectiveness of different
feature selection methods. sors 2018, 18, x
13 Figure 5. Pool and superior features against 16 flight states. Figure 5. Pool and superior features against 16 flight states. igure 5. Pool and superior features against 16 flight stat
Figure 5. Pool and superior features against 16 flight states. The last column MDV in Table 4 is an improvement of MDE for preventing high collinearity
amine the effects of the proposed algorithm, Correlation analysis is conducted for MDV and M
shown in Figure 6. The last column MDV in Table 4 is an improvement of MDE for preventing high collinearity. To examine the effects of the proposed algorithm, Correlation analysis is conducted for MDV and MDE
as shown in Figure 6. It is obvious that the top 10 features selected by MDE are highly correlated with each other. In comparison, the overall collinearity of the features in MDV is much lower except for the small
region of the top three. To visualize the feature selection performance by MDV, t-Distributed Stochastic Neighbor
Embedding (t-SNE) is employed which is a relatively new method of dimension reduction particularly
suitable for non-linear and high-dimensional datasets. It is a kind of manifold learning technique by
mapping to probability distributions through affine transformation. For detailed algorithm, please
refer to [45]. The 3D visualization by t-SNE is shown in Figure 7. The left figure is the visualization
using the entire feature pool while the right figure uses only top six features obtained by MDV. It can Sensors 2018, 18, 1379 14 of 21 14 of 21 be seen that the feature subset through MDV selection exhibits better classification effects compared to
the entire feature pool. p
p
g
g
y
examine the effects of the proposed algorithm, Correlation analysis is conducted for MDV and MDE
as shown in Figure 6. (a)
(b)
Figure 6. Correlation between features by MDV (a) and MDE (b). Figure 6. Correlation between features by MDV (a) and MDE (b). 2018, 18, x
1
ned by MDV. It can be seen that the feature subset through MDV selection exhibits
fication effects compared to the entire feature pool. (a)
018, 18, x (b) (b) (a) Figure 6. Correlation between features by MDV (a) and MDE (b). Figure 6. ai
pee
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a
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increase occurs approximately afte
5.1. General Flight State Identification Cross-validation is used in each model and the average accuracy va
ve tests is computed to reduce the unbalance influence between training and testing samples
uld be noted that since the objective of the case study is to compare the effects of different feat
ction methods instead of obtaining the optimized parameter setting for each machine learn
del to achieve the highest accuracy level, default parameter settings in Python scikit-learn pack
LR, SVM, NB and RF are used and remain the same for all feature selection methods while
the parameter setting is as follows: {hidden layer size = 20 solver = ‘lbfgs’ activation functio
Further, machine learning techniques are used to quantify the flight state identification process. For each feature selection method, the most salient 6 features are obtained as model inputs and the
16 flight states are set as model outputs. Five supervised learning models are employed including
Logistic Regression (LR), Support Vector Machine (SVM), Naïve Bayes (NB), Random Forest (RF), and
Neural Network (NN). Cross-validation is used in each model and the average accuracy value of five
tests is computed to reduce the unbalance influence between training and testing samples. It should
be noted that since the objective of the case study is to compare the effects of different feature selection
methods instead of obtaining the optimized parameter setting for each machine learning model to
achieve the highest accuracy level, default parameter settings in Python scikit-learn package for LR,
SVM, NB and RF are used and remain the same for all feature selection methods while for NN, the
parameter setting is as follows: {hidden layer size = 20, solver = ‘lbfgs’, activation function = ’relu’,
learning rate = 0.001, maximum iteration = 100}. The identification results are shown in Figure 8. 15 of 21 Sensors 2018, 18, 1379
’relu’, learning
Fi
8 Figure 8. Identification accuracy against different feature selection methods. Figure 8. Identification accuracy against different feature selection methods. Figure 8. Identification accuracy against different feature selection methods. Figure 8. Identification accuracy against different feature selection methods. It can be observed that our proposed method MDV achieves the highest identification accuracy
in all five machine learning models and particularly, there is a significant improvement in Logistic
It can be observed that our proposed method MDV achieves the highest identification accuracy
in all five machine learning models and particularly, there is a significant improvement in Logistic
Regression. ai
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a
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increase occurs approximately afte
5.1. General Flight State Identification This demonstrates the superior effectiveness of MDV. The comparison between MDV
and MDE shows that a group of individually powerful features with low collinearity can lead to
better results. 5.2. Stall Detection and Alerting So far, the developed MDV algorithm has achieved the best performance in feature selection and
the final flight state identification accuracy is up to 100%. Herein, the second dataset with higher
resolution is used for the application of stall detection and alerting. Similarly, totally 47 features as
discussed in Section 3 are extracted and the most salient 6 features are selected by MDV as model
inputs. A neural network is employed with the same parameter settings as the first case. The split rule
is 80% samples for training and 20% samples for testing. The classification report is shown in Table 6 including three criteria: Precision, Recall and
F1-score. Precision is the ratio of correctly predicted positive observations to the total predicted
positive observations while Recall is the ratio of correctly predicted positive observations to the all
observations in the actual class. F1-Score is the weighted average of Precision and Recall: F1-Score =
2 * (Recall * Precision)/(Recall + Precision) [46]. Safe, Alert, and Stall regions are divided with
corresponding flight states. The overall identification accuracy is 98%. Table 6. Classification report. States ID
AoA (deg)
Speed (m/s)
Precision
Recall
F1-Score
Safe
1
11
10
0.92
1.00
0.96
2
11
13
0.92
1.00
0.96
3
11
16
1.00
0.92
0.96
4
11
19
1.00
0.92
0.96
Alert
5
12
10
1.00
1.00
1.00
6
12
13
1.00
0.92
0.96
7
12
16
0.92
1.00
0.96
8
12
19
1.00
1.00
1.00
Stall
9
13
10
1.00
1.00
1.00
10
13
13
1.00
1.00
1.00
11
13
16
1.00
1.00
1.00
12
13
19
1.00
1.00
1.00 Table 6. Classification report. Table 6. Classification report. Sensors 2018, 18, 1379 16 of 21 16 of 21 To facilitate detailed analysis, a normalized confusion matrix is presented in Figure 9. Each row
of the matrix represents the test samples in a true class label while each column indicates the samples
in a predicted class label [47]. As can be observed from Table 6, for stall states (ID: 9, 10, 11, 12), Recall
values all equal to 100%, meaning that all the critical states can be successfully identified and there is
no safety risk. In terms of alert states (ID: 5, 6, 7, 8), Recall value of State 6 is 0.92, which means 92% samples in
State 6 are correctly predicted. 5.2. Stall Detection and Alerting By examining the 6th row in the confusion matrix, the rest 8% samples
are misclassified as State 1, which is in the safe region. This situation may lead to dangerous results
since the wing is already in the alert states yet there is no warning. From the other perspective, the
precision value of State 7 is 0.92, which indicates that among all samples predicted as State 7, there are
8% samples actually belonging to State 4 as shown in the 7th column of the confusion matrix. This
value can be interpreted as the false-alarm ratio that the wing flying in the safe region yet receives
a false alert. For safe states (ID: 1, 2, 3, 4), the misclassified samples are for State 3 and State 4, in which 8%
samples of State 3 are predicted as State 2 while 8% samples of State 4 are identified as State 7, which
is the false alarm. Sensors 2018, 18, x
16 of 21 For safe states (ID: 1, 2, 3, 4), the misclassified samples are for State 3 and State 4, in which 8%
samples of State 3 are predicted as State 2 while 8% samples of State 4 are identified as State 7, which
is the false alarm. Sensors 2018, 18, x
16 of 21 Figure 9. Confusion matrix of flight state identification. Figure 9. Confusion matrix of flight state identification. Figure 9. Confusion matrix of flight state identification. Figure 9. Confusion matrix of flight state identification. Further, we select the different number of features from the modified distance evaluation (MDE)
method and use the same neural network structure for training and testing. The comparison on the
overall identification accuracy between MDV and various MDE is shown in Figure 10. The x axis
denotes number of top ranked features selected. Further, we select the different number of features from the modified distance evaluation (MDE)
method and use the same neural network structure for training and testing. The comparison on the
overall identification accuracy between MDV and various MDE is shown in Figure 10. The x axis
denotes number of top ranked features selected. It can be seen that if we use the same number of input as MDV, features selected by MDE lead to
a pool result of 0.33. The identification accuracy reaches the same level as MDV until the number of
top ranked features selected from MDE increases to 20. 5.2. Stall Detection and Alerting This shows that our proposed method MDV is
able to address the collinearity problem and uses fewer features to achieve superior performance with
a considerable model complexity reduction. 17 of 21
the
axis Sensors 2018, 18, 1379
method and us
overall identifi Figure 10. Identification accuracy between MDV and various MDE. Figure 10. Identification accuracy between MDV and various MDE. Figure 10. Identification accuracy between MDV and various MDE. Figure 10. Identification accuracy between MDV and various MDE. to a pool res
f
k
6. Conclusions of top ranked features selected from MDE increases to 20. This shows that our proposed method
MDV is able to address the collinearity problem and uses fewer features to achieve superior
performance with a considerable model complexity reduction. This paper focuses on the feature engineering in structural vibration signals obtained from
a self-sensing composite wing through wind tunnel experiments. In addition to common statistical
features from the time domain and frequency domain, complex features from the information domain
inspired by electroencephalograph analysis and mechanical fault diagnosis are also extracted, some of
which exhibit good classification ability. A novel filter feature selection method (MDV) is proposed by
combining the modified distance evaluation (MDE) algorithm and the variance inflation factor (VIF). MDE is able to select individually powerful features but cannot address high collinearity. VIF is then
applied for each top ranked feature to remove highly correlated elements. Results from both general
flight state identification and stall detection & alerting demonstrate that this method can reduce the
model complexity with fewer features while maintain a high identification accuracy. Knowledge can
be gained by calculating the limited important features obtained by MDV efficiently for flight state
identification using light-weight machine learning models. This would save considerable efforts in
feature extraction and feature selection by manpower and has the potential to provide autonomous
control with real-time flight state monitoring. For multi-sensor utilizations, this method can be
applied to each sensor and ensemble methods can be developed to fuse multi-source results for more
robust identification. Author Contributions: X.C. analyzed the data and developed the feature selection method; F.K. and F.-K.C. designed the self-sensing wing and performed the wind tunnel experiments; Q.W. provides feature extraction
algorithms; H.R. and F.-K.C. coordinated the research and revised the manuscript. Funding: This research was funded by the National Natural Science Foundation of China (Grant No. 51705242),
Shanghai Sailing Program (Grant No. 16YF1404900), and the U.S. Air Force Office of Scientific Research (AFOSR)
program “Avian-Inspired Multi-functional Morphing Vehicles” under grant FA9550-16-1-0087 with Program
Manager Byung-Lip (“Les”) Lee. Acknowledgments: The authors would like to thank Pengchuan Wang, Ravi Gondaliya, Jun Wu and Shaobo Liu
for their help during the wind tunnel experiments. Also, the authors would like to acknowledge the support of
Lester Su and John Eaton in the wind tunnel facility at Stanford University. Conflicts of Interest: The authors declare no conflict of interest. References 1. NASA Fly-By-Feel Systems Represent The Next Revolution In Aircraft Controls. Available online:
https://www.nasa.gov/centers/dryden/news/X-Press/aerovations/2011/fly-by-feel.html (accessed on
16 May 2017). 2. Kopsaftopoulos, F.; Nardari, R.; Li, Y.H.; Chang, F.K. A stochastic global identification framework for
aerospace structures operating under varying flight states. Mech. Syst. Signal Process. 2018, 98, 425–447. [CrossRef] 3. Brenner, M.J. Controller Performance Evaluation of Fluy-by-feel (FBF) Technology. Available online:
https://nari.arc.nasa.gov/node/448 (accessed on 16 May 2017). 4. Mangalam, A.S.; Brenner, M.J. Fly-by-Feel Sensing and Control: Aeroservoelasticity. AIAA Atmos
Mech. Conf. 2014. [CrossRef] 5. Suh, P.; Chin, A.; Mavris, D. Virtual Deformation Control of the X-56A Model with Simulated Fiber Optic
Sensors. AIAA Atmos. Flight Mech. Conf. 2013. [CrossRef] 6. Suh, P.M.; Chin, A.; Mavris, D.N. Robust Modal Filtering and Control of the X-56A Model with Simulated
Fiber Optic Sensor Failures. AIAA Atmos. Flight Mech. Conf. 2014. [CrossRef] 7. Lanzara, G.; Feng, J.; Chang, F.-K. Design of Micro-Scaled Highly Expandable Networks of Polymer Based
Substrates for Macro-Scale Applications. Smart Mater. Struct. 2010, 19, 045013. [CrossRef] 8. Salowitz, N.; Guo, Z.; Li, Y.H.; Kim, K.; Lanzara, G.; Chang, F.K. Bio-inspired stretchable network-based
intelligent composites. J. Compos. Mater. 2013, 47, 97–105. [CrossRef] 9. Salowitz, N.; Guo, Z.; Roy, S.; Nardari, R.; Li, Y.H.; Kim, S.J.; Kopsaftopoulos, F.; Chang, F.K. Recent advancements and vision toward stretchable bio-inspired networks for intelligent structures. Struct. Heal. Monit. 2014, 13, 609–620. [CrossRef] 10. Guo, Z.; Aboudi, U.; Peumans, P.; Howe, R.T.; Chang, F.K. A Super Stretchable Organic Thin-Film Diodes
Network That Can Be Embedded into Carbon Fiber Composite Materials for Sensor Network Applications. J. Microelectromech. Syst. 2016, 25, 524–532. [CrossRef] 11. Kopsaftopoulos, F.P.; Nardari, R.; Li, Y.-H.; Wang, P.; Ye, B.; Chang, F.-K. Experimental identification of
structural dynamics and aeroelastic properties of a self-sensing smart composite wing. In Proceedings of the
10th International Workshop on Structural Health Monitoring, Stanford, CA, USA, 1–3 September 2015. 12. Roy, S.; Lonkar, K.; Janapati, V.; Chang, F.-K. A novel physics-based temperature compensation model for
structural health monitoring using ultrasonic guided waves. Struct. Heal. Monit. Int. J. 2014, 13, 321–342. [CrossRef] 13. Huang, R.; Zhao, Y.; Hu, H. Wind-Tunnel Tests for Active Flutter Control and Closed-Loop Flutter
Identification. AIAA J. 2016, 54, 1–11. [CrossRef] 14. Pang, Z.Y.; Cesnik, C.E.S. Strain state estimation of very flexible unmanned aerial vehicle. In Proceedings of
the 57th AIAA/ASCE/AHS/ASC Structures, Structural Dynamics, and Materials Conference, San Diego,
CA, USA, 4–8 January 2016. 15. Appendix A The expressions of selected features by different feature selection methods are shown in Table A1. 18 of 21 Sensors 2018, 18, 1379 Table A1. Top 10 ranking feature expressions. R
UFS_m
SVM_L1
GBDT
STAB
MDE
MDV
1
t25 = ∑N
n=1 (x(n)−t1)9
N·t29
I3 = MSE[3]
I9 = HST
I9 = HST
f10 = f6
f5
f10 = f6
f5
2
f9 =
∑K
k=1 frk2y(k)
√
∑K
k=1 y(k)∑K
k=1 frk4y(k)
I5 = PFD
I2 = MSE[2]
t12 = ∑N
n=1|x(n)−t1|
N
f1 = ∑K
k=1 y(k)
N
f5 = ∑K
k=1 ( frk·y(k))
∑K
k=1 y(k)
3
t6 =
q
∑N
n=1 (x(n))2
N
I1 = MSE[1]
I8 = ApEn
t21 = ∑N
n=1 (x(n)−t1)5
N·t25
t2 =
q
∑N
n=1(x(n)−t1)2
N
t5 = ∑N
n=1(x(n)−t1)2
N
4
t2 =
q
∑N
n=1(x(n)−t1)2
N
t25 = ∑N
n=1 (x(n)−t1)9
N·t29
t14 = t6
t8
t20 = ∑N
n=1 (x(n)−t1)4
N·t24
t6 =
q
∑N
n=1 (x(n))2
N
f3 = ∑K
k=1 (y(k)−f1)3
K(√
f2)
3
5
t5 = ∑N
n=1(x(n)−t1)2
N
I8 = ApEn
I1 = MSE[1]
t19 = ∑N
n=1 (x(n)−t1)3
N·t23
f6 =
q
∑K
k=1 ( f rk−f5)2y(k)
K
I4 = PMMSE
6
t4 = ∑N
n=1 (x(n)−t1)4
N
t19 = ∑N
n=1 (x(n)−t1)3
N·t23
I6 = HFD
t18 = t4
t64
f3 = ∑K
k=1 (y(k)−f1)3
K(√
f2)
3
I7 = FI
7
I2 = MSE[2]
f8 =
r
∑K
k=1 f rk4y(k)
∑K
k=1 f rk2y(k)
I3 = MSE[3]
t17 = t3
t63
t12 = ∑N
n=1|x(n)−t1|
N
I3 = MSE[3]
8
t23 = ∑N
n=1 (x(n)−t1)7
N·t27
t13 = t9
t6
t1 = ∑N
n=1 x(n)
N
t16 = t9
t7
t8 = ∑N
n=1|x(n)|
N
I8 = ApEn
9
I4 = PMMSE
I6 = HFD
t21 = ∑N
n=1 (x(n)−t1)5
N·t25
t15 = t9
t8
f11 = ∑K
k=1 ( f rk−f5)3y(k)
K· f63
t14 = t6
t8
10
t17 = t3
t63
t10 = min(x(n))
I7 = FI
t14 = t6
t8
t10 = min(x(n))
t23 = ∑N
n=1 (x(n)−t1)7
N·t27 19 of 21 19 of 21 Sensors 2018, 18, 1379 References Kohavi, R.; John, G.H. Wrappers for feature subset selection. Artif. Intell. 1997, 97, 273–324. [CrossRef]
26
Saeys, Y; Inza, I ; Larrañaga, P A review of feature selection techniques in bioinformatics Bioinformatics 26. Saeys, Y.; Inza, I.; Larrañaga, P. A review of feature selection techniques in bioinformatics. Bioinformatics
2007, 23, 2507–2517. [CrossRef] [PubMed] 27. Chen, X.; Kopsaftopoulos, F.; Cao, H.; Chang, F.-K. Intelligent flight state identification of a self-sensing wing
through neural network modelling. In Proceedings of the 11th International Workshop on Structural Health
Monitoring, IWSHM 2017, Stanford, CA, USA, 12–14 September 2017. 28. Welch, P.D. The Use of Fast Fourier Transform for the Estimation of Power Spectral: A Method Based on Time
Averaging Over Short Modified Periodograms. IEEE Trans. Audio Electroacoust. 1967, 15, 70–73. [CrossRef] 28. Welch, P.D. The Use of Fast Fourier Transform for the Estimation of Power Spectral: A Method Based on Time
Averaging Over Short Modified Periodograms. IEEE Trans. Audio Electroacoust. 1967, 15, 70–73. [CrossRef]
29. He, Y.; Huang, J.; Zhang, B. Approximate entropy as a nonlinear feature parameter for fault diagnosis in 29. He, Y.; Huang, J.; Zhang, B. Approximate entropy as a nonlinear feature parameter for fault diagnosis in
rotating machinery. Meas. Sci. Technol. 2012, 23, 045603. [CrossRef] 30. Petrosian, A. Kolmogorov complexity of finite sequences and recognition of different preictal EEG patterns. In Proceedings of the Eighth IEEE Symposium on Computer-Based Medical Systems, Lubbock, TX, USA,
9–10 June 1995. 31. Costa, M.; Goldberger, A.L.; Peng, C. Multiscale Entropy Analysis of Complex Physiologic Time Series. Phys. Rev. Lett. 2002, 89, 6–9. [CrossRef] [PubMed] 32. Jiang, W.; Dong, K.; Zhu, Y.; Wang, H. Fault Feature Identification Based on Partial Mean of Multi-scale
Entropy for Hydraulic Pump. Hydraul. Pneum. 2016, 4, 518–522. [CrossRef] 33. Falconer, K. Fractal geometry: Mathematical foundations and applications, 2nd ed.; John Wiley & Sons:
West Sussex, UK, 2003. 34. Higuchi, T. Approach to an irregular time series on the basis of the fractal theory. Phys. D Nonlinear Phenom. 1988, 31, 277–283. [CrossRef] 35. James, C.J.; Lowe, D. Extracting multisource brain activity from a single electromagnetic channel. Artif. Intell. Med. 2003, 28, 89–104. [CrossRef] 36. Pincus, S.M.; Gladstone, I.M.; Ehrenkranz, R. A regularity statistic for medical data analysis. J. Clin. Monit. 1991, 7, 335–345. [CrossRef] [PubMed] 37. Balli, T.; Palaniappan, R. A combined linear & nonlinear approach for classification of epileptic EEG signals. Meinshausen, N. Stability selection (Slides). J. R. Stat. Soc. Ser. B Stat. Methodol. 2009, 72, 1–30. [CrossRef] References Sodja, J.; Werter, N.; Dillinger, J.K.; De Breuker, R. Dynamic Response of Aeroelastically Tailored Composite
Wing: Analysis and Experiment. In Proceedings of the 57th AIAA/ASCE/AHS/ASC Structures, Structural
Dynamics, and Materials Conference, San Diego, CA, USA, 4–8 January 2016. 16. Kopsaftopoulos, F.; Nardari, R.; Li, Y.-H.; Wang, P.; Chang, F.-K. Stochastic global identification of
a bio-inspired self-sensing composite UAV wing via wind tunnel experiments. In Proceedings of the
Health Monitoring of Structural and Biological Systems 2016. International Society for Optics and Photonics,
Las Vegas, NV, USA, 2016. 17. Kopsaftopoulos, F.P.; Fassois, S.D. Vector-dependent functionally pooled ARX models for the identification
of systems under multiple operating conditions. IFAC Proc. Vol. 2012, 16, 310–315. [CrossRef] 18. Guyon, I. Feature Extraction Foundations and Applications; Springer: Berlin, Germany, 2006; Volume 207,
ISBN 9783540354871. 19. Samanta, B. Gear fault detection using artificial neural networks and support vector machines with genetic
algorithms. Mech. Syst. Signal Process. 2004, 18, 625–644. [CrossRef] 20. Shen, Z.; Chen, X.; Zhang, X.; He, Z. A novel intelligent gear fault diagnosis model based on EMD and
multi-class TSVM. Meas. J. Int. Meas. Confed. 2012, 45, 30–40. [CrossRef] 20 of 21 20 of 21 Sensors 2018, 18, 1379 21. Xi, Y.L.; Xi, Z.H.; Xi, Y.Z. Fault Diagnosis of Rotating Machinery Based on Multiple ANFIS Combination
with Gas Fault diagnosis of rotating machinery based on multiple ANFIS combination with GAs. Mech. Syst. Signal Process. 2007, 21, 2280–2294. [CrossRef] Guyon, I.; Elisseeff, A. An Introduction to Variable and Feature Selection. J. Mach. Learn. Res. 2003, 3
1157–1182. [CrossRef] 23. Zhou, L.; Si, Y.W.; Fujita, H. Predicting the listing statuses of Chinese-listed companies using decision trees
combined with an improved filter feature selection method. Knowl.-Based Syst. 2017, 128, 93–101. [CrossRef] 23. Zhou, L.; Si, Y.W.; Fujita, H. Predicting the listing statuses of Chinese-listed companies using decision trees
combined with an improved filter feature selection method. Knowl.-Based Syst. 2017, 128, 93–101. [CrossRef]
24. Chandrashekar, G.; Sahin, F. A survey on feature selection methods. Comput. Electr. Eng. 2014, 40, 16–28. [CrossRef] combined with an improved filter feature selection method. Knowl.-Based Syst. 2017, 128, 93–101. [CrossRef]
24. Chandrashekar, G.; Sahin, F. A survey on feature selection methods. Comput. Electr. Eng. 2014, 40, 16–28. [CrossRef] 24. Chandrashekar, G.; Sahin, F. A survey on feature selection methods. Comput. Electr. Eng. 2014, 40, 16–28. [CrossRef] 25. Kohavi, R.; John, G.H. Wrappers for feature subset selection. Artif. Intell. 1997, 97, 273–324. [CrossRef] 25. References In Proceedings of the 2009 4th International IEEE/EMBS Conference on Neural Engineering, Antalya, Turkey,
29 April–2 May 2009; pp. 714–717. 38. Yang, B.; Kim, K. Application of Dempster–Shafer theory in fault diagnosis of induction motors using
vibration and current signals. Mech. Syst. Signal Process. 2006, 20, 403–420. [CrossRef] 39. Dormann, C.F.; Elith, J.; Bacher, S.; Buchmann, C.; Carl, G.; Carré, G.; Marquéz, J.R.G.; Gruber, B.;
Lafourcade, B.; Leitão, P.J.; et al. Collinearity: A review of methods to deal with it and a simulation
study evaluating their performance. Ecography 2013, 36, 27–46. [CrossRef] 40. Peng, H.; Long, F.; Ding, C. Feature selection based on mutual information: Criteria of max-dependency. IEEE Trans. Pattern Anal. Mach. Intell. 2005, 27, 1226–1238. [CrossRef] [PubMed] 41. Burges, C.J.C. A tutorial on support vector machines for pattern. Data Min. Knowl. Discov. 1998, 2, 955–974. [CrossRef] 42. Ogutu, J.O.; Schulz-Streeck, T.; Piepho, H.-P. Genomic selection using regularized linear regression models:
Ridge regression, lasso, elastic net and their extensions. BMC Proc. 2012, 6, S10. [CrossRef] [PubMed] Ridge regression, lasso, elastic net and their extensions. BMC Proc. 2012, 6, S10. [CrossRef] [PubMed]
43. Natekin, A.; Knoll, A. Gradient boosting machines, a tutorial. Front. Neurorobot. 2013, 7, 21. [CrossRef]
[PubMed] 43. Natekin, A.; Knoll, A. Gradient boosting machines, a tutorial. Front. Neurorobot. 2013, 7, 21. [CrossRef]
[PubMed] 43. Natekin, A.; Knoll, A. Gradient boosting machines, a tutorial. Front. Neurorobot. 2013, 7, 21. [CrossRef]
[PubMed] 44. Meinshausen, N. Stability selection (Slides). J. R. Stat. Soc. Ser. B Stat. Methodol. 2009, 72, 1–30. [CrossRef]
45. Van Der Maaten, L.; Hinton, G. Visualizing Data using t-SNE. J. Mach. Learn. Res. 2008, 620, 267–284. [CrossRef] 44. Meinshausen, N. Stability selection (Slides). J. R. Stat. Soc. Ser. B Stat. Methodol. 2009, 72, 1–30. [CrossRef] 44. Meinshausen, N. Stability selection (Slides). J. R. Stat. Soc. Ser. B Stat. Methodol. 2009, 72, 1–30. [CrossRef]
45
V
D
M
t
L
Hi t
G Vi
li i
D t
i
t SNE J M
h
L
R
2008 620 267 284 45. Van Der Maaten, L.; Hinton, G. Visualizing Data using t-SNE. J. Mach. Learn. Res. 2008, 620, 267–284. [CrossRef] 21 of 21 Sensors 2018, 18, 1379 46. Davis, J.; Goadrich, M. The relationship between Precision-Recall and ROC curves. In Proceedings of the
23rd International Conference on Machine Learning—ICML ’06, New York, NY, USA, 25–29 June 2006;
pp. 233–240. 46. Davis, J.; Goadrich, M. © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). 46.
Davis, J.; Goadrich, M. The relationship between Precision-Recall and ROC curves. In Proceedings of the
23rd International Conference on Machine Learning—ICML ’06, New York, NY, USA, 25–29 June 2006;
pp. 233–240. pp
47.
Powers, D.M.W. Evaluation: From Precision, Recall and F-Measure To Roc, Informedness, Markedness &
Correlation. J. Mach. Learn. Technol. 2011, 2, 37–63. g
pp. 233–240.
47.
Powers, D.M.W. Evaluation: From Precision, Recall and F-Measure To Roc, Informedness, Markedness &
Correlation. J. Mach. Learn. Technol. 2011, 2, 37–63. References The relationship between Precision-Recall and ROC curves. In Proceedings of the
23rd International Conference on Machine Learning—ICML ’06, New York, NY, USA, 25–29 June 2006;
pp. 233–240. 7. Powers, D.M.W. Evaluation: From Precision, Recall and F-Measure To Roc, Informedness, Markedne
Correlation. J. Mach. Learn. Technol. 2011, 2, 37–63. © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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